US20100035236A1 - Enrichment method for variant proteins with altered binding properties - Google Patents
Enrichment method for variant proteins with altered binding properties Download PDFInfo
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- US20100035236A1 US20100035236A1 US12/508,859 US50885909A US2010035236A1 US 20100035236 A1 US20100035236 A1 US 20100035236A1 US 50885909 A US50885909 A US 50885909A US 2010035236 A1 US2010035236 A1 US 2010035236A1
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
- C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
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- C12N2795/00—Bacteriophages
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- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/14011—Details ssDNA Bacteriophages
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- C12N2795/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/61—Growth hormones [GH] (Somatotropin)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/802—Protein-bacteriophage conjugates
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S930/00—Peptide or protein sequence
- Y10S930/01—Peptide or protein sequence
- Y10S930/12—Growth hormone, growth factor other than t-cell or b-cell growth factor, and growth hormone releasing factor; related peptides
Definitions
- This invention relates to the preparation and systematic selection of novel binding proteins having altered binding properties for a target molecule. Specifically, this invention relates to methods for producing foreign polypeptides mimicking the binding activity of naturally occurring binding partners. In preferred embodiments, the invention is directed to the preparation of therapeutic or diagnostic compounds that mimic proteins or nonpeptidyl molecules such a hormones, drugs and other small molecules, particularly biologically active molecules such as growth hormone.
- Binding partners are substances that specifically bind to one another, usually through noncovalent interactions. Examples of binding partners include ligand-receptor, antibody-antigen, drug-target, and enzyme-substrate interactions. Binding partners are extremely useful in both therapeutic and diagnostic fields.
- Binding partners have been produced in the past by a variety of methods including; harvesting them from nature (e.g., antibody-antigen, and ligand-receptor pairings) and by adventitious identification (e.g. traditional drug development employing random screening of candidate molecules). In some instances these two approaches have been combined. For example, variants of proteins or polypeptides, such as polypeptide fragments, have been made that contain key functional residues that participate in binding. These polypeptide fragments, in turn, have been derivatized by methods akin to traditional drug development. An example of such derivitization would include strategies such as cyclization to conformationally constrain a polypeptide fragment to produce a novel candidate binding partner.
- Geysen In an attempt to overcome these problems, Geysen (Geysen, Immun. Today, 6:364-369 [1985]); and (Geysen et al., Mol. Immun., 23:709-715 [1986]) has proposed the use of polypeptide synthesis to provide a framework for systematic iterative binding partner identification and preparation.
- short polypeptides such as dipeptides
- the most active dipeptides are then selected for an additional round of testing comprising linking, to the starting dipeptide, an additional residue (or by internally modifying the components of the original starting dipeptide) and then screening this set of candidates for the desired activity. This process is reiterated until the binding partner having the desired properties is identified.
- the Geysen et al. method suffers from the disadvantage that the chemistry upon which it is based, peptide synthesis, produces molecules with ill-defined or variable secondary and tertiary structure.
- random interactions accelerate among the various substituent groups of the polypeptide so that a true random population of interactive molecules having reproducible higher order structure becomes less and less attainable.
- interactions between side chains of amino acids, which are sequentially widely separated but which are spatially neighbors freely occur.
- sequences that do not facilitate conformationally stable secondary structures provide complex peptide-sidechain interactions which may prevent sidechain interactions of a given amino acid with the target molecule.
- Such complex interactions are facilitated by the flexibility of the polyamide backbone of the polypeptide candidates.
- candidates may exist in numerous conformations making it difficult to identify the conformer that interacts or binds to the target with greatest affinity or specificity complicating rational drug design.
- a final problem with the iterative polypeptide method of Geysen is that, at present, there are no practical methods with which a great diversity of different peptides can be produced, screened and analyzed.
- the total number of all combinations of hexapeptides that must be synthesized is 64,000,000.
- Even having prepared such a diversity of peptides there are no methods available with which mixtures of such a diversity of peptides can be rapidly screened to select those peptides having a high affinity for the target molecule.
- each “adherent” peptide must be recovered in amounts large enough to carry out protein sequencing.
- fusion phage have been shown to be useful for displaying short mutated peptide sequences for identifying peptides that may react with antibodies (Scott et al., Science 249: 386-390, [1990]) and Cwirla et al., Proc. Natl. Acad. U.S.A 87: 6378-6382, [1990]). or a foreign protein (Devlin et al., Science, 249: 404-406 [1990]).
- fusion phage there are, however, several important limitations in using such “fusion phage” to identify altered peptides or proteins with new or enhanced binding properties.
- prior art methods have been unable to select peptides from a library having the highest binding affinity for a target molecule.
- Ladner WO 90/02802 discloses a method for selecting novel binding proteins displayed on the outer surface of cells and viral particles where it is contemplated that the heterologous proteins may have up to 164 amino acid residues. The method contemplates isolating and amplifying the displayed proteins to engineer a new family of binding proteins having desired affinity for a target molecule. More specifically, Ladner discloses a “fusion phage” displaying proteins having “initial protein binding domains” ranging from 46 residues (crambin) to 164 residues (T4 lysozyme) fused to the M13 gene III coat protein.
- Ladner teaches the use of proteins “no larger than necessary” because it is easier to arrange restriction sites in smaller amino acid sequences and prefers the 58 amino acid residue bovine pancreatic trypsin inhibitor (BPTI).
- Small fusion proteins such as BPTI
- BPTI small fusion proteins
- T4 lysozyme small target molecules
- the preferred protein, BPTI is proposed to be fused to gene III at the site disclosed by Smith et al. or de la Cruz et al., J. Biol.
- hGH Human growth hormone
- hGH is a member of a family of homologous hormones that include placental lactogens, prolactins, and other genetic and species variants or growth hormone (Nicoll, C. S., et al., (1986) Endocrine Reviews 7, 169). hGH is unusual among these in that it exhibits broad species specificity and binds to either the cloned somatogenic (Leung, D. W., et al., [I987 ] Nature 330, 537) or prolactin receptor (Boutin, J. M., et al., [I988 ] Ce; 53, 69).
- the cloned gene for hGH has been expressed in a secreted form in Escherichia coli (Chang, C. N., et al., [I987 ] Gene 55, I89) and its DNA and amino acid sequence has been reported (Goeddel, et al., [I979 ] Nature 281, 544; Gray, et al., [I985 ] Gene 39, 247).
- the three-dimensional structure of hGH is not available. However, the three-dimensional folding pattern for porcine growth hormone (pGH) has been reported at moderate resolution and refinement (Abdel-Meguid, S. S., et al., [I987 ] Proc. Natl. Acad. Sci.
- hGH Human growth hormone
- It is another object of this invention to prepare candidate binding substances comprising fusion proteins of a phage coat protein and a heterologous polypeptide where the polypeptide is greater than 100 amino acids in length and may be more than one subunit and is displayed on a phagemid particle where the polypeptide is encoded by the phagemid genome.
- Still another object of the invention is the production of growth hormone variants that exhibit stronger affinity for growth hormone receptor and binding protein.
- a method for selecting novel binding polypeptides comprising: (a) constructing a replicable expression vector comprising a first gene encoding a polypeptide, a second gene encoding at least a portion of a natural or wild-type phage coat protein wherein the first and second genes are heterologous, and a transcription regulatory element operably linked to the first and second genes, thereby forming a gene fusion encoding a fusion protein; (b) mutating the vector at one or more selected positions within the first gene thereby forming a family of related plasmids; (c) transforming suitable host cells with the plasmids; (d) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein; (e) culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that
- the method for selecting novel binding proteins where the proteins are composed of more than one subunit is achieved by selecting novel binding peptides comprising constructing a replicable expression vector comprising a transcription regulatory element operably linked to DNA encoding a protein of interest containing one or more subunits, wherein the DNA encoding at least one of the subunits is fused to the DNA encoding at least a portion of a phage coat protein; mutating the DNA encoding the protein of interest at one or more selected positions thereby forming a family of related vectors; transforming suitable host cells with the vectors; infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein; culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface
- the plasmid is under tight control of the transcription regulatory element, and the culturing conditions are adjusted so that the amount or number of phagemid particles displaying more than one copy of the fusion protein on the surface of the particle is less than about 1%. Also preferably, amount of phagemid particles displaying more than one copy of the fusion protein is less than 10% the amount of phagemid particles displaying a single copy of the fusion protein. Most preferably the amount is less than 20%.
- the expression vector will further contain a secretory signal sequences fused to the DNA encoding each subunit of the polypeptide, and the transcription regulatory element will be a promoter system.
- Preferred promoter systems are selected from; Lac Z, ⁇ PL , TAC, T 7 polymerase, tryptophan, and alkaline phosphatase promoters and combinations thereof.
- the first gene will encode a mammalian protein, preferably the protein will be selected from; human growth hormone (hGH), N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin A-chain, insulin B-chain, proinsulin, relaxin A-chain, relaxin B-chain, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and leutinizing hormone (LH), glycoprotein hormone receptors, calcitonin, glucagon, factor VIII, an antibody, lung surfactant, urokinase, streptokinase, human tissue-type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hemopoietic growth factor, tumor necrosis factor-alpha and -beta, enkephalinase, human serum albumin, mullerian-inhibiting substance, mouse gonadotropin-associated peptide,
- the first gene will encode a polypeptide of one or more subunits containing more than about 100 amino acid residues and will be folded to form a plurality of rigid secondary structures displaying a plurality of amino acids capable of interacting with the target.
- the first gene will be mutated at codons corresponding to only the amino acids capable of interacting with the target so that the integrity of the rigid secondary structures will be preserved.
- helper phage selected from; 13 KO 7, M13R408, M13-VCS, and Phi X 174.
- the preferred helper phage is M13KO7
- the preferred coat protein is the M13 Phage gene III coat protein.
- the preferred host is E. coli , and protease deficient strains of E. coli . Novel hGH variants selected by the method of the present invention have been detected.
- Phagemid expression vectors were constructed that contain a suppressible termination codon functionally located between the nucleic acids encoding the polypeptide and the phage coat protein.
- FIG. 1 Strategy for displaying large proteins on the surface of filamentous phage and enriching for altered receptor binding properties.
- a plasmid, phGH-M13gIII was constructed that fuses the entire coding sequence of hGH to the carboxyl terminal domain of M13 gene III. Transcription of the fusion protein is under control of the lac promoter/operator sequence, and secretion is directed by the stII signal sequence.
- Phagemid particles are produced by infection with the “helper” phage, M13KO7, and particles displaying hGH can be enriched by binding to an affinity matrix containing the hGH receptor.
- the wild-type gene III (derived from the M13KO7 phage) is diagramed by 4-5 copies of the multiple arrows on the tip of the phage, and the fusion protein (derived from the phagemid, phGH-M13gIII) is indicated schematically by the folding diagram of hGH replacing the arrow head.
- FIG. 2 Immunoblot of whole phage particles shows that hGH comigrates with phage.
- Phagemid particles purified in a cesium chloride gradient were loaded into duplicate wells and electrophoresed through a 1% agarose gel in 375 mM Tris, 40 mM glycine pH 9.6 buffer. The gel was soaked in transfer buffer (25 mM Tris, pH 8.3, 200 mM glycine, 20% methanol) containing 2% SDS and 2% ⁇ -mercaptoethanol for 2 hours, then rinsed in transfer buffer for 6 hours. The proteins in the gel were then electroblotted onto immobilon membranes (Millipore).
- the membrane containing one set of samples was stained with Coomassie blue to show the position of the phage proteins (A).
- the duplicate membrane was immuno-stained for hGH by reacting the membrane with polyclonal rabbit anti-hGH antibodies followed by reaction with horseradish peroxidase conjugated goat anti-rabbit IgG antibodies (B).
- Lane 1 contains the M13KO7 parent phage and is visible only in the Coomassie blue stained membrane, since it lacks hGH.
- Lanes 2 and 3 contain separate preparations of the hormone phagemid particles which is visible both by Coomassie and hGH immuno-staining.
- the difference in migration distance between the parent M13KO7 phage and hormone phagemid particles reflects the different size genomes that are packaged within (8.7 kb vs. 5.1 kb, respectively).
- FIG. 3 Summary diagram of steps in the selection process for an hGH-phage library randomized at codons 172, 174, 176, and 178.
- the template molecules, pH0415, containing a unique KpnI restriction site and the hGH(R178G, I179T) gene was mutagenized as described in the text and electrotransformed into E. coli strain WJM101 to obtain the initial phagemid library, Library 1.
- An aliquot (approximately 2%) from Library 1 was used directly in an initial selection round as described in the text to yield Library 1G.
- dsDNA double-stranded DNA (dsDNA) was prepared from Library I, digested with restriction enzyme KpnI to eliminate template background, and electrotransformed into WJM101 to yield Library 2.
- FIG. 4 Structural model of hGH derived from a 2.8 ⁇ folding diagram of porcine growth hormone determined crystallographically. Location of residues in hGH that strongly modulate its binding to the hGH-binding protein are within the shaded circle. Alanine substitutions that cause a greater than tenfold reduction ( ⁇ ), a four- to tenfold reduction ( ⁇ ), or increase ( ⁇ ), or a two- to fourfold reduction ( ⁇ ), in binding affinity are indicated.
- Helical wheel projections in the regions of ⁇ -helix reveal their amphipathic quality. Blackened, shaded, or nonshaded residues are charged, polar, or nonpolar, respectively. In helix-4 the most important residues for mutation are on the hydrophilic face.
- FIG. 5 Amino acid substitutions at positions 172, 174, 176 and 178 of hGH (The notation, e.g. KSYR, denotes hGH mutant 172K/174S/176Y/178R) found after sequencing a number of clones from rounds 1 and 3 of the selection process for the pathways indicated (hGH elution; Glycine elution; or Glycine elution after pre-adsorption).
- Non-functional sequences i.e. vector background, or other prematurely terminated and/or frame-shifted mutants
- Functional sequences which contained a non-silent, spurious mutation i.e. outside the set of target residues
- Protein sequences which appeared more than once among all the sequenced clones, but with different DNA sequences, are marked with a “#”. Protein sequences which appeared more than once among the sequenced clones and with the same DNA sequence are marked with a “*”. Note that after three rounds of selection, 2 different contaminating sequences were found; these clones did not correspond to cassette mutants, but to previously constructed hormone phage.
- the pS0643 contaminant corresponds to wild-type hGH-phage (hGH “KEFR” (SEQ ID NO:44)).
- the pH0457 contaminant which dominates the third-round glycine-selected pool of phage, corresponds to a previously identified mutant of hGH, “KSYR.”
- K a previously identified mutant of hGH
- the amplification of these contaminants emphasizes the ability of the hormone-phage selection process to select for rarely occurring mutants.
- the convergence of sequences is also striking in all three pathways: R or K occurs most often at positions 172 and 178; Y or F occurs most often at position 176; and S, T, A, and other residues occur at position 174.
- FIG. 6 Sequences from phage selected on hPRLbp-beads in the presence of zinc. The notation is as described in FIG. 5 . Here, the convergence of sequences is not predictable, but there appears to be a bias towards hydrophobic sequences under the most stringent (Glycine) selection conditions; L, W and P residues are frequently found in this pool.
- Glycine Glycine
- FIG. 7 Sequences from phage selected on hPRLbp-beads in the absence of zinc. The notation is as described in FIG. 5 . In contrast to the sequences of FIG. 6 , these sequences appear more hydrophilic. After 4 rounds of selection using hGH elution, two clones (ANHQ (SEQ ID NO:45), and TLDT/171V (SEQ ID NO:108)) dominate the pool.
- FIG. 8 Sequences from phage selected on blank beads. The notation is as described in FIG. 5 . After three rounds of selection with glycine elution, no siblings were observed and a background level of non-functional sequences remained.
- FIG. 9 Construction of phagemid fl ori from pHO415.
- This vector for cassette mutagenesis and expression of the hGH-gene III fusion protein was constructed as follows. Plasmid pS0643 was constructed by oligonucleotide-directed mutagenesis of pS0132, which contains pBR322 and f1 origins of replication and expresses an hGH-gene III fusion protein (hGH residues 1-191, followed by a single Gly residue, fused to Pro-198 of gene III) under the control of the E. coli phoA promoter.
- Mutagenesis was carried out with the oligonucleotide 5′-GGC-AGC-TGT-GGC-T TC-TAG-A GT-GGC-GGC-GGC-TCT-GGT-3′ (SEQ ID NO:1), which introduced a XbaI site (underlined) and an amber stop codon (TAG) following Phe-191 of hGH.
- FIG. 10 A. Diagram of plasmid pDH188 insert containing the DNA encoding the light chain and heavy chain (variable and constant domain 1) of the F ab humanized antibody directed to the HER-2 receptor.
- V L and V H are the variable regions for the light and heavy chains, respectively.
- C k is the constant region of the human kappa light chain.
- CH1 G1 is the first constant region of the human gamma 1 chain. Both coding regions start with the bacterial st II signal sequence.
- B A schematic diagram of the entire plasma pDH188 containing the insert described in 5A. After transformation of the plasmid into E. coli SR101 cells and the addition of helper phage, the plasmid is packaged into phage particles. Some of these particles display the F ab -p III fusion (where p III is the protein encoded by the M13 gene III DNA). The segments in the plasmid figure correspond to the insert shown in 5A.
- FIG. 11 A through C are collectively referred to here as FIG. 11 .
- the amino acid sequence of the light chain is also shown (Seq. ID No: 25), as is the amino acid sequence of the heavy chain p III fusion (Seq. ID No:26).
- FIG. 12 Enrichment of wild-type 4D5 F ab phagemid from variant F ab phagemid.
- Mixtures of wild-type phagemid and variant 4D5 F ab phagemid in a ratio of 1:1,000 were selected on plates coated with the extra-cellular domain protein of the HER-2 receptor. After each round of selection, a portion of the eluted phagemid were infected into E. coli and plasmid DNA was prepared. This plasmid DNA was then digested with Eco RV and Pst I, separated on a 5% polyacrylamide gel, and stained with ethidium bromide. The bands were visualized under UV light. The bands due to the wild-type and variant plasmids are marked with arrows.
- the first round of selection was eluted only under acid conditions; subsequent rounds were eluted with either an acid elution (left side of Figure) or with a humanized 4D5 antibody wash step prior to acid elution (right side of Figure) using methods described in Example VIII.
- H91A amino acid histidine at position 91 on the V L chain mutated to alanine; indicated as ‘A’ lanes in Figure
- Y49A amino acid tyrosine at position 49 on the V L chain mutated to alanine; indicated as ‘B’ lanes in the Figure
- Y92A amino acid tyrosine at position 92 on the V L chain mutated to alanine; indicated as ‘C’ lanes in the Figure.
- Amino acid position numbering is according to Kabat et al., ( Sequences of proteins of immunological interest, 4th ed., U.S. Dept of Health and Human Services, Public Health Service, Nat'l. Institute of Health, Bethesda, Md. [1987]).
- FIG. 13 The Scatchard analysis of the RIA affinity determination described in Experimental Protocols is shown here.
- the amount of labeled ECD antigen that is bound is shown on the x-axis while the amount that is bound divided by the amount that is free is shown on the y-axis.
- the slope of the line indicates the K a ; the calculated K d is 1/K a .
- the method of the instant invention comprises a method for selecting novel binding polypeptides, such as protein ligands, having a desired, usually high, affinity for a target molecule from a library of structurally related binding polypeptides.
- the library of structurally related polypeptides, fused to a phage coat protein, is produced by mutagenesis and, preferably, a single copy of each related polypeptide is displayed on the surface of a phagemid particle containing DNA encoding that polypeptide.
- These phagemid particles are then contacted with a target molecule and those particles having the highest affinity for the target are separated from those of lower affinity.
- the high affinity binders are then amplified by infection of a bacterial host and the competitive binding step is repeated. This process is reiterated until polypeptides of the desired affinity are obtained.
- novel binding polypeptides or ligands produced by the method of this invention are useful per se as diagnostics or therapeutics (eg. agonists or antagonists) used in treatment of biological organisms. Structural analysis of the selected polypeptides may also be used to facilitate rational drug design.
- binding polypeptide as used herein is meant any polypeptide that binds with a selectable affinity to a target molecule.
- the polypeptide will be a protein that most preferably contains more than about 100 amino acid residues.
- the polypeptide will be a hormone or an antibody or a fragment thereof.
- high affinity as used herein is meant an affinity constant (K d ) of ⁇ 10 ⁇ 5 M and preferably ⁇ 10 ⁇ 7 M under physiological conditions.
- target molecule as used herein is meant any molecule, not necessarily a protein, for which it is desirable to produce a ligand.
- the target will be a protein and most preferably the target will be a receptor, such as a hormone receptor.
- humanized antibody as used herein is meant an antibody in which the complementarity-determining regions (CDRs) of a mouse or other non-human antibody are grafted onto a human antibody framework.
- human antibody framework is meant the entire human antibody excluding the CDRs.
- the first step in the method of this invention is to choose a polypeptide having rigid secondary structure exposed to the surface of the polypeptide for display on the surface of a phage.
- polypeptide as used herein is meant any molecule whose expression can be directed by a specific DNA sequence.
- the polypeptides of this invention may comprise more than one subunit, where each subunit is encoded by a separate DNA sequence.
- rigid secondary structure any polypeptide segment exhibiting a regular repeated structure such as is found in; ⁇ -helices, 3 10 helices, ⁇ -helices, parallel and antiparallel ⁇ -sheets, and reverse turns.
- Certain “non-ordered” structures that lack recognizable geometric order are also included in the definition of rigid secondary structure provided they form a domain or “patch” of amino acid residues capable of interaction with a target and that the overall shape of the structure is not destroyed by replacement of an amino acid within the structure. It is believed that some non-ordered structures are combinations of reverse turns.
- the geometry of these rigid secondary structures is well defined by ⁇ and ⁇ torsional angles about the ⁇ -carbons of the peptide “backbone”.
- the requirement that the secondary structure be exposed to the surface of the polypeptide is to provide a domain or “patch” of amino acid residues that can be exposed to and bind with a target molecule. It is primarily these amino acid residues that are replaced by mutagenesis that form the “library” of structurally related (mutant) binding polypeptides that are displayed on the surface of the phage and from which novel polypeptide ligands are selected. Mutagenesis or replacement of amino acid residues directed toward the interior of the polypeptide is generally avoided so that the overall structure of the rigid secondary structure is preserved. Some replacement of amino acids on the interior region of the rigid secondary structures, especially with hydrophobic amino acid residues, may be tolerated since these conservative substitutions are unlikely to distort the overall structure of the polypeptide.
- hGH amino acids 167, 171, 175 and 179 were phagemid selected.
- hGH amino acids 10, 14, 18 and 21 were phagemid selected.
- Optimum amino acid changes from a previous cycle may be incorporated into the polypeptide before the next cycle of selection. For example, hGH amino acids substitution 174 (serine) and 176 (tyrosine) were incorporated into the hGH before the phagemid selection of hGH amino acids 167, 171, 175 and 179.
- the amino acid residues that form the binding domain of the polypeptide will not be sequentially linked and may reside on different subunits of the polypeptide. That is, the binding domain tracks with the particular secondary structure at the binding site and not the primary structure.
- mutations will be introduced into codons encoding amino acids within a particular secondary structure at sites directed away from the interior of the polypeptide so that they will have the potential to interact with the target.
- FIG. 2 shows the location of residues in hGH that are known to strongly modulate its binding to the hGH-binding protein (Cunningham et al., Science 247:1461-1465 [1990]).
- representative sites suitable for mutagenesis would include residues 172, 174, 176, and 178 on helix-4, as well as residue 64 located in a “non-ordered” secondary structure.
- polypeptide chosen as a ligand to a target normally bind to that target.
- a glycoprotein hormone such as TSH can be chosen as a ligand for the FSH receptor and a library of mutant TSH molecules are employed in the method of this invention to produce novel drug candidates.
- polypeptides that binds to a target molecule, and includes antibodies.
- Preferred polypeptides are those that have pharmaceutical utility. More preferred polypeptides include; a growth hormone, including human growth hormone, des-N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroid stimulating hormone; thyroxine; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; leutinizing hormone; glucagon; factor VIII; an antibody; lung surfactant; a plasminogen activator, such as urokinase or human tissue-type plasminogen activator (t-PA); bombesin; factor IX, thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; enkephalinase; a serum albumin such as human serum albumin; mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prore
- polypeptides of this invention are human growth hormone and atrial naturetic peptides A, B, and C, endotoxin, subtilisin, trypsin and other serine proteases.
- polypeptide hormones that can be defined as any amino acid sequence produced in a first cell that binds specifically to a receptor on the same cell type (autocrine hormones) or a second cell type (non-autocrine) and causes a physiological response characteristic of the receptor-bearing cell.
- polypeptide hormones include cytokines, lymphokines, neurotrophic hormones and adenohypophyseal polypeptide hormones such as growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, thyrotropin, chorionic gonadotropin, corticotropin, or ⁇ -melanocyte-stimulating hormone, ⁇ -lipotropin gamma-lipotropin and the endorphins; hypothalmic release-inhibiting hormones such as corticotropin-release factor, growth hormone release-inhibiting hormone, growth hormone-release factor; and other polypeptide hormones such as atrial natriuretic peptides A, B or C.
- cytokines such as growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, thyrotropin, chorionic gonadotropin, corticotropin, or ⁇ -melanocyte-
- the gene encoding the desired polypeptide i.e., a polypeptide with a rigid secondary structure
- the DNA encoding the gene may be chemically synthesized (Merrfield, J. Am. Chem. Soc., 85:2149 [1963]).
- the sequence of the gene is not known, or if the gene has not previously been isolated, it may be cloned from a cDNA library (made from RNA obtained from a suitable tissue in which the desired gene is expressed) or from a suitable genomic DNA library.
- probes include monoclonal or polyclonal antibodies (provided that the cDNA library is an expression library), oligonucleotides, and complementary or homologous cDNAs or fragments thereof.
- the probes that may be used to isolate the gene of interest from genomic DNA libraries include cDNAs or fragments thereof that encode the same or a similar gene, homologous genomic DNAs or DNA fragments, and oligonucleotides. Screening the cDNA or genomic library with the selected probe is conducted using standard procedures as described in chapters 10-12 of Sambrook et al., supra.
- PCR polymerase chain reaction methodology
- the gene may be inserted into a suitable vector (preferably a plasmid) for amplification, as described generally in Sambrook et al., supra.
- a suitable vector preferably a plasmid
- Plasmid vectors are the preferred vectors for use herein, as they may be constructed with relative ease, and can be readily amplified. Plasmid vectors generally contain a variety of components including promoters, signal sequences, phenotypic selection genes, origin of replication sites, and other necessary components as are known to those of ordinary skill in the art.
- Promoters most commonly used in prokaryotic vectors include the lac Z promoter system, the alkaline phosphatase pho A promoter, the bacteriophage ⁇ PL promoter (a temperature sensitive promoter), the tac promoter (a hybrid trp - lac promoter that is regulated by the lac repressor), the tryptophan promoter, and the bacteriophage T7 promoter.
- the lac Z promoter system the alkaline phosphatase pho A promoter
- the bacteriophage ⁇ PL promoter a temperature sensitive promoter
- the tac promoter a hybrid trp - lac promoter that is regulated by the lac repressor
- tryptophan promoter a hybrid trp - lac promoter that is regulated by the lac repressor
- the tryptophan promoter a hybrid trp - lac promoter that is regulated by the lac repressor
- the tryptophan promoter the tryptophan promoter
- Preferred promoters for practicing this invention are those that can be tightly regulated such that expression of the fusion gene can be controlled. It is believed that the problem that went unrecognized in the prior art was that display of multiple copies of the fusion protein on the surface of the phagemid particle lead to multipoint attachment of the phagemid with the target. It is believed this effect, referred to as the “chelate effect”, results in selection of false “high affinity” polypeptides when multiple copies of the fusion protein are displayed on the phagemid particle in close proximity to one another so that the target was “chelated”. When multipoint attachment occurs, the effective or apparent Kd may be as high as the product of the individual Kds for each copy of the displayed fusion protein. This effect may be the reason Cwirla and coworkers supra were unable to separate moderate affinity peptides from higher affinity peptides.
- Preferred promoters used to practice this invention are the lac Z promoter and the pho A promoter.
- the lac Z promoter is regulated by the lac repressor protein lac i, and thus transcription of the fusion gene can be controlled by manipulation of the level of the lac repressor protein.
- the phagemid containing the lac Z promoter is grown in a cell strain that contains a copy of the lac i repressor gene, a repressor for the lac Z promoter.
- Exemplary cell strains containing the lac i gene include JM 101 and XL1-blue.
- the host cell can be cotransfected with a plasmid containing both the repressor lac i and the lac Z promoter.
- phagemid particles containing the lac Z promoter are grown in cell strains containing the lac i gene and the cell strains are cotransfected with a plasmid containing both the lac Z and lac i genes.
- an inducer such as isopropylthiogalactoside (IPTG).
- IPTG isopropylthiogalactoside
- the number of fusion proteins per phagemid particle is about 0.1 (number of bulk fusion proteins/number of phagemid particles).
- the most preferred promoter used to practice this invention is pho A. This promoter is believed to be regulated by the level of inorganic phosphate in the cell where the phosphate acts to down-regulate the activity of the promoter. Thus, by depleting cells of phosphate, the activity of the promoter can be increased. The desired result is achieved by growing cells in a phosphate enriched medium such as 2YT or LB thereby controlling the expression of the gene III fusion.
- One other useful component of vectors used to practice this invention is a signal sequence.
- This sequence is typically located immediately 5′ to the gene encoding the fusion protein, and will thus be transcribed at the amino terminus of the fusion protein. However, in certain cases, the signal sequence has been demonstrated to be located at positions other 5′ to the gene encoding the protein to be secreted. This sequence targets the protein to which it is attached across the inner membrane of the bacterial cell.
- the DNA encoding the signal sequence may be obtained as a restriction endonuclease fragment from any gene encoding a protein that has a signal sequence.
- Suitable prokaryotic signal sequences may be obtained from genes encoding, for example, LamB or OmpF (Wong et al., Gene, 68:193 [1983]), MalE, PhoA and other genes.
- a preferred prokaryotic signal sequence for practicing this invention is the E. coli heat-stable enterotoxin II (STII) signal sequence as described by Chang et al., Gene, 55: 189 [1987].
- STII enterotoxin II
- phenotypic selection genes are those encoding proteins that confer antibiotic resistance upon the host cell.
- amp ampicillin resistance gene
- tet tetracycline resistance gene
- Suitable vectors comprising the aforementioned components as well as the gene encoding the desired polypeptide (gene 1) are prepared using standard recombinant DNA procedures as described in Sambrook et al. supra. Isolated DNA fragments to be combined to form the vector are cleaved, tailored, and ligated together in a specific order and orientation to generate the desired vector.
- the DNA is cleaved using the appropriate restriction enzyme or enzymes in a suitable buffer.
- a suitable buffer In general, about 0.2-1 ⁇ g of plasmid or DNA fragments is used with about 1-2 units of the appropriate restriction enzyme in about 20 ⁇ l of buffer solution.
- Appropriate buffers, DNA concentrations, and incubation times and temperatures are specified by the manufacturers of the restriction enzymes. Generally, incubation times of about one or two hours at 37° C. are adequate, although several enzymes require higher temperatures. After incubation, the enzymes and other contaminants are removed by extraction of the digestion solution with a mixture of phenol and chloroform, and the DNA is recovered from the aqueous fraction by precipitation with ethanol.
- the ends of the DNA fragments must be compatible with each other. In some cases, the ends will be directly compatible after endonuclease digestion. However, it may be necessary to first convert the sticky ends commonly produced by endonuclease digestion to blunt ends to make them compatible for ligation. To blunt the ends, the DNA is treated in a suitable buffer for at least 15 minutes at 15° C. with 10 units of the Klenow fragment of DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates. The DNA is then purified by phenol-chloroform extraction and ethanol precipitation.
- the cleaved DNA fragments may be size-separated and selected using DNA gel electrophoresis.
- the DNA may be electrophoresed through either an agarose or a polyacrylamide matrix. The selection of the matrix will depend on the size of the DNA fragments to be separated.
- the DNA is extracted from the matrix by electroelution, or, if low-melting agarose has been used as the matrix, by melting the agarose and extracting the DNA from it, as described in sections 6.30-6.33 of Sambrook et al., supra.
- the DNA fragments that are to be ligated together are put in solution in about equimolar amounts.
- the solution will also contain ATP, ligase buffer and a ligase such as T4 DNA ligase at about 10 units per 0.5 ⁇ g of DNA.
- the vector is at first linearized by cutting with the appropriate restriction endonuclease(s).
- the linearized vector is then treated with alkaline phosphatase or calf intestinal phosphatase. The phosphatasing prevents self-ligation of the vector during the ligation step.
- Prokaryotes are the preferred host cells for this invention.
- Suitable prokaryotic host cells include E. coli strain JM101, E. coli K12 strain 294 (ATCC number 31,446), E. coli strain W3110 (ATCC number 27,325), E. coli X1776 (ATCC number 31,537), E. coli XL-1 Blue (stratagene), and E. coli B; however many other strains of E. coli , such as HB101, NM522, NM538, NM539, and many other species and genera of prokaryotes may be used as well.
- E. coli strain JM101 E. coli K12 strain 294
- E. coli strain W3110 ATCC number 27,325
- E. coli X1776 ATCC number 31,537
- E. coli XL-1 Blue stratagene
- E. coli B E. coli B
- many other strains of E. coli such as HB101, NM522, NM53
- bacilli such as Bacillus subtilis
- enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans
- various Pseudomonas species may all be used as hosts.
- Transformation of prokaryotic cells is readily accomplished using the calcium chloride method as described in section 1.82 of Sambrook et al., supra.
- electroporation may be used to transform these cells.
- the transformed cells are selected by growth on an antibiotic, commonly tetracycline (tet) or ampicillin (amp), to which they are rendered resistant due to the presence of tet and/or amp resistance genes on the vector.
- tet tetracycline
- amp amp
- Plasmid DNA can be isolated using methods known in the art. Two suitable methods are the small scale preparation of DNA and the large-scale preparation of DNA as described in sections 1.25-1.33 of Sambrook et al., supra. The isolated DNA can be purified by methods known in the art such as that described in section 1.40 of Sambrook et al., supra. This purified plasmid DNA is then analyzed by restriction mapping and/or DNA sequencing. DNA sequencing is generally performed by either the method of Messing et al. Nucleic Acids Res., 9:309 [1981] or by the method of Maxam et al. Meth. Enzymol., 65: 499 [1980].
- This invention contemplates fusing the gene enclosing the desired polypeptide (gene 1) to a second gene (gene 2) such that a fusion protein is generated during transcription.
- Gene 2 is typically a coat protein gene of a phage, and preferably it is the phage M13 gene III coat protein, or a fragment thereof. Fusion of genes 1 and 2 may be accomplished by inserting gene 2 into a particular site on a plasmid that contains gene 1, or by inserting gene 1 into a particular site on a plasmid that contains gene 2.
- Insertion of a gene into a plasmid requires that the plasmid be cut at the precise location that the gene is to be inserted. Thus, there must be a restriction endonuclease site at this location (preferably a unique site such that the plasmid will only be cut at a single location during restriction endonuclease digestion).
- the plasmid is digested, phosphatased, and purified as described above.
- the gene is then inserted into this linearized plasmid by ligating the two DNAs together. Ligation can be accomplished if the ends of the plasmid are compatible with the ends of the gene to be inserted.
- the DNAs can be ligated together directly using a ligase such as bacteriophage T4 DNA ligase and incubating the mixture at 16° C. for 1-4 hours in the presence of ATP and ligase buffer as described in section 1.68 of Sambrook et al., supra. If the ends are not compatible, they must first be made blunt by using the Klenow fragment of DNA polymerase I or bacteriophage T4 DNA polymerase, both of which require the four deoxyribonucleotide triphosphates to fill-in overhanging single-stranded ends of the digested DNA.
- a ligase such as bacteriophage T4 DNA ligase
- the ends may be blunted using a nuclease such as nuclease S1 or mung-bean nuclease, both of which function by cutting back the overhanging single strands of DNA.
- the DNA is then religated using a ligase as described above.
- oligonucleotide linkers may be used. The linkers serve as a bridge to connect the plasmid to the gene to be inserted. These linkers can be made synthetically as double stranded or single stranded DNA using standard methods.
- the linkers have one end that is compatible with the ends of the gene to be inserted; the linkers are first ligated to this gene using ligation methods described above.
- the other end of the linkers is designed to be compatible with the plasmid for ligation.
- care must be taken to not destroy the reading frame of the gene to be inserted or the reading frame of the gene contained on the plasmid.
- it may be necessary to design the linkers such that they code for part of an amino acid, or such that they code for one or more amino acids.
- termination codons are UAG (amber), UAA (ocher) and UGA (opel). (Microbiology, Davis et al. Harper & Row, New York, 1980, pages 237, 245-47 and 274).
- the termination codon expressed in a wild type host cell results in the synthesis of the gene 1 protein product without the gene 2 protein attached.
- growth in a suppressor host cell results in the synthesis of detectable quantities of fused protein.
- Such suppressor host cells contain a tRNA modified to insert an amino acid in the termination codon position of the mRNA thereby resulting in production of detectable amounts of the fusion protein.
- suppressor host cells are well known and described, such as E. coli suppressor strain (Bullock et al., BioTechniques 5, 376-379 [1987]). Any acceptable method may be used to place such a termination codon into the mRNA encoding the fusion polypeptide.
- the suppressible codon may be inserted between the first gene encoding a polypeptide, and a second gene encoding at least a portion of a phage coat protein.
- the suppressible termination codon may be inserted adjacent to the fusion site by replacing the last amino acid triplet in the polypeptide or the first amino acid in the phage coat protein.
- the polypeptide When the phagemid is grown in a non-suppressor host cell, the polypeptide is synthesized substantially without fusion to the phage coat protein due to termination at the inserted suppressible triplet encoding UAG, UAA, or UGA. In the non-suppressor cell the polypeptide is synthesized and secreted from the host cell due to the absence of the fused phage coat protein which otherwise anchored it to the host cell.
- Gene 1 encoding the desired polypeptide may be altered at one or more selected codons.
- An alteration is defined as a substitution, deletion, or insertion of one or more codons in the gene encoding the polypeptide that results in a change in the amino acid sequence of the polypeptide as compared with the unaltered or native sequence of the same polypeptide.
- the alterations will be by substitution of at least one amino acid with any other amino acid in one or more regions of the molecule.
- the alterations may be produced be a variety of methods known in the art. These methods include but are not limited to oligonucleotide-mediated mutagenesis and cassette mutagenesis.
- Oligonucleotide-mediated mutagenesis is preferred method for preparing substitution, deletion, and insertion variants of gene 1. This technique is well known in the art as described by Zoller et al. Nucleic Acids Res. 10: 6487-6504 [1987]. Briefly, gene 1 is altered by hybridizing an oligonucleotide encoding the desired mutation to a DNA template, where the template is the single-stranded form of the plasmid containing the unaltered or native DNA sequence of gene 1. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template will thus incorporate the oligonucleotide primer, and will code for the selected alteration in gene 1.
- oligonucleotides of at least 25 nucleotides in length are used.
- An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single-stranded DNA template molecule.
- the oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al. Proc. Nat'l. Acad. Sci. USA, 75: 5765 [1978].
- the DNA template can only be generated by those vectors that are either derived from bacteriophage M13 vectors (the commercially available M13mp18 and M13mp19 vectors are suitable), or those vectors that contain a single-stranded phage origin of replication as described by Viera et al. Meth. Enzymol., 153: 3 [1987]. Thus, the DNA that is to be mutated must be inserted into one of these vectors in order to generate single-stranded template. Production of the single-stranded template is described in sections 4.21-4.41 of Sambrook et al., supra.
- the oligonucleotide is hybridized to the single stranded template under suitable hybridization conditions.
- a DNA polymerizing enzyme usually the Klenow fragment of DNA polymerase I, is then added to synthesize the complementary strand of the template using the oligonucleotide as a primer for synthesis.
- a heteroduplex molecule is thus formed such that one strand of DNA encodes the mutated form of gene 1, and the other strand (the original template) encodes the native, unaltered sequence of gene 1.
- This heteroduplex molecule is then transformed into a suitable host cell, usually a prokaryote such as E. Coli JM101. After growing the cells, they are plated onto agarose plates and screened using the oligonucleotide primer radiolabelled with 32-Phosphate to identify the bacterial colonies that contain the mutated DNA.
- the method described immediately above may be modified such that a homoduplex molecule is created wherein both strands of the plasmid contain the mutation(s).
- the modifications are as follows:
- the single-stranded oligonucleotide is annealed to the single-stranded template as described above.
- a mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), and deoxyribothymidine (dTTP) is combined with a modified thio-deoxyribocytosine called dCTP-(aS) (which can be obtained from Amersham). This mixture is added to the template-oligonucleotide complex.
- this new strand of DNA Upon addition of DNA polymerase to this mixture, a strand of DNA identical to the template except for the mutated bases is generated.
- this new strand of DNA will contain dCTP-(aS) instead of dCTP, which serves to protect it from restriction endonuclease digestion.
- the template strand can be digested with ExolII nuclease or another appropriate nuclease past the region that contains the site(s) to be mutagenized. The reaction is then stopped to leave a molecule that is only partially single-stranded.
- a complete double-stranded DNA homoduplex is then formed using DNA polymerase in the presence of all four deoxyribonucleotide triphosphates, ATP, and DNA ligase.
- This homoduplex molecule can then be transformed into a suitable host cell such as E. coli JM101, as described above.
- Mutants with more than one amino acid to be substituted may be generated in one of several ways. If the amino acids are located close together in the polypeptide chain, they may be mutated simultaneously using one oligonucleotide that codes for all of the desired amino acid substitutions. If, however, the amino acids are located some distance from each other (separated by more than about ten amino acids), it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed.
- a separate oligonucleotide is generated for each amino acid to be substituted.
- the oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions.
- the alternative method involves two or more rounds of mutagenesis to produce the desired mutant.
- the first round is as described for the single mutants: wild-type DNA is used for the template, an oligonucleotide encoding the first desired amino acid substitution(s) is annealed to this template, and the heteroduplex DNA molecule is then generated.
- the second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template.
- this template already contains one or more mutations.
- the oligonucleotide encoding the additional desired amino acid substitution(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis.
- This resultant DNA can be used as a template in a third round of mutagenesis, and so on.
- This method is also a preferred method for preparing substitution, deletion, and insertion variants of gene 1.
- the method is based on that described by Wells et al. Gene, 34:315 [1985].
- the starting material is the plasmid (or other vector) comprising gene 1, the gene to be mutated.
- the codon(s) in gene 1 to be mutated are identified.
- a double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques.
- This double-stranded oligonucleotide is referred to as the cassette.
- This cassette is designed to have 3′ and 5′ ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid.
- This plasmid now contains the mutated DNA sequence of gene 1.
- this invention contemplates production of variants of a desired protein containing one or more subunits.
- Each subunit is typically encoded by separate gene.
- Each gene encoding each subunit can be obtained by methods known in the art (see, for example, Section II). In some instances it may be necessary to obtain the gene encoding the various subunits using separate techniques selected from any of the methods described in Section II.
- all subunits can be regulated by the same promoter, typically located 5′ to the DNA encoding the subunits, or each may be regulated by separate promoter suitably oriented in the vector so that each promoter is operably linked to the DNA it is intended to regulate. Selection of promoters is carried out as described in Section III above.
- FIG. 10 In constructing a replicable expression vector containing DNA encoding the protein of interest having multiple subunits, the reader is referred to FIG. 10 where, by way of illustration, a vector is diagrammed showing DNA encoding each subunit of an antibody fragment.
- a vector is diagrammed showing DNA encoding each subunit of an antibody fragment.
- This figure shows that, generally, one of the subunits of the protein of interest will be fused to a phage coat protein such as M13 gene III. This gene fusion generally will contain its own signal sequence. A separate gene encodes the other subunit or subunits, and it is apparent that each subunit generally has its own signal sequence.
- FIG. 10 also shows that a single promoter can regulate the expression of both subunits. Alternatively, each subunit may be independently regulated by a different promoter.
- the protein of interest subunit-phage coat protein fusion construct can be made as described in Section IV above.
- DNA encoding each subunit in the vector may mutated in one or more positions in each subunit.
- preferred sites of mutagenesis correspond to codons encoding amino acid residues located in the complementarity-determining regions (CDR) of either the light chain, the heavy chain, or both chains.
- CDRs are commonly referred to as the hypervariable regions.
- Target proteins such as receptors
- glycoprotein hormone receptors may be prepared by the technique described by McFarland et al., Science 245:494-499 [1989]
- nonglycosylated forms expressed in E. coli are described by Fuh et al. J. Biol. Chem 265:3111-3115 [1990]
- Other receptors can be prepared by standard methods.
- the purified target protein may be attached to a suitable matrix such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic copolymers, hydroxylalkyl methacrylate gels, polyacrylic and polymethacrylic copolymers, nylon, neutral and ionic carriers, and the like. Attachment of the target protein to the matrix may be accomplished by methods described in Methods in Enzymology 44 [1976], or by other means known in the art.
- the immobilized target is contacted with the library of phagemid particles under conditions suitable for binding of at least a portion of the phagemid particles with the immobilized target.
- the conditions including pH, ionic strength, temperature and the like will mimic physiological conditions.
- Binders having high affinity for the immobilized target are separated from those having a low affinity (and thus do not bind to the target) by washing. Binders may be dissociated from the immobilized target by a variety of methods. These methods include competitive dissociation using the wild-type ligand, altering pH and/or ionic strength, and methods known in the art.
- Suitable host cells are infected with the binders and helper phage, and the host cells are cultured under conditions suitable for amplification of the phagemid particles. The phagemid particles are then collected and the selection process is repeated one or more times until binders having the desired affinity for the target molecule are selected.
- the library of phagemid particles may be sequentially contacted with more than one immobilized target to improve selectivity for a particular target.
- a ligand such as hGH has more than one natural receptor.
- both the growth hormone receptor and the prolactin receptor bind the hGH ligand. It may be desirable to improve the selectivity of hGH for the growth hormone receptor over the prolactin receptor. This can be achieved by first contacting the library of phagemid particles with immobilized prolactin receptor, eluting those with a low affinity (i.e.
- hGH mutant having a lower affinity for the prolactin receptor would have therapeutic utility even if the affinity for the growth hormone receptor were somewhat lower than that of wild type hGH.
- This same strategy may be employed to improve selectivity of a particular hormone or protein for its primary function receptor over its clearance receptor.
- an improved substrate amino acid sequence can be obtained. These may be useful for making better “cut sites” for protein linkers, or for better protease substrates/inhibitors.
- an immobilizable molecule e.g. hGH-receptor, biotin-avidin, or one capable of covalent linkage with a matrix
- the linker will preferably be from 3 to 10 amino acids in length and will act as a substrate for a protease.
- a phagemid will be constructed as described above where the DNA encoding the linker region is randomly mutated to produce a randomized library of phagemid particles with different amino acid sequences at the linking site.
- the library of phagemid particles are then immobilized on a matrix and exposed to a desired protease.
- Phagemid particles having preferred or better substrate amino acid sequences in the liner region for the desired protease will be eluted, first producing an enriched pool of phagemid particles encoding preferred linkers.
- These phagemid particles are then cycled several more times to produce an enriched pool of particles encoding consense sequence(s) (see examples XIII and XIV).
- the cloned gene for hGH has been expressed in a secreted form in Eschericha coli (Chang, C. N>, et al., [1987 ] Gene 55, 189) and its DNA and amino acid sequence has been reported (Goeddel, et al. [1979 ] Nature 281, 544; Gray et al., [1985 ] Gene 39, 247).
- the present invention describes novel hGH variants produced using the phagemid selection methods. Human growth hormone variants containing substitutions at positions 10, 14, 18, 21, 167, 171, 172, 174, 175, 176, 178 and 179 have been described. Those having higher binding affinities are described in Tables VII, XIII and XIV.
- Growth hormone variants may be administered and formulated in the same manner as regular growth hormone.
- the growth hormone variants of the present invention may be expressed in any recombinant system which is capable of expressing native or met hGH.
- Therapeutic formulations of hGH for therapeutic administration are prepared for storage by mixing hGH having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers ( Remington's Pharmaceutical Sciences, 16th edition, Osol, A., Ed., (1980), in the form of lyophilized cake or aqueous solutions.
- Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; divalent metal ions such as zinc, cobalt or copper; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG).
- buffers such as phosphate, citrate, and other
- Formulations of the present invention may additionally contain a pharmaceutically acceptable buffer, amino acid, bulking agent and/or non-ionic surfactant.
- a pharmaceutically acceptable buffer include, for example, buffers, chelating agents, antioxidants, preservatives, cosolvents, and the like; specific examples of these could include, trimethylamaine salts (“Tris buffer”), and disodium edetate.
- Tris buffer trimethylamaine salts
- the phagemids of the present invention may be used to produce quantities of the hGH variants free of the phage protein.
- pS0643 and derivatives can simply be grown in a non-suppressor strain such as 16C9. In this case, the amber codon (TAG) leads to termination of translation, which yields free hormone, without the need for an independent DNA construction.
- the hGH variant is secreted from the host and may be isolated from the culture medium.
- One or more of the eight hGH amino acids F10, M14, H18, H21, R167, D171, T175 and I179 may be replaced by any amino acid other than the one found in that position in naturally occurring hGH as indicated. Therefore, 1, 2, 3, 4, 5, 6, 7, or all 8 of the indicated amino acids, F10, M14, H18, H21, R167, D171, T175 and I179, may be replaced by any of the other 19 amino acids out of the 20 amino acids listed below. In a preferred embodiment, all eight listed amino acids are replaced by another amino acid. The most preferred eight amino acids to be substituted are indicated in Table XIV in Example XII.
- the plasmid phGH-M13gIII ( FIG. 1 ), was constructed from M13KO7 7 and the hGH producing plasmid, pBO473 (Cunningham, B. C., et al., Science, 243:1330-1336, [1989]).
- a synthetic oligonucleotide 5′-AGC-TGT-GGC-TTC- GGG-CCC -TTA-GCA-TTT-AAT-GCG-GTA-3′ (SEQ ID NO:2) was used to introduce a unique ApaI restriction site (underlined) into pBO473 after the final Phe191 codon of hGH.
- the oligonucleotide 5′-TTC-ACA-AAC-GAA- GGG-CCC -CTA-ATT-AAA-GCC-AGA-3′ was used to introduce a unique ApaI restriction site (underlined), and a Glu197-to-amber stop codon (bold lettering) into M13KO7 gene III.
- the oligonucleotide 5′-CAA-TAA-TAA-CGG- GCT-AGC -CAA-AAG-AAC-TGG-3′ introduces a unique NheI site (underlined) after the 3′ end of the gene III coding sequence.
- a 138 bp EcoRI-XbaI fragment containing the lac promoter, operator, and Cap binding site was produced by PCR of plasmid pUC119 using the oligonucleotides 5′-CACGACA GAATTC CCGACTGGAAA-3′ (SEQ ID NO:5) and 5′-CTGTT TCTAGA GTGAAATTGTTA-3′ (SEQ ID NO:6) that flank the desired lac sequences and introduce the EcoRI and XbaI restriction sites (underlined).
- This lac fragment was gel purified and ligated into the large EcoRI-XbaI fragment of pSO132 to create the plasmid, phGH-M13gIII.
- the sequences of all tailored DNA junctions were verified by the dideoxy sequence method (Sanger, F., et al. Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467, [1977]).
- the R64A variant hGH phagemid was constructed as follows: the NsiI-BglII mutated fragment of hGH (Cunningham et al. supra) encoding the Arg64 to Ala substitution (R64A) (Cunningham, B. C., Wells, J.
- Plasmids were transformed into a male strain of E. coli (JM101) and selected on carbenicillin plates. A single transformant was grown in 2 ml 2YT medium for 4 h at 37° C. and infected with 50 ⁇ l of M13KO7 helper phage. The infected culture was diluted into 30 ml 2YT, grown overnight, and phagemid particles were harvested by precipitation with polyethylene glycol (Vierra, J., Messing, J., Methods in Enzymology, 153:3-11, [1987]). Typical phagemid particle titers ranged from 2 to 5 ⁇ 10 11 cfu/ml. The particles were purified to homogeneity by CsCl density centrifugation (Day, L. A. J. Mol. Biol., 39:265-277, [1969]) to remove any fusion protein not attached to virions.
- Rabbit polyclonal antibodies to hGH were purified with protein A, and coated onto microtiter plates (Nunc) at a concentration of 2 ⁇ g/ml in 50 mM sodium carbonate buffer (pH 10) at 4° C. for 16-20 hours. After washing in PBS containing 0.05% Tween 20, hGH or hGH-phagemid particles were serially diluted from 2.0-0.002 nM in buffer A (50 mM Tris (pH 7.5), 50 mM NaCl, 2 mM EDTA, 5 mg/ml bovine serum albumin, and 0.05% Tween 20). After 2 hours at room temperature (rt), the plates were washed well and the indicated Mab (Cunningham et al.
- Oxirane polyacrylamide beads (Sigma) were conjugated to the purified extracellular domain of the hGH receptor (hGHbp) (Fuh, G., et al., J. Biol. Chem., 265:3111-3115 [1990]) containing an extra cysteine residue introduced by site-directed mutagenesis at position 237 that does not affect binding of hGH (J. Wells, unpublished).
- the hGHbp was conjugated as recommended by the supplier to a level of 1.7 pmol hGHbp/mg dry oxirane bead, as measured by binding of [ 125 I] hGH to the resin. Subsequently, any unreacted oxirane groups were blocked with BSA and Tris.
- BSA was similarly coupled to the beads.
- Buffer for adsorption and washing contained 10 mM Tris.HCl (pH 7.5), 1 mM EDTA, 50 mM NaCl, 1 mg/ml BSA, and 0.02% Tween 20.
- Elution buffers contained wash buffer plus 200 nM hGH or 0.2 M glycine (pH 2.1).
- Parental phage M13KO7 was mixed with hGH phagemid particles at a ratio of nearly 3000:1 (original mixture) and tumbled for 8-12 h with a 5 ⁇ l aliquot (0.2 mg of acrylamide beads) of either absorbent in a 50 ⁇ l volume at room temperature.
- the beads were pelleted by centrifugation and the supernate carefully removed.
- the beads were resuspended in 200 ⁇ l wash buffer and tumbled at room temperature for 4 hours (wash 1). After a second wash (wash 2), the beads were eluted twice with 200 nM hGH for 6-10 hours each (eluate 1, eluate 2).
- the final elution was with a glycine buffer (pH 2.1) for 4 hours to remove remaining hGH phagemid particles (eluate 3).
- Each fraction was diluted appropriately in 2YT media, mixed with fresh JM101, incubated at 37° C. for 5 minutes, and plated with 3 ml of 2YT soft agar on LB or LB carbenicillin plates.
- the gene III protein is composed of 410 residues divided into two domains that are separated by a flexible linker sequence (Armstrong, J., et al., FEBS Lett., 135:167-172, [1981]).
- the amino-terminal domain is required for attachment to the pili of E. coli , while the carboxyl-terminal domain is imbedded in the phage coat and required for proper phage assembly (Crissman, J. W., Smith, G. P., Virology, 132:445-455, [1984]).
- the signal sequence and amino-terminal domain of gene III was replaced with the stII signal and entire hGH gene (Chang et al.
- FIG. 1 by fusion to residue 198 in the carboxyl-terminal domain of gene III ( FIG. 1 ).
- the hGH-gene III fusion was placed under control of the lac promoter/operator in a plasmid (phGH-M13gIII; FIG. 1 ) containing the pBR322 ⁇ -lactamase gene and Col E1 replication origin, and the phage f1 intergenic region.
- the vector can be easily maintained as a small plasmid vector by selection on carbenicillin, which avoids relying on a functional gene III fusion for propagation.
- the plasmid can be efficiently packaged into virions (called phagemid particles) by infection with helper phage such as M13KO7 (Viera et al. supra) which avoids problems of phage assembly.
- helper phage such as M13KO7 (Viera et al. supra) which avoids problems of phage assembly.
- Phagemid infectivity titers based upon transduction to carbenicillin resistance in this system varied from 2-5 ⁇ 10 11 colony forming units (cfu)/ml.
- the titer of the M13KO7 helper phage in these phagemid stocks is ⁇ 10 10 plaque forming units (pfu)/ml.
- the titer of fusion phage displaying the hGH gene III fusion is about 2-5 ⁇ 10 10 /ml. This number is much greater than the titer of E. coli ( ⁇ 10 8 to 10 9 /ml) in the culture from which they are derived. Thus, on average every E. coli cell produces 10-100 copies of phage decorated with an hGH gene III fusion protein.
- FIG. 2 Immunoblot analysis ( FIG. 2 ) of the hGH-gene III phagemid show that hGH cross-reactive material comigrates with phagemid particles in agarose gels. This indicates that the hGH is tightly associated with phagemid particles.
- the hGH-gene III fusion protein from the phagemid particles runs as a single immuno-stained band showing that there is little degradation of the hGH when it is attached to gene III. Wild-type gene III protein is clearly present because about 25% of the phagemid particles are infectious. This is comparable to specific infectivity estimates made for wild-type M13 phage that are similarly purified (by CsCl density gradients) and concentrations estimated by UV absorbance (Smith, G. P. supra and Parmley, Smith supra) Thus, both wild-type gene III and the hGH-gene III fusion proteins are displayed in the phage pool.
- hGHbp The extracellular domain of the hGH receptor (hGHbp) (Fuh et al., supra) containing a free cysteino residue was efficiently coupled to these beads and phagemid particles showed very low non-specific binding to beads coupled only to bovine serum albumin (Table II).
- a fusion phagemid was constructed with an hGH mutant in which Arg64 was substituted with Ala (R64A).
- the R64A variant hormone is about 20-fold reduced in receptor binding affinity compared to hGH (Kd values of 7.1 nM and 0.34 nM, respectively [Cunningham, Wells, supra]).
- the titers of the R64A hGH-gene III fusion phagemid were comparable to those of wild-type hGH phagemid.
- the wild-type hGH phagemid was enriched from a mixture of the two phagemids plus M13KO7 by 8-fold relative to the phagemid R64A, and ⁇ 10 4 relative to M13KO7 helper phage.
- Binding selections were carried out using beads linked with BSA (control beads) or with the hGHbp (hGHbp beads) as described in Table II and the Materials and Methods After each step, plasmid DNA was isolated (Birnboim, H. C., Doly, J., Nucleic Acids Res., 7: 1513-1523, [1979]) from carbenicillin resistant colonies and analyzed by restriction analysis to determine if it contained the wild-type hGH or the R64A hGH gene III fusion.
- hGH a 22 kD protein
- Protein-protein and antibody-antigen interactions are dominated by discontinuous epitopes (Janin, J., et al., J. Mol. Biol., 204:155-164, [1988]; Argos, P., Prot. Eng., 2:101-113, [1988]; Barlow, D. J., et al., Nature, 322:747-748, [1987]; and Davies, D. R., et al., J. Biol. Chem., 263:10541-10544, [1988]); that is the residues directly involved in binding are close in tertiary structure but separated by residues not involved in binding.
- the screening system presented here should allow one to analyze more conveniently protein-receptor interactions and isolate discontinuous epitopes in proteins with new and high affinity binding properties.
- a mutant of the hGH-gene III fusion protein was constructed using the method of Kunkel, et al. Meth. Enzymol. 154, 367-382 [1987].
- Template DNA was prepared by growing the plasmid pS0132 (containing the natural hGH gene fused to the carboxy-terminal half of M13 gene III, under control of the alkaline phosphatase promoter) in CJ236 cells with M13-K07 phage added as helper.
- Single-stranded, uracil-containing DNA was prepared for mutagenesis to introduce (1) a mutation in hGH which would greatly reduce binding to the hGH binding protein (hGHbp); and (2) a unique restriction site (KpnI) which could be used for assaying for—and selecting against—parental background phage.
- Oligonucleotide-directed mutagenesis was carried out using T7 DNA polymerase and the following oligodeoxy-nucleotide (SEQ ID NO:7):
- hGH codon Gly Thr 178 179 5′-G ACA TTC CTG G G T A C C GTG CAG T-3′ ⁇ KpnI >
- This oligo introduces the KpnI site as shown, along with mutations (R178G, I179T) in hGH. These mutations are predicted to reduce binding of hGH to hGHbp by more than 30-fold.
- Clones from the mutagenesis were screened by KpnI digestion and confirmed by dideoxy DNA sequencing. The resulting construct, to be used as a template for random mutagenesis, was designated pHO415.
- Codons 172, 174, 176, 178 were targeted for random mutagenesis in hGH, again using the method of Kunkel.
- Single-stranded template from pH0415 was prepared as above and mutagenesis was carried out using the following pool of oligos (SEQ ID NO:8):
- hGH codon 172 174 5′-GC TTC AGG AAG GAC ATG GAC NNS GTC NNS ACA-- Ile 176 178 179 NNS CTG NNS A T C GTG CAG TGC CGC TCT GTG G-3′
- NNS random codons
- the mutagenesis products were extracted twice with phenol:chloroform (50:50) and ethanol precipitated with an excess of carrier tRNA to avoid adding salt that would confound the subsequent electroporation step.
- dsDNA double-stranded DNA
- pLIB1 double-stranded DNA
- the supernatant was spun again to remove any remaining cells, and the phage, designated phage pool ⁇ 1, were PEG-precipitated and resuspended in 1 mL STE buffer (10 mM Tris, pH 7.6, 1 mM EDTA, 50 mM NaCl).
- Phage titers were measured as colony-forming units (CFU) for the recombinant phagemid containing hGH-g3p gene III fusion (hGH-g 3 ) plasmid, and plaque-forming units (PFU) for the M13-K07 helper phage.
- CFU colony-forming units
- PFU plaque-forming units
- BINDING An aliquot of phage pool ⁇ 1 (6 ⁇ 10 9 CFU, 6 ⁇ 10 7 PFU) was diluted 4.5-fold in buffer A (Phosphate-buffered saline, 0.5% BSA, 0.05% Tween-20, 0.01% thimerosal) and mixed with a 5 ⁇ L suspension of oxirane-polyacrylamide beads coupled to the hGHbp containing a Ser237 Cys mutation (350 fmols) in a 1.5 mL silated polypropylene tube.
- buffer A Phosphate-buffered saline, 0.5% BSA, 0.05% Tween-20, 0.01% thimerosal
- an equivalent aliquot of phage were mixed in a separate tube with beads that had been coated with BSA only.
- the phage were allowed to bind to the beads by incubating 3 hours at room temperature (23° C.) with slow rotation (approximately 7 RPM). Subsequent steps were
- hGH ELUTION Phage/phagemid binding weakly to the beads were removed by stepwise elution with hGH. In the first step, the beads were rotated with buffer A containing 2 nM hGH. After 17 hours, the beads were pelleted and resuspended in buffer A containing 20 nM hGH and rotated for 3 hours, then pelleted. In the final hGH wash, the beads were suspended in buffer A containing 200 nM hGH and rotated for 3 hours then pelleted.
- GLYCINE ELUTION To remove the tightest-binding phagemid (i.e. those still bound after the hGH washes), beads were suspended in Glycine buffer (1 M Glycine, pH 2.0 with HCl), rotated 2 hours and pelleted. The supernatant (fraction “G”; 200 ⁇ L) was neutralized by adding 30 ⁇ L of 1 M Tris base.
- the hGHbp-beads yielded 14 times as many CFU's. This reflects the enrichment of tight-binding hGH-displaying phagemid over nonspecifically-binding phagemid.
- Phage from library 1G ( FIG. 3 ) were selected for binding to hGHbp beads as described above. Fraction G eluted from hGHbp beads contained 30 times as many CFU's as fraction G eluted from BSA-beads in this selection. Again, an aliquot of fraction G was propagated in WJM101 cells to yield library 1G 2 (indicating that this library had been twice selected by glycine elution). Double-stranded DNA (pLIB 1G 2 ) was also prepared from this culture.
- phage Library 2 ( FIG. 3 ).
- Phagemid binding, elution, and propagation were carried out in successive rounds for phagemid derived from both pLIB 2 and pLIB 3 ( FIG. 3 ) as described above, except that (1) an excess (10-fold over CFU) of purified K07 phage (not displaying hGH) was added in the bead-binding cocktail, and (2) the hGH stepwise elutions were replaced with brief washings of buffer A alone. Also, in some cases, XL1-Blue cells were used for phagemid propagation.
- hGH codon 172 174 176 178 5′-AAG GTC TCC ACA TAC CTG AGG ATC-3′
- Residue 172 in these clones is Lys as in wild-type.
- the codon selected for 172 is also identical to wild-type hGH. This is not surprising since AAG is the only lysine-codon possible from a degenerate “NNS” codon set.
- Residue 178-Arg is also the same as wild-type, but here, the codon selected from the library was AAG instead of CGC as is found in wild-type hGH, even though the latter codon is also possible using the “NNS” codon set.
- the multiplicity of infection of K07 infection is an important parameter in the propagation of recombinant phagemids.
- the K07 multiplicity of infection must be high enough to insure that virtually all cells transformed or transfected with phagemid are able to package new phagemid particles.
- the concentration of wild-type gene III in each cell should be kept high to reduce the possibility of multiple hGH-gene III fusion molecules being displayed on each phagemid particle, thereby reducing chelate effects in binding.
- the K07 multiplicity of infection is too high, the packaging of K07 will compete with that of recombinant phagemid. We find that acceptable phagemid yields, with only 1-10% background K07 phage, are obtained when the K07 multiplicity of infection is 100.
- Phage pools are labeled as shown ( FIG. 3 ).
- the multiplicity of infection (moi) refers to the multiplicity of K07 infection (PFU/cells) in the propagation of phagemid.
- the enrichment of CFU over PFU is shown in those cases where purified K07 was added in the binding step.
- the ratio of CFU eluting from hGHbp-beads over CFU eluting from BSA-beads is shown.
- the fraction of KpnI-containing template (i.e., pH0415) remaining in the pool was determined by digesting dsDNA with KpnI plus EcoRI, running the products on a 1% agarose gel, and laser-scanning a negative of the ethidium bromide-stained DNA.
- hGH codon 172 174 176 178 Lys Ser Tyr Arg 5′-ATG GAC AAG GT G TC G ACA T A C CTG CGC ATC GTG-3′
- the resulting construct, pH0458B was transformed into E. coli strain 16C9 for expression of the mutant hormone. Scatchard analysis of competitive binding of hGH(E174S, F176Y) versus 125 I-hGH to hGHbp indicated that the (E174S, F176Y) mutant has a binding affinity at least 5.0-fold tighter than that of wild-type hGH.
- Human growth hormone variants were produced by the method of the present invention using the phagemid described in FIG. 9 .
- Plasmid pS0643 was constructed by oligonucleotide-directed mutagenesis (Kunkel et al., Methods Enzymol. 154, 367-382 [1987]) of pS0132, which contains pBR322 and f1 origins of replication and expresses an hGH-gene III fusion protein (hGH residues 1-191, followed by a single Gly residue, fused to Pro-198 of gene III) under the control of the E. coli phoA promoter (Bass et al., Proteins 8, 309-314 [1990])( FIG. 9 ).
- pS0643 and derivatives can be grown in a amber-suppressor strain of E. coli , such as JM101 or XL1-Blue (Bullock et al., BioTechniques 5, 376-379 [1987]). Shown above is substitution of Glu at the amber codon which occurs in supE suppressor strains. Suppression with other amino acids is also possible in various available strains of E. coli well known and publically available.
- pS0643 and derivatives can simply be grown in a non-suppressor strain such as 16C9.
- the amber codon (TAG) leads to termination of translation, which yields free hormone, without the need for an independent DNA construction.
- pS0643 was mutated with the oligonucleotides (1) 5′-CGG-ACT-GGG-C AG-ATA-T TC-AAG-CAG-ACC-3′ (SEQ ID NO:13), which destroys the unique BglII site of pS0643; (2) 5′-CTC-AAG-AAC-TAC-G GG-TTA-CC C-TGA-CTG-CTT-CAG-GAA-GG-3′ (SEQ ID NO:14), which inserts a unique BstEII site, a single-base frameshift, and a non-amber stop codon (TGA); and (3) 5′-CGC-ATC-GTG-CAG-TGC- AGA-TCT -GTG-GAG-GGC-3′ (SEQ ID NO:15), which introduces a new BglII site, to yield the starting vector, pH0509.
- Codons 172, 174, 176 and 178 of hGH were targeted for random mutagenesis because they all lie on or near the surface of hGH and contribute significantly to receptor-binding (Cunningham and Wells, Science 244, 1081-1085 [1989]); they all lie within a well-defined structure, occupying 2 “turns” on the same side of helix 4; and they are each substituted by at least one amino acid among known evolutionary variants of hGH.
- TAG amber
- the vector was prepared by digesting pH0509 with BstEII followed by BglII . The products were run on a 1% agarose gel and the large fragment excised, phenol-extracted, and ethanol precipitated. This fragment was treated with calf intestinal phosphatase (Boehringer), then phenol:chloroform extracted, ethanol precipitated, and resuspended for ligation with the mutagenic cassette.
- Approximately 150 ng (45 fmols) of DNA was electroporated into XL1-Blue cells (1.8 ⁇ 10 9 cells in 0.045 mL) in a 0.2 cm cuvette at a voltage setting of 2.49 kV with a single pulse (time constant 4.7 msec.).
- dsDNA double-stranded DNA
- pH0529E the initial library
- phage pool ⁇ H0529E the initial library of phage
- Phage titers were measured as colony-forming units (CFU) for the recombinant phagemid containing hGH-g3p. Approximately 4.5 ⁇ 10 13 CFU were obtained from the starting library.
- hGHbp-beads Immobilized hGHbp (“hGHbp-beads”) was prepared as described (Bass et al., Proteins 8, 309-314 [1990]), except that wild-type hGHbp (Fuh et al., J. Biol. Chem. 265, 3111-3115 [1990]) was used.
- hPRLbp-beads Immobilized hPRLbp (“hPRLbp-beads”) was prepared as above, using the 211-residue extracellular domain of the prolactin receptor (Cunningham et al., Science 250, 1709-1712 [1990]).
- “Blank beads” were prepared by treating the oxirane-acrylamide beads with 0.6 M ethanolamine (pH 9.2) for 15 hours at 4° C.
- Buffer A PBS, 0.5% BSA, 0.05% Tween 20, 0.01% thimerosal
- Buffer B 50 m M tris pH 7.5, 10 m M MgCl 2 , 0.5% BSA, 0.05% Tween 20, 100 m M ZnCl 2
- Buffer C PBS, 0.5% BSA, 0.05% Tween 20, 0.01% thimerosal, 10 m M EDTA
- Binding selections were carried out according to each of the following paths: (1) binding to blank beads, (2) binding to hGHbp-beads, (3) binding to hPRLbp-beads (+Zn 2+ ), (4) binding to hPRLbp-beads (+EDTA), (5) pre-adsorbing twice with hGHbp beads then binding the non-adsorbed fraction to hPRLbp-beads (“ ⁇ hGHbp, +hPRLbp” selection), or (6) pre-adsorbing twice with hPRLbp-beads then binding the non-adsorbed fraction to hGHbp-beads (“ ⁇ hPRLbp, +hGHbp” selection).
- Binding and elution of phage was carried out in each cycle as follows:
- BINDING An aliquot of hormone phage (typically 10 9 -10 10 CFU) was mixed with an equal amount of non-hormone phage (pCAT), diluted into the appropriate buffer (A, B, or C), and mixed with a 10 mL suspension of hGHbp, hPRLbp or blank beads in a total volume of 200 mL in a 1.5 mL polypropylene tube. The phage were allowed to bind to the beads by incubating 1 hour at room temperature (23° C.) with slow rotation (approximately 7 RPM). Subsequent steps were carried out with a constant volume of 200 ⁇ L and at room temperature.
- pCAT non-hormone phage
- WASHES The beads were spun 15 sec., and the supernatant was removed. To reduce the number of phage not specifically bound, the beads were washed 5 times by resuspending briefly in the appropriate buffer, then pelleting.
- hGH ELUTION Phage binding weakly to the beads were removed by elution with hGH. The beads were rotated with the appropriate buffer containing 400 n M hGH for 15-17 hours. The supernatant was saved as the “hGH elution” and the beads. The beads were washed by resuspending briefly n buffer and pelleting.
- GLYCINE ELUTION To remove the tightest-binding phage (i.e. those still bound after the hGH wash), beads were suspended in Glycine buffer (Buffer A plus 0.2 M Glycine, pH 2.0 with HCl), rotated 1 hour and pelleted. The supernatant (“Glycine elution”; 200 ⁇ L) was neutralized by adding 30 mL of 1 M Tris base and stored at 4° C.
- Phage binding, elution, and propagation were carried out in successive rounds, according to the cycle described above.
- the phage amplified from the hGH elution from hGHbp-beads were again selected on hGHbp-beads and eluted with hGH, then used to infect a new culture of XL1-Blue cells.
- Three to five rounds of selection and propagation were carried out for each of the selection procedures described above.
- Mutants of hGH were prepared from osmotically shocked cells by ammonium sulfate precipitation as described for hGH (Olson et al., Nature 293, 408-411 [1981]), and protein concentrations were measured by laser densitomoetry of Coomassie-stained SDS-polyacrylamide gel electrophoresis gels, using hGH as standard (Cunningham and Wells, Science 244, 1081-1085 [1989]).
- the binding affinity of each mutant was determined by displacement of 125 I hGH as described (Spencer et al., J. Biol. Chem. 263, 7862-7867 [1988]; Fuh et al., J. Biol. Chem. 265, 3111-3115 [1990]), using an anti-receptor monoclonal antibody (Mab263).
- Binding assays may be carried out for mutants selected for hPRLbp-binding.
- substitution of a particular amino acid has essentially the same effect independent of surrounding residues.
- substitution of F176Y in the background of 172R/174S reduces binding affinity by 2.0-fold (RSFR (SEQ ID NO:85) vs. RSYR (SEQ ID NO:88)).
- RSFR SEQ ID NO:85
- RSYR SEQ ID NO:88
- the binding affinity of the F176Y mutant is 2.9-fold weaker than the corresponding 176F mutant (KAFR; Cunningham and Wells, 1989).
- the binding constants determined for several selected mutants of hGH demonstrate non-additive effects of some amino acid substitutions at residues 172, 174, 176, and 178.
- the substitution E174S results in a mutant (KSYR (SEQ ID NO:84)) which binds hGHbp 3.7-fold tighter than the corresponding mutant containing E174A (KAYR (SEQ ID NO:89)).
- the effects of these E174 substitutions are reversed.
- the E174A mutant (RAYR (SEQ ID NO:86)
- RYR SEQ ID NO:88
- Example VIII Using the methods described in Example VIII, we targeted another region of hGH involved in binding to the hGHbp and/or hPRLbp, helix 1 residues 10, 14, 18, 21, for random mutagenesis in the phGHam-g3p vector (also known as pS0643; see Example VIII).
- phGHam-g3p the “amber” hGH-g3 construct
- Phage produced from both pS0132 S. Bass, R. Greene, J. A. Wells, Proteins 8, 309 (1990)
- phGHam-g3 were tested with three antibodies (Medix 2, 1B5.G2, and 5B7.C10) that are known to have binding determinants near the carboxyl-terminus of hGH [B. C. Cunningham, P. Jhurani, P. Ng, J. A.
- Phagemid particles from phGHam-g3 reacted much more strongly with antibodies Medix 2, 1B5.G2, and 5B7.C10 than did phagemid particles from pS0132.
- binding of pS0132 particles was reduced by >2000-fold for both Medix 2 and 5B7.C10 and reduced by >25-fold for 1B5.G2 compared to binding to Medix 1.
- binding of phGHam-g3 phage was weaker by only about 1.5-fold, 1.2-fold, and 2.3-fold for the Medix 2, 1B5.G2, and 5B7.C10 antibodies, respectively, compared with binding to MEDIX 1.
- This library was constructed by cassette mutagenesis that fully mutated four residues at a time (see Example VIII) which utilized a mutated version of phGHam-g3 into which unique KpnI (at hGH codon 27) and XhoI (at hGH codon 6) restriction sites (underlined below) had been inserted by mutagenesis [T. A. Kunkel, J. D. Roberts, R. A. Zakour, Methods Enzymol.
- the later oligo also introduced a +1 frameshift (italicized) to terminate translation from the starting vector and minimize wild-type background in the phagemid library. This strating vector was designated pH0508B.
- the helix 1 library which mutated hGH residues 10, 14, 18, 21, was constructed by ligating to the large XhoI-KpnI fragment of pH0508B a cassette made from the complementary oligonucleotides 5′-pTCG AGG CTC NNS GAC AAC GCG NNS CTG CGT GCT NNS CGT CTT NNS CAG CTG GCC TTT GAC ACG TAC-3′ (SEQ ID NO:20) and 5′-pGT GTC AAA GGC CAG CTG SNN AAG ACG SNN AGC ACG CAG SNN CGC GTT GTC SNN GAG CC-3′ (SEQ ID NO:21).
- the KpnI site was destroyed in the junction of the ligation product so that restriction enzyme digestion could be used for analysis of non-mutated background.
- the library contained at least 10 7 independent transformants so that if the library were absolutely random (10 6 different combinations of codons) we would have an average of about 10 copies of each possible mutated hGH gene. Restriction analysis using KpnI indicated that at least 80% of helix 1 library constructs contained the inserted cassette.
- Binding enrichments of hGH-phage from the libraries was carried out using hGHbp immobilized on oxirane-polyacrylamide beads (Sigma Chemical Co.) as described (Example VIII).
- hGHbp immobilized on oxirane-polyacrylamide beads
- Table VIII Four residues in helix 1 (F10, M14, H18, and H21) were similarly mutated and after 4 and 6 cycles a non-wild-type consensus developed (Table VIII).
- Position 10 on the hydrophobic face of helix 1 tended to be hydrophobic whereas positions 21 and 18 on the hydrophillic face tended were dominated by Asn; no obvious consensus was evident for position 14 (Table IX).
- the binding constants for these mutants of hGH to hGHbp was determined by expressing the free hormone variants in the non-suppressor E. coli strain 16C9, purifying the protein, and assaying by competitive displacement of labelled wt-hGH from hGHbp (see Example VIII). As indicated, several mutants bind tighter to hGHbp than does wt-hGH.
- the number P indicates the fractional occurrence of each mutant among all the clones sequenced after one or more rounds of selection.
- the helix 4b library was constructed in an attempt to further improve the helix 4 double mutant (E174S/F176Y) selected from the helix 4a library that we found bound tighter to the hGH receptor (see Example VIII). With the E174S/F176Y hGH mutant as the background starting hormone, residues were mutated that surrounded positions 174 and 176 on the hydrophilic face of helix 4 (R167, D171, T175 and I179).
- Binding enrichments of hGH-phage from the libraries was carried out using hGHbp immobilized on oxirane-polyacrylamide beads (Sigma Chemical Co.) as described (Example VIII). After 6 cycles of binding a reasonably clear consensus developed (Table XI). Interestingly, all positions tended to contain polar residues, notably Ser, Thr and Asn (XII).
- the binding constants for some of these mutants of hGH to hGHbp was determined by expressing the free hormone variants in the non-suppressor E. coli strain 16C9, purifying the protein, and assaying by competitive displacement of labelled wt-hGH from hGHbp (see Example VIII). As indicated, the binding affinities of several helix-4b mutants for hGHbp were tighter than that of wt-hGH Table XIII).
- the E174S/F176Y mutant binds 200-fold weaker to the hPRLbp than hGH.
- the E174T/F176Y/R178K and R167N/D171S/E174S/F176Y/I179T mutants each bind >500-fold weaker to the hPRLbp than hGH.
- Hormone-Phagemid Selection Identifies the Information-Content of Particular Residues
- mutations learned through hormone-phagemid enrichment to improve binding can be combined by simple cutting and ligation of restriction fragments or mutagenesis to yield cumulatively optimized mutants of hGH.
- hormone phagemid enrichment can be carried out by one of several variations on the iterative enrichment approach (1) random DNA libraries can be generated in each of two (or perhaps more) regions of the molecule by cassette or another mutagenesis method.
- a combined library can be created by ligation of restriction fragments from the two DNA libraries; (2) an hGH variant, optimized for binding by mutation in one region of the molecule, can be randomly mutated in a second region of the molecule as in the helix-4b library example; (3) two or more random libraries can be partially selected for improved binding by hormone-phagemid enrichment; after this “roughing-in” of the optimized binding site, the still-partially-diverse libraries can be recombined by ligation of restriction fragments to generate a single library, partially diverse in two or more regions of the molecules, which in turn can be further selected for optimized binding using hormone-phagemid enrichment.
- the number P indicates the fractional occurrence of each mutant among all the clones sequenced after one or more rounds of selection.
- the helix 4b mutations (*) are in the background of hGH(E174S/F176Y).
- the E174S/F176Y mutant (*) with wt residues at 167, 171, 175, 179, is shown in bold.
- Plasmid pDH 188 contains the DNA encoding the F ab portion of a humanized IgG antibody, called 4D5, that recognizes the HER-2 receptor. This plasmid is contained in E. coli strain SR 101, and has been deposited with the ATCC in Rockville, Md.
- the plasmid was prepared as follows: the starting plasmid was pS0132, containing the alkaline phosphatase promoter as described above.
- the DNA encoding human growth hormone was excised and, after a series of manipulations to make the ends of the plasmid compatible for ligation, the DNA encoding 4D5 was inserted.
- the 4D5 DNA contains two genes. The first gene encodes the variable and constant regions of the light chain, and contains at its 5′ end the DNA encoding the st II signal sequence. The second gene contains four portions: first, at its 5′ end is the DNA encoding the st II signal sequence.
- PEG polyethylene glycol
- electroporation Both polyethylene glycol (PEG) and electroporation were used to transform plasmids into SR101 cells.
- PEG competent cells were prepared and transformed according to the method of Chung and Miller (Nucleic Acids Res. 16:3580 [1988]). Cells that were competent for electroporation were prepared, and subsequently transformed via electroporation according to the method of Zabarovsky and Winberg ( Nucleic Acids Res. 18:5912 [1990]). After placing the cells in 1 ml of the SOC media (described in Sambrook et al., supra), they were grown for 1 hour at 37° C. with shaking. At this time, the concentration of the cells was determined using light scattering at OD 600 .
- a titered KO7 phage stock was added to achieve an multiplicity of infection (MOI) of 100, and the phage were allowed to adhere to the cells for 20 minutes at room temperature. This mixture was then diluted into 25 mls of 2YT broth (described in Sambrook et al., supra) and incubated with shaking at 37° C. overnight. The next day, cells were pelleted by centrifugation at 5000 ⁇ g for 10 minutes, the supernatant was collected, and the phage particles were precipitated with 0.5 M NaCl and 4% PEG (final concentration) at room temperature for 10 minutes.
- MOI multiplicity of infection
- Phage particles were pelleted by centrifugation at 10,000 ⁇ g for 10 minutes, resuspended in 1 ml of TEN (10 mM Tris, pH 7.6, 1 mM EDTA, and 150 mM NaCl), and stored at 4° C.
- TEN 10 mM Tris, pH 7.6, 1 mM EDTA, and 150 mM NaCl
- Approximately 10 9 phage particles were mixed with a 100-fold excess of KO7 helper phage and 1 ml of buffer A. This mixture was divided into two 0.5 ml aliquots; one of which was applied to ECD coated wells, and the other was applied to BSA coated wells. The plates were incubated at room temperature while shaking for one to three hours, and were then washed three times over a period of 30 minutes with 1 ml aliquots of buffer A.
- Elution of the phage from the plates was done at room temperature by one of two methods: 1) an initial overnight incubation of 0.025 mg/ml purified Mu4D5 antibody (murine) followed by a 30 minute incubation with 0.4 ml of the acid elution buffer (0.2 M glycine, pH 2.1, 0.5% BSA, and 0.05% Tween-20), or 2) an incubation with the acid elution buffer alone. Eluates were then neutralized with 1 M Tris base, and a 0.5 ml aliquot of TEN was added. These samples were then propagated, titered, and stored at 4° C.
- the acid elution buffer 0.2 M glycine, pH 2.1, 0.5% BSA, and 0.05% Tween-20
- the affinity of h4D5 F ab fragments and F ab phage for the ECD antigen was determined using a competitive receptor binding RIA (Burt, D. R., Receptor Binding in Drug Research . O'Brien, R. A. (Ed.). pp. 3-29, Dekker, New York [1986]).
- the ECD antigen was labeled with 125 -Iodine using the sequential chloramine-T method (De Larco, J. E. et al., J. Cell. Physiol. 109:143-152 [1981]) which produced a radioactive tracer with a specific activity of 14 ⁇ Ci/ ⁇ g and incorporation of 0.47 moles of Iodine per mole of receptor.
- a series of 0.2 ml solutions containing 0.5 ng (by ELISA) of F ab or F ab phage, 50 nCi of 125 I ECD tracer, and a range of unlabeled ECD amounts (6.4 ng to 3277 ng) were prepared and incubated at room temperature overnight.
- the labeled ECD-F ab or ECD-F ab phage complex was separated from the unbound labeled antigen by forming an aggregate complex induced by the addition of an anti-human IgG (Fitzgerald 40-GH23) and 6% PEG 8000.
- the complex was pelleted by centrifugation (15,000 ⁇ g for 20 minutes) and the amount of labeled ECD (in cpm) was determined by a gamma counter.
- the dissociation constant (K d ) was calculated by employing a modified version of the program LIGAND (Munson, P. and Rothbard, D., Anal. Biochem. 107:220-239 [1980]) which utilizes Scatchard analysis (Scatchard, G., Ann. N.Y. Acad. Sci. 51:660-672 [1949]).
- the Kd values are shown in FIG. 13 .
- Murine 4D5 antibody was labeled with 125-I to a specific activity of 40-50 ⁇ Ci/ ⁇ g using the Iodogen procedure. Solutions containing a constant amount of labeled antibody and increasing amounts of unlabeled variant Fab were prepared and added to near confluent cultures of SK-BR-3 cells grown in 96-well microtiter dishes (final concentration of labeled antibody was 0.1 nM). After an overnight incubation at 4° C., the supernatant was removed, the cells were washed and the cell associated radioactivity was determined in a gamma counter. K d values were determined by analyzing the data using a modified version of the program LIGAND (Munson, P. and Rothbard, D., supra)
- hGH-phagemid double-stranded DNA (dsDNA) from each of the one-helix variants was isolated and digested with the restriction enzymes EcoRI and BstXI. The large fragment from each helix-4b variant was then isolated and ligated with the small fragment from each helix-1 variant to yield the new two-helix variants shown in Table XIII. All of these variants also contained the mutations E174S/F176Y obtained in earlier hGH-phage binding selections (see Example X for details).
- hGH-phagemid double-stranded DNA from each of the one-helix library pools (selected for 0, 2, or 4 rounds) was isolated and digested with the restriction enzymes AccI and BstXI.
- the large fragment from each helix-1 variant pool was then isolated and ligated with the small fragment from each helix-4b variant pool to yield the three combinatorial libraries pH0707A (unselected helix 1 and helix 4b pools, as described in examples IX and X), pH0707B (twice-selected helix-1 pool with twice-selected helix-4b pool), and pH0707C (4-times selected helix-1 pool with 4-times selected helix-4b pool).
- Duplicate ligations were also set up with less DNA and designated as pH0707D, pH0707E, and pH0707F, corresponding to the 0-, 2-, and 4-round starting libraries respectively. All of these variant pools also contained the mutations E174S/F176Y obtained in earlier hGH-phage binding selections (see Example X for details).
- the ligation products pH0707A-F were processed and electro-transformed into XL1-Blue cells as described (Example VIII). Based on colony-forming units (CFU), the number of transformants obtained from each pool was as follows: 2.4 ⁇ 10 6 from pH0707A, 1.8 ⁇ 10 6 from pH0707B, 1.6 ⁇ 10 6 from pH0707C, 8 ⁇ 10 5 from pH0707D, 3 ⁇ 10 5 from pH0707E, and 4 ⁇ 10 5 from pH0707F.
- hGH-phagemid particles were prepared and selected for hGHbp-binding over 2 to 7 cycles as described in Example VIII.
- the pharmacological properties of a protein may be dependent on binding affinity or on k on or k off , depending on the detailed mechanism of action.
- phagemid particles from the pH0707B pool were incubated with immobilized hGHbp for only 1 minute, then washed six times with 1 mL of binding buffer; the hGH-wash step was omitted; and the remaining hGH-phagemid particles were eluted with a pH2 (0.2M glycine in binding buffer) wash. Enrichment of hGH-phagemid particles over non-displaying particles indicated that even with a short binding period and no cognate-ligand (hGH) challenge, hGH-phagemid binding selection sorts tight-binding variants out of a randomized pool.
- the binding constants for some of these mutants of hGH to hGHbp was determined by expressing the free hormone variants in the non-suppressor E. coli strain 16C9 or 34B8, purifying the protein, and assaying by competitive displacement of labelled wt-hGH from hGHbp (see Example VIII) in a radio-immunoprecipitation assay.
- Table XIII below, all the variants have glutamate 174 replaced by serine 174 and phenylalanine 176 replaced by tyrosine 176 (E174S and F1176Y) plus the additional substitutions as indicated at hGH amino acid positions 10, 14, 18, 21, 167, 171, 175 and 179.
- hGH variants were selected from combinatorial libraries by the phagemid binding selection process. All hGH variants in Table XIV contain two background mutations (E174S/F176Y). hGH-phagemid pools from the libraries pH0707A (Part A), pH0707B and pH0707E (Part B), or pH0707C (Part C) were sorted for 2 to 7 cycles for binding to hGHbp. The number P indicates the fractional occurrence of each variant type among the set of clones sequenced from each pool.
- hGH variants were selected from combinatorial libraries by the phagemid binding selection process. All hGH variants in Table XV contain two background mutations (E174S/F176Y). The number P is the fractional occurrence of a given variant among all clones sequenced after 4 cycles of rapid-binding selection.
- binding constants were measured by competitive displacement of 125 I-labelled hormone H0650BD or labelled hGH using hGHbp (1-238) and either Mab5 or Mab263.
- the variant H0650BD appears bind more than 30-fold tighter than wild-type hGH.
- the plasmid pS0132 contains the gene for hGH fused to the residue Pro198 of the gene III protein with the insertion of an extra glycine residue.
- This plasmid may be used to produce hGH-phage particles in which the hGH-gene III fusion product is displayed monovalently on the phage surface (Example IV).
- the fusion protein comprises the entire hGH protein fused to the carboxy terminal domain of gene III via a flexible linker sequence.
- subtilisin BPN′ a genetically engineered variant of subtilisin BPN′.
- A64SAL subtilisin contains the following mutations: Ser24Cys, His64Ala, Glu156Ser, Gly169Ala and Tyr217Leu. Since this enzyme lacks the essential catalytic residue His64, its substrate specificity is greatly restricted so that certain histidine-containing substrates are preferentially hyrdrolysed (Carter et al., Science 237:394-399 (1987)).
- the sequence of the linker region in pS0132 was mutated to create a substrate sequence for A64SAL subtilisin, using the oligonucleotide 5′-TTC-GGG-CCC-TTC-GCT-GCT-CAC-TAT-ACG-CGT-CAG-TCG-ACT-GAC-CTG-CCT-3′ (SEQ ID NO:27).
- Phagemid particles derived from pS0132 and pS0640 were constructed as described in Example I.
- a 10 ⁇ l aliquot of each phage pool was separately mixed with 30 ⁇ l of oxirane beads (prepared as described in Example II) in 100 ⁇ l of buffer comprising 20 mM Tris-HCl pH 8.6 and 2.5M NaCl.
- the binding and washing steps were performed as described in example VII.
- the beads were then resuspended in 400 ⁇ l of the same buffer, with or without 50 nM of A64SAL subtilisin. Following incubation for 10 minutes, the supernatants were collected and the phage titres (cfu) measured.
- Table XVII shows that approximately 10 times more substrate-containing phagemid particles (pS0640) were eluted in the presence of enzyme than in the absence of enzyme, or than in the case of the non-substrate phagemids (pS0132) in the presence or absence of enzyme. Increasing the enzyme, phagemid or bead concentrations did not improve this ratio.
- hGH human growth hormone
- a tight-binding variant of hGH was introduced in place of the wild-type hGH gene in pS0132 and pS0640.
- the hGH variant used was as described in example XI (pH0650bd) and contains the mutations Phe10Ala, Met14Trp, His18Asp, His21Asn, Arg167Asn, Asp171Ser, Glu174Ser, Phe176Tyr and Ile179Thr.
- Binding COSTAR 12-well tissue culture plates were coated for 16 hours with 0.5 ml/well 2 ug/ml hGHbp in sodium carbonate buffer pH 10.0. The plates were then incubated with 1 ml/well of blocking buffer (phosphate buffered saline (PBS) containing 0.1% w/v bovine serum albumen) for 2 hours and washed in an assay buffer containing 10 mM Tris-HCl pH 7.5, 1 mM EDTA and 100 mM NaCl. Phagemids were again prepared as described in Example I: the phage pool was diluted 1:4 in the above assay buffer and 0.5 ml of phage incubated per well for 2 hours.
- blocking buffer phosphate buffered saline (PBS) containing 0.1% w/v bovine serum albumen
- Table XVII shows that there was a dramatic increase in the ratio of specifically eluted substrate-phagemid particles compared to the method previously described for pS0640 and pS0132. It is likely that this is due to the fact that the tight-binding hGH mutant has a significantly slower off-rate for binding to hGH binding protein compared to wild-type hGH.
- Example XIII We sought to employ the selective enrichment procedure described in Example XIII to identify good substrate sequences from a library of random substrate sequences.
- This new construct was designated pDM0253 (The actual sequence of pDM0253 is 5′-AGC-TGT-GGC-TTC-GGG-CCC-GCC- C CC-GCG-TCG-ACT-GGC-GGT-GGC-TCT-3′ (SEQ ID NO:29), where the underlined base substitution is due to a spurious error in the mutagenic oligonucleotide).
- the tight-binding hGH variant described in example was introduced by exchanging a fragment from pDM0411 (example XIII).
- the resulting library vector was designated pDM0454.
- pDM0454 was digested with ApaI followed by SalI, then precipitated with 13% PEG 8000+10 mM MgCl 2 , washed twice in 70% ethanol and resuspended This efficiently precipitates the vector but leaves the small Apa-Sal fragment in solution (Pai thankar, K. R. and Prasad, K. S. N., Nucleic Acids Research 19:1346).
- the product was run on a 1% agarose gel and the ApaI-SalI digested vector excised, purified using a Bandprep kit (Pharmacia) and resuspended for ligation with the mutagenic cassette.
- the cassette to be inserted contained a DNA sequence similar to that in the linker region of pS0640 and pDM0411, but with the codons for the histidine and tyrosine residues in the substrate sequence replaced by randomised codons.
- the oligonucleotides used in the mutagenic cassettes were: 5′-C-TTC-GCT-GCT-NNS-NNS-ACC-CGG-CAA-3′ (coding strand) (SEQ ID NO:30) and 5′-T-CGA-TTG-CCG-GGT-SNN-SNN-AGC-AGC-GAA-GGG-CC-3′ (non-coding strand) (SEQ ID NO:31).
- This cassette also destroys the SalI site, so that digestion with SalI may be used to reduce the vector background.
- oligonucleotides were not phosphorylated before insertion into the Apa-Sal cassette site, as it was feared that subsequent oligomerisation of a small population of the cassettes may lead to spurious results with multiple cassette inserts.
- the reaction products were phenol:chloroform extracted, ethanol precipitated and resuspended in water. Initially, no digestion with SalI to reduce the background vector was performed. Approximately 200 ng was electroporated into XL-1 blue cells and a phagemid library was prepared as described in example VIII.
- the selection procedure used was identical to that described for pDM0411 and pDM0390 in example XIII. After each round of selection, the eluted phage were propagated by transducing a fresh culture of XL-1 blue cells and propagating a new phagemid library as described for hGH-phage in example VIII. The progress of the selection procedure was monitored by measuring eluted phage titres and by sequencing individual clones after each round of selection.
- Table A shows the successive phage titres for elution in the presence and absence of enzyme after 1, 2 and 3 rounds of selection.
- Tables B1 and B2 shows the sequences of isolates obtained after round 2 and round 3 of selection. After 2 rounds of selection, there is clearly a high incidence of histidine residues. This is exactly what is expected: as described in example XIII, A64SAL subtilisin requires a histidine residue in the substrate as it employs a substrate-assisted catalytic mechanism. After 3 rounds of selection, each of the 10 clones sequenced has a histidine in the randomised cassette. Note, however, that 2 of the sequences are of pDM0411, which was not present in the starting library and is therefore a contaminant.
Abstract
A method for selecting novel proteins such as growth hormone and antibody fragment variants having altered binding properties for their respective receptor molecules is provided. The method comprises fusing a gene encoding a protein of interest to the carboxy terminal domain of the gene III coat protein of the filamentous phage M13. The gene fusion is mutated to form a library of structurally related fusion proteins that are expressed in low quantity on the surface of a phagemid particle. Biological selection and screening are employed to identify novel ligands useful as drug candidates. Disclosed are preferred phagemid expression vectors and selected human growth hormone variants.
Description
- This application is a continuation application of Ser. No. 11/761,180 filed Jun. 11, 2007, which is a continuation of Ser. No. 11/199,062 filed Aug. 8, 2005, now abandoned; which is a continuation of application Ser. No. 09/717,641 filed Nov. 21, 2000, now abandoned; which is a continuation of application Ser. No. 08/922,345 filed Sep. 3, 1997, now abandoned; which is a continuation of application Ser. No. 08/463,587 filed Jun. 5, 1995, now issued as U.S. Pat. No. 5,821,047; which is a divisional of application Ser. No. 08/050,058 filed Apr. 30, 1993, now issued as U.S. Pat. No. 5,750,373; which is a 371 of International Application No. PCT/US91/09133 filed Dec. 3, 1991, which is a continuation-in-part of application Ser. No. 07/743,614 filed Aug. 9, 1991, now abandoned; which is a continuation-in-part of application Ser. No. 07/715,300 filed Jun. 14, 1991, now abandoned; which is a continuation-in-part of application Ser. No. 07/683,400 filed Apr. 10, 1991, now abandoned; which is a continuation-in-part of application Ser. No. 07/621,667 filed Dec. 3, 1990, now abandoned. The contents of these applications are incorporated herein by reference.
- This invention relates to the preparation and systematic selection of novel binding proteins having altered binding properties for a target molecule. Specifically, this invention relates to methods for producing foreign polypeptides mimicking the binding activity of naturally occurring binding partners. In preferred embodiments, the invention is directed to the preparation of therapeutic or diagnostic compounds that mimic proteins or nonpeptidyl molecules such a hormones, drugs and other small molecules, particularly biologically active molecules such as growth hormone.
- Binding partners are substances that specifically bind to one another, usually through noncovalent interactions. Examples of binding partners include ligand-receptor, antibody-antigen, drug-target, and enzyme-substrate interactions. Binding partners are extremely useful in both therapeutic and diagnostic fields.
- Binding partners have been produced in the past by a variety of methods including; harvesting them from nature (e.g., antibody-antigen, and ligand-receptor pairings) and by adventitious identification (e.g. traditional drug development employing random screening of candidate molecules). In some instances these two approaches have been combined. For example, variants of proteins or polypeptides, such as polypeptide fragments, have been made that contain key functional residues that participate in binding. These polypeptide fragments, in turn, have been derivatized by methods akin to traditional drug development. An example of such derivitization would include strategies such as cyclization to conformationally constrain a polypeptide fragment to produce a novel candidate binding partner.
- The problem with prior art methods is that naturally occurring ligands may not have proper characteristics for all therapeutic applications. Additionally, polypeptide ligands may not even be available for some target substances. Furthermore, methods for making non-naturally occurring synthetic binding partners are often expensive and difficult, usually requiring complex synthetic methods to produce each candidate. The inability to characterize the structure of the resulting candidate so that rational drug design methods can be applied for further optimization of candidate molecules further hampers these methods.
- In an attempt to overcome these problems, Geysen (Geysen, Immun. Today, 6:364-369 [1985]); and (Geysen et al., Mol. Immun., 23:709-715 [1986]) has proposed the use of polypeptide synthesis to provide a framework for systematic iterative binding partner identification and preparation. According to Geysen et al., Ibid, short polypeptides, such as dipeptides, are first screened for the ability to bind to a target molecule. The most active dipeptides are then selected for an additional round of testing comprising linking, to the starting dipeptide, an additional residue (or by internally modifying the components of the original starting dipeptide) and then screening this set of candidates for the desired activity. This process is reiterated until the binding partner having the desired properties is identified.
- The Geysen et al. method suffers from the disadvantage that the chemistry upon which it is based, peptide synthesis, produces molecules with ill-defined or variable secondary and tertiary structure. As rounds of iterative selection progress, random interactions accelerate among the various substituent groups of the polypeptide so that a true random population of interactive molecules having reproducible higher order structure becomes less and less attainable. For example, interactions between side chains of amino acids, which are sequentially widely separated but which are spatially neighbors, freely occur. Furthermore, sequences that do not facilitate conformationally stable secondary structures provide complex peptide-sidechain interactions which may prevent sidechain interactions of a given amino acid with the target molecule. Such complex interactions are facilitated by the flexibility of the polyamide backbone of the polypeptide candidates. Additionally, candidates may exist in numerous conformations making it difficult to identify the conformer that interacts or binds to the target with greatest affinity or specificity complicating rational drug design.
- A final problem with the iterative polypeptide method of Geysen is that, at present, there are no practical methods with which a great diversity of different peptides can be produced, screened and analyzed. By using the twenty naturally occurring amino acids, the total number of all combinations of hexapeptides that must be synthesized is 64,000,000. Even having prepared such a diversity of peptides, there are no methods available with which mixtures of such a diversity of peptides can be rapidly screened to select those peptides having a high affinity for the target molecule. At present, each “adherent” peptide must be recovered in amounts large enough to carry out protein sequencing.
- To overcome many of the problems inherent in the Geysen approach, biological selection and screening was chosen as an alternative. Biological selections and screens are powerful tools to probe protein function and to isolate variant proteins with desirable properties (Shortle, Protein Engineering, Oxender and Fox, eds., A.R. Liss, Inc., NY, pp. 103-108 [1988]) and Bowie et al., Science, 247:1306-1310 [1990)]. However, a given selection or screen is applicable to only one or a small number of related proteins.
- Recently, Smith and coworkers (Smith, Science, 228: 1315-1317 [1985]) and Parmley and Smith, Gene, 73:305-318 [1985] have demonstrated that small protein fragments (10-50 amino acids) can be “displayed” efficiently on the surface of filamentous phage by inserting short gene fragments into gene III of the fd phage (“fusion phage”). The gene III minor coat protein (present in about 5 copies at one end of the virion) is important for proper phage assembly and for infection by attachment to the pili of E. coli (see Rasched et al., Microbiol. Rev., 50: 401-427 [1986]). Recently, “fusion phage” have been shown to be useful for displaying short mutated peptide sequences for identifying peptides that may react with antibodies (Scott et al., Science 249: 386-390, [1990]) and Cwirla et al., Proc. Natl. Acad. U.S.A 87: 6378-6382, [1990]). or a foreign protein (Devlin et al., Science, 249: 404-406 [1990]).
- There are, however, several important limitations in using such “fusion phage” to identify altered peptides or proteins with new or enhanced binding properties. First, it has been shown (Parmley et al., Gene, 73: 305-318, [1988]) that fusion phage are useful only for displaying proteins of less than 100 and preferably less than 50 amino acid residues, because large inserts presumably disrupt the function of gene III and therefore phage assembly and infectivity. Second, prior art methods have been unable to select peptides from a library having the highest binding affinity for a target molecule. For example, after exhaustive panning of a random peptide library with an anti-β endorphin monoclonal antibody, Cwirla and co-workers could not separate moderate affinity peptides (Kd˜10 μM) from higher affinity peptides (Kd˜0.4 μM) fused to phage. Moreover, the parent β-endorphin peptide sequence which has very high affinity (Kd˜7 nM), was not panned from the epitope library.
- Ladner WO 90/02802 discloses a method for selecting novel binding proteins displayed on the outer surface of cells and viral particles where it is contemplated that the heterologous proteins may have up to 164 amino acid residues. The method contemplates isolating and amplifying the displayed proteins to engineer a new family of binding proteins having desired affinity for a target molecule. More specifically, Ladner discloses a “fusion phage” displaying proteins having “initial protein binding domains” ranging from 46 residues (crambin) to 164 residues (T4 lysozyme) fused to the M13 gene III coat protein. Ladner teaches the use of proteins “no larger than necessary” because it is easier to arrange restriction sites in smaller amino acid sequences and prefers the 58 amino acid residue bovine pancreatic trypsin inhibitor (BPTI). Small fusion proteins, such as BPTI, are preferred when the target is a protein or macromolecule, while larger fusion proteins, such as T4 lysozyme, are preferred for small target molecules such as steroids because such large proteins have clefts and grooves into which small molecules can fit. The preferred protein, BPTI, is proposed to be fused to gene III at the site disclosed by Smith et al. or de la Cruz et al., J. Biol. Chem., 263: 4318-4322 [1988], or to one of the terminii, along with a second synthetic copy of gene III so that “some” unaltered gene III protein will be present. Ladner does not address the problem of successfully panning high affinity peptides from the random peptide library which plagues the biological selection and screening methods of the prior art.
- Human growth hormone (hGH) participates in much of the regulation of normal human growth and development. This 22,000 dalton pituitary hormone exhibits a multitude of biological effects including linear growth (somatogenesis), lactation, activation of macrophages, insulin-like and diabetogenic effects among others (Chawla, R, K. (1983) Ann. Rev. Med. 34, 519; Edwards, C. K. et al. (1988) Science 239, 769; Thorner, M. O., et al. (1988) J. Clin. Invest. 81 745). Growth hormone deficiency in children leads to dwarfism which has been successfully treated for more than a decade by exogenous administration of hGH. hGH is a member of a family of homologous hormones that include placental lactogens, prolactins, and other genetic and species variants or growth hormone (Nicoll, C. S., et al., (1986) Endocrine Reviews 7, 169). hGH is unusual among these in that it exhibits broad species specificity and binds to either the cloned somatogenic (Leung, D. W., et al., [I987]
Nature 330, 537) or prolactin receptor (Boutin, J. M., et al., [I988] Ce; 53, 69). The cloned gene for hGH has been expressed in a secreted form in Escherichia coli (Chang, C. N., et al., [I987]Gene 55, I89) and its DNA and amino acid sequence has been reported (Goeddel, et al., [I979] Nature 281, 544; Gray, et al., [I985] Gene 39, 247). The three-dimensional structure of hGH is not available. However, the three-dimensional folding pattern for porcine growth hormone (pGH) has been reported at moderate resolution and refinement (Abdel-Meguid, S. S., et al., [I987] Proc. Natl. Acad. Sci. USA 84, 6434). Human growth hormone's receptor and antibody epitopes have been identified by homolog-scanning mutagenesis (Cunningham et al., Science 243: 1330, 1989). The structure of novel amino terminal methionyl bovine growth hormone containing a spliced-in sequence of human growthhormone including histidine 18 and histidine 21 has been shown (U.S. Pat. No. 4,880,910) - Human growth hormone (hGH) causes a variety of physiological and metabolic effects in various animal models including linear bone growth, lactation, activation of macrophages, insulin-like and diabetogenic effects and others (R. K. Chawla et al., Annu. Rev. Med. 34, 519 (1983); O. G. P. Isaksson et al., Annu. Rev. Physiol. 47, 483 (1985); C. K. Edwards et al., Science 239, 769 (1988); M. O. Thorner and M. L. Vance, J. Clin. Invest 82, 745 (1988); J. P. Hughes and H. G. Friesen, Ann. Rev. Physiol. 47, 469 (1985)). These biological effects derive from the interaction between hGH and specific cellular receptors.
- Accordingly, it is an object of this invention to provide a rapid and effective method for the systematic preparation of candidate binding substances.
- It is another object of this invention to prepare candidate binding substances displayed on surface of a phagemid particle that are conformationally stable.
- It is another object of this invention to prepare candidate binding substances comprising fusion proteins of a phage coat protein and a heterologous polypeptide where the polypeptide is greater than 100 amino acids in length and may be more than one subunit and is displayed on a phagemid particle where the polypeptide is encoded by the phagemid genome.
- It is a further object of this invention to provide a method for the preparation and selection of binding substances that is sufficiently versatile to present, or display, all peptidyl moieties that could potentially participate in a noncovalent binding interaction, and to present these moieties in a fashion that is sterically confined.
- Still another object of the invention is the production of growth hormone variants that exhibit stronger affinity for growth hormone receptor and binding protein.
- It is yet another object of this invention to produce expression vector phagemids that contain a suppressible termination codon functionally located between the heterologous polypeptide and the phage coat protein such that detectable fusion protein is produced in a host suppressor cell and only the heterologous polypeptide is produced in a non-suppressor host cell.
- Finally, it is an object of this invention to produce a phagemid particle that rarely displays more than one copy of candidate binding proteins on the outer surface of the phagemid particle so that efficient selection of high affinity binding proteins can be achieved.
- These and other objects of this invention will be apparent from consideration of the invention as a whole.
- These objectives have been achieved by providing a method for selecting novel binding polypeptides comprising: (a) constructing a replicable expression vector comprising a first gene encoding a polypeptide, a second gene encoding at least a portion of a natural or wild-type phage coat protein wherein the first and second genes are heterologous, and a transcription regulatory element operably linked to the first and second genes, thereby forming a gene fusion encoding a fusion protein; (b) mutating the vector at one or more selected positions within the first gene thereby forming a family of related plasmids; (c) transforming suitable host cells with the plasmids; (d) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein; (e) culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface of the particle; (f) contacting the phagemid particles with a target molecule so that at least a portion of the phagemid particles bind to the target molecule; and (g) separating the phagemid particles that bind from those that do not. Preferably, the method further comprises transforming suitable host cells with recombinant phagemid particles that bind to the target molecule and repeating steps (d) through (g) one or more times.
- Additionally, the method for selecting novel binding proteins where the proteins are composed of more than one subunit is achieved by selecting novel binding peptides comprising constructing a replicable expression vector comprising a transcription regulatory element operably linked to DNA encoding a protein of interest containing one or more subunits, wherein the DNA encoding at least one of the subunits is fused to the DNA encoding at least a portion of a phage coat protein; mutating the DNA encoding the protein of interest at one or more selected positions thereby forming a family of related vectors; transforming suitable host cells with the vectors; infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein; culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface of the particle; contacting the phagemid particles with a target molecule so that at least a portion of the phagemid particles bind to the target molecule; and separating the phagemid particles that bind from those that do not.
- Preferably in the method of this invention the plasmid is under tight control of the transcription regulatory element, and the culturing conditions are adjusted so that the amount or number of phagemid particles displaying more than one copy of the fusion protein on the surface of the particle is less than about 1%. Also preferably, amount of phagemid particles displaying more than one copy of the fusion protein is less than 10% the amount of phagemid particles displaying a single copy of the fusion protein. Most preferably the amount is less than 20%.
- Typically, in the method of this invention, the expression vector will further contain a secretory signal sequences fused to the DNA encoding each subunit of the polypeptide, and the transcription regulatory element will be a promoter system. Preferred promoter systems are selected from; Lac Z, λPL, TAC, T 7 polymerase, tryptophan, and alkaline phosphatase promoters and combinations thereof.
- Also typically, the first gene will encode a mammalian protein, preferably the protein will be selected from; human growth hormone (hGH), N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin A-chain, insulin B-chain, proinsulin, relaxin A-chain, relaxin B-chain, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and leutinizing hormone (LH), glycoprotein hormone receptors, calcitonin, glucagon, factor VIII, an antibody, lung surfactant, urokinase, streptokinase, human tissue-type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hemopoietic growth factor, tumor necrosis factor-alpha and -beta, enkephalinase, human serum albumin, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, a microbial protein, such as betalactamase, tissue factor protein, inhibin, activin, vascular endothelial growth factor, receptors for hormones or growth factors; integrin, thrombopoietin, protein A or D, rheumatoid factors, nerve growth factors such as NGF-β platelet-growth factor, transforming growth factors (TGF) such as TGF-alpha and TGF-beta, insulin-like growth factor-I and -II, insulin-like growth factor binding proteins, CD-4, DNase, latency associated peptide, erythropoietin, osteoinductive factors, interferons such as interferon-alpha, -beta, and -gamma, colony stimulating factors (CSFs) such as M-CSF, GM-CSF, and G-CSF, interleukins (ILs) such as IL-1, IL-2, IL-3, IL-4, superoxide dismutase; decay accelerating factor, viral antigen, HIV envelope proteins such as GP120, GP140, atrial natriuretic peptides A, B or C, immunoglobulins, and fragments of any of the above-listed proteins.
- Preferably the first gene will encode a polypeptide of one or more subunits containing more than about 100 amino acid residues and will be folded to form a plurality of rigid secondary structures displaying a plurality of amino acids capable of interacting with the target. Preferably the first gene will be mutated at codons corresponding to only the amino acids capable of interacting with the target so that the integrity of the rigid secondary structures will be preserved.
- Normally, the method of this invention will employ a helper phage selected from; 13
KO 7, M13R408, M13-VCS, andPhi X 174. The preferred helper phage is M13KO7, and the preferred coat protein is the M13 Phage gene III coat protein. The preferred host is E. coli, and protease deficient strains of E. coli. Novel hGH variants selected by the method of the present invention have been detected. Phagemid expression vectors were constructed that contain a suppressible termination codon functionally located between the nucleic acids encoding the polypeptide and the phage coat protein. -
FIG. 1 . Strategy for displaying large proteins on the surface of filamentous phage and enriching for altered receptor binding properties. A plasmid, phGH-M13gIII was constructed that fuses the entire coding sequence of hGH to the carboxyl terminal domain of M13 gene III. Transcription of the fusion protein is under control of the lac promoter/operator sequence, and secretion is directed by the stII signal sequence. Phagemid particles are produced by infection with the “helper” phage, M13KO7, and particles displaying hGH can be enriched by binding to an affinity matrix containing the hGH receptor. The wild-type gene III (derived from the M13KO7 phage) is diagramed by 4-5 copies of the multiple arrows on the tip of the phage, and the fusion protein (derived from the phagemid, phGH-M13gIII) is indicated schematically by the folding diagram of hGH replacing the arrow head. -
FIG. 2 . Immunoblot of whole phage particles shows that hGH comigrates with phage. Phagemid particles purified in a cesium chloride gradient were loaded into duplicate wells and electrophoresed through a 1% agarose gel in 375 mM Tris, 40 mM glycine pH 9.6 buffer. The gel was soaked in transfer buffer (25 mM Tris, pH 8.3, 200 mM glycine, 20% methanol) containing 2% SDS and 2% β-mercaptoethanol for 2 hours, then rinsed in transfer buffer for 6 hours. The proteins in the gel were then electroblotted onto immobilon membranes (Millipore). The membrane containing one set of samples was stained with Coomassie blue to show the position of the phage proteins (A). The duplicate membrane was immuno-stained for hGH by reacting the membrane with polyclonal rabbit anti-hGH antibodies followed by reaction with horseradish peroxidase conjugated goat anti-rabbit IgG antibodies (B).Lane 1 contains the M13KO7 parent phage and is visible only in the Coomassie blue stained membrane, since it lacks hGH.Lanes -
FIG. 3 . Summary diagram of steps in the selection process for an hGH-phage library randomized atcodons Library 1. An aliquot (approximately 2%) fromLibrary 1 was used directly in an initial selection round as described in the text to yieldLibrary 1G. Meanwhile, double-stranded DNA (dsDNA) was prepared from Library I, digested with restriction enzyme KpnI to eliminate template background, and electrotransformed into WJM101 to yieldLibrary 2. Subsequent rounds of selection (or KpnI digestion, shaded boxes) followed by phagemid propagation were carried out as indicated by the arrows, according to the procedure described in the text. Four independent clones fromLibrary 4G4 and four independent clones fromLibrary 5G6 were sequenced by dideoxy sequencing. All of these clones had the identical DNA sequence, corresponding to the hGH mutant (Glu 174 Ser,Phe 176 Tyr). -
FIG. 4 . Structural model of hGH derived from a 2.8 Å folding diagram of porcine growth hormone determined crystallographically. Location of residues in hGH that strongly modulate its binding to the hGH-binding protein are within the shaded circle. Alanine substitutions that cause a greater than tenfold reduction (), a four- to tenfold reduction (), or increase (◯), or a two- to fourfold reduction (), in binding affinity are indicated. Helical wheel projections in the regions of α-helix reveal their amphipathic quality. Blackened, shaded, or nonshaded residues are charged, polar, or nonpolar, respectively. In helix-4 the most important residues for mutation are on the hydrophilic face. -
FIG. 5 . Amino acid substitutions atpositions rounds positions position 176; and S, T, A, and other residues occur atposition 174. -
FIG. 6 . Sequences from phage selected on hPRLbp-beads in the presence of zinc. The notation is as described inFIG. 5 . Here, the convergence of sequences is not predictable, but there appears to be a bias towards hydrophobic sequences under the most stringent (Glycine) selection conditions; L, W and P residues are frequently found in this pool. -
FIG. 7 . Sequences from phage selected on hPRLbp-beads in the absence of zinc. The notation is as described inFIG. 5 . In contrast to the sequences ofFIG. 6 , these sequences appear more hydrophilic. After 4 rounds of selection using hGH elution, two clones (ANHQ (SEQ ID NO:45), and TLDT/171V (SEQ ID NO:108)) dominate the pool. -
FIG. 8 . Sequences from phage selected on blank beads. The notation is as described inFIG. 5 . After three rounds of selection with glycine elution, no siblings were observed and a background level of non-functional sequences remained. -
FIG. 9 . Construction of phagemid fl ori from pHO415. This vector for cassette mutagenesis and expression of the hGH-gene III fusion protein was constructed as follows. Plasmid pS0643 was constructed by oligonucleotide-directed mutagenesis of pS0132, which contains pBR322 and f1 origins of replication and expresses an hGH-gene III fusion protein (hGH residues 1-191, followed by a single Gly residue, fused to Pro-198 of gene III) under the control of the E. coli phoA promoter. Mutagenesis was carried out with theoligonucleotide 5′-GGC-AGC-TGT-GGC-TTC-TAG-AGT-GGC-GGC-GGC-TCT-GGT-3′ (SEQ ID NO:1), which introduced a XbaI site (underlined) and an amber stop codon (TAG) following Phe-191 of hGH. -
FIG. 10 . A. Diagram of plasmid pDH188 insert containing the DNA encoding the light chain and heavy chain (variable and constant domain 1) of the Fab humanized antibody directed to the HER-2 receptor. VL and VH are the variable regions for the light and heavy chains, respectively. Ck is the constant region of the human kappa light chain. CH1G1 is the first constant region of thehuman gamma 1 chain. Both coding regions start with the bacterial st II signal sequence. B. A schematic diagram of the entire plasma pDH188 containing the insert described in 5A. After transformation of the plasmid into E. coli SR101 cells and the addition of helper phage, the plasmid is packaged into phage particles. Some of these particles display the Fab-p III fusion (where p III is the protein encoded by the M13 gene III DNA). The segments in the plasmid figure correspond to the insert shown in 5A. -
FIG. 11 . A through C are collectively referred to here asFIG. 11 . The nucleotide (Seq. ID No: 24) sequence of the DNA encoding the 4D5 Fab molecule expressed on the phagemid surface. The amino acid sequence of the light chain is also shown (Seq. ID No: 25), as is the amino acid sequence of the heavy chain p III fusion (Seq. ID No:26). -
FIG. 12 . Enrichment of wild-type 4D5 Fab phagemid from variant Fab phagemid. Mixtures of wild-type phagemid and variant 4D5 Fab phagemid in a ratio of 1:1,000 were selected on plates coated with the extra-cellular domain protein of the HER-2 receptor. After each round of selection, a portion of the eluted phagemid were infected into E. coli and plasmid DNA was prepared. This plasmid DNA was then digested with Eco RV and Pst I, separated on a 5% polyacrylamide gel, and stained with ethidium bromide. The bands were visualized under UV light. The bands due to the wild-type and variant plasmids are marked with arrows. The first round of selection was eluted only under acid conditions; subsequent rounds were eluted with either an acid elution (left side of Figure) or with a humanized 4D5 antibody wash step prior to acid elution (right side of Figure) using methods described in Example VIII. Three variant 4D5 Fab molecules were made: H91A (amino acid histidine at position 91 on the VL chain mutated to alanine; indicated as ‘A’ lanes in Figure), Y49A (amino acid tyrosine at position 49 on the VL chain mutated to alanine; indicated as ‘B’ lanes in the Figure), and Y92A (amino acid tyrosine at position 92 on the VL chain mutated to alanine; indicated as ‘C’ lanes in the Figure). Amino acid position numbering is according to Kabat et al., (Sequences of proteins of immunological interest, 4th ed., U.S. Dept of Health and Human Services, Public Health Service, Nat'l. Institute of Health, Bethesda, Md. [1987]). -
FIG. 13 . The Scatchard analysis of the RIA affinity determination described in Experimental Protocols is shown here. The amount of labeled ECD antigen that is bound is shown on the x-axis while the amount that is bound divided by the amount that is free is shown on the y-axis. The slope of the line indicates the Ka; the calculated Kd is 1/Ka. - The following discussion will be best understood by referring to
FIG. 1 . In its simplest form, the method of the instant invention comprises a method for selecting novel binding polypeptides, such as protein ligands, having a desired, usually high, affinity for a target molecule from a library of structurally related binding polypeptides. The library of structurally related polypeptides, fused to a phage coat protein, is produced by mutagenesis and, preferably, a single copy of each related polypeptide is displayed on the surface of a phagemid particle containing DNA encoding that polypeptide. These phagemid particles are then contacted with a target molecule and those particles having the highest affinity for the target are separated from those of lower affinity. The high affinity binders are then amplified by infection of a bacterial host and the competitive binding step is repeated. This process is reiterated until polypeptides of the desired affinity are obtained. - The novel binding polypeptides or ligands produced by the method of this invention are useful per se as diagnostics or therapeutics (eg. agonists or antagonists) used in treatment of biological organisms. Structural analysis of the selected polypeptides may also be used to facilitate rational drug design.
- By “binding polypeptide” as used herein is meant any polypeptide that binds with a selectable affinity to a target molecule. Preferably the polypeptide will be a protein that most preferably contains more than about 100 amino acid residues. Typically the polypeptide will be a hormone or an antibody or a fragment thereof.
- By “high affinity” as used herein is meant an affinity constant (Kd) of <10−5 M and preferably <10−7M under physiological conditions.
- By “target molecule” as used herein is meant any molecule, not necessarily a protein, for which it is desirable to produce a ligand. Preferably, however, the target will be a protein and most preferably the target will be a receptor, such as a hormone receptor.
- By “humanized antibody” as used herein is meant an antibody in which the complementarity-determining regions (CDRs) of a mouse or other non-human antibody are grafted onto a human antibody framework. By human antibody framework is meant the entire human antibody excluding the CDRs.
- The first step in the method of this invention is to choose a polypeptide having rigid secondary structure exposed to the surface of the polypeptide for display on the surface of a phage.
- By “polypeptide” as used herein is meant any molecule whose expression can be directed by a specific DNA sequence. The polypeptides of this invention may comprise more than one subunit, where each subunit is encoded by a separate DNA sequence.
- By “rigid secondary structure” as used herein is meant any polypeptide segment exhibiting a regular repeated structure such as is found in; α-helices, 310 helices, π-helices, parallel and antiparallel β-sheets, and reverse turns. Certain “non-ordered” structures that lack recognizable geometric order are also included in the definition of rigid secondary structure provided they form a domain or “patch” of amino acid residues capable of interaction with a target and that the overall shape of the structure is not destroyed by replacement of an amino acid within the structure. It is believed that some non-ordered structures are combinations of reverse turns. The geometry of these rigid secondary structures is well defined by φ and ψ torsional angles about the α-carbons of the peptide “backbone”.
- The requirement that the secondary structure be exposed to the surface of the polypeptide is to provide a domain or “patch” of amino acid residues that can be exposed to and bind with a target molecule. It is primarily these amino acid residues that are replaced by mutagenesis that form the “library” of structurally related (mutant) binding polypeptides that are displayed on the surface of the phage and from which novel polypeptide ligands are selected. Mutagenesis or replacement of amino acid residues directed toward the interior of the polypeptide is generally avoided so that the overall structure of the rigid secondary structure is preserved. Some replacement of amino acids on the interior region of the rigid secondary structures, especially with hydrophobic amino acid residues, may be tolerated since these conservative substitutions are unlikely to distort the overall structure of the polypeptide.
- Repeated cycles of “polypeptide” selection are used to select for higher and higher affinity binding by the phagemid selection of multiple amino acid changes which are selected by multiple selection cycles. Following a first round of phagemid selection, involving a first region or selection of amino acids in the ligand polypeptide, additional rounds of phagemid selection in other regions or amino acids of the ligand polypeptide are conducted. The cycles of phagemid selection are repeated until the desired affinity properties of the ligand polypeptide are achieved. To illustrate this process, Example VIII phagemid selection of hGH was conducted in cycles. In the first cycle
hGH amino acids hGH amino acids hGH amino acids amino acids - From the forgoing it will be appreciated that the amino acid residues that form the binding domain of the polypeptide will not be sequentially linked and may reside on different subunits of the polypeptide. That is, the binding domain tracks with the particular secondary structure at the binding site and not the primary structure. Thus, generally, mutations will be introduced into codons encoding amino acids within a particular secondary structure at sites directed away from the interior of the polypeptide so that they will have the potential to interact with the target. By way of illustration,
FIG. 2 shows the location of residues in hGH that are known to strongly modulate its binding to the hGH-binding protein (Cunningham et al., Science 247:1461-1465 [1990]). Thus representative sites suitable for mutagenesis would includeresidues - There is no requirement that the polypeptide chosen as a ligand to a target normally bind to that target. Thus, for example, a glycoprotein hormone such as TSH can be chosen as a ligand for the FSH receptor and a library of mutant TSH molecules are employed in the method of this invention to produce novel drug candidates.
- This invention thus contemplates any polypeptide that binds to a target molecule, and includes antibodies. Preferred polypeptides are those that have pharmaceutical utility. More preferred polypeptides include; a growth hormone, including human growth hormone, des-N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroid stimulating hormone; thyroxine; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; leutinizing hormone; glucagon; factor VIII; an antibody; lung surfactant; a plasminogen activator, such as urokinase or human tissue-type plasminogen activator (t-PA); bombesin; factor IX, thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; enkephalinase; a serum albumin such as human serum albumin; mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbial protein, such as betalactamase; tissue factor protein; inhibin; activin; vascular endothelial growth factor; receptors for hormones or growth factors; integrin; thrombopoietin; protein A or D; rheumatoid factors; nerve growth factor such as NGF-β; platelet-derived growth factor; fibroblast growth factor such as aFGF and bFGF; epidermal growth factor; transforming growth factor (TGF) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; insulin-like growth factor binding proteins; CD-4; DNase; latency associated peptide; erythropoietin; osteoinductive factors; an interferon such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1, IL-2, IL-3, IL-4, etc.; superoxide dismutase; decay accelerating factor; atrial natriuretic peptides A, B or C; viral antigen such as, for example, a portion of the HIV envelope; immunoglobulins; and fragments of any of the above-listed polypeptides. In addition, one or more predetermined amino acid residues on the polypeptide may be substituted, inserted, or deleted, for example, to produce products with improved biological properties. Further, fragments of these polypeptides, especially biologically active fragments, are included. Yet more preferred polypeptides of this invention are human growth hormone and atrial naturetic peptides A, B, and C, endotoxin, subtilisin, trypsin and other serine proteases.
- Still more preferred are polypeptide hormones that can be defined as any amino acid sequence produced in a first cell that binds specifically to a receptor on the same cell type (autocrine hormones) or a second cell type (non-autocrine) and causes a physiological response characteristic of the receptor-bearing cell. Among such polypeptide hormones are cytokines, lymphokines, neurotrophic hormones and adenohypophyseal polypeptide hormones such as growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, thyrotropin, chorionic gonadotropin, corticotropin, or β-melanocyte-stimulating hormone, β-lipotropin gamma-lipotropin and the endorphins; hypothalmic release-inhibiting hormones such as corticotropin-release factor, growth hormone release-inhibiting hormone, growth hormone-release factor; and other polypeptide hormones such as atrial natriuretic peptides A, B or C.
- The gene encoding the desired polypeptide (i.e., a polypeptide with a rigid secondary structure) can be obtained by methods known in the art (see generally, Sambrook et al., Molecular Biology: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. [1989]). If the sequence of the gene is known, the DNA encoding the gene may be chemically synthesized (Merrfield, J. Am. Chem. Soc., 85:2149 [1963]). If the sequence of the gene is not known, or if the gene has not previously been isolated, it may be cloned from a cDNA library (made from RNA obtained from a suitable tissue in which the desired gene is expressed) or from a suitable genomic DNA library. The gene is then isolated using an appropriate probe. For cDNA libraries, suitable probes include monoclonal or polyclonal antibodies (provided that the cDNA library is an expression library), oligonucleotides, and complementary or homologous cDNAs or fragments thereof. The probes that may be used to isolate the gene of interest from genomic DNA libraries include cDNAs or fragments thereof that encode the same or a similar gene, homologous genomic DNAs or DNA fragments, and oligonucleotides. Screening the cDNA or genomic library with the selected probe is conducted using standard procedures as described in chapters 10-12 of Sambrook et al., supra.
- An alternative means to isolating the gene encoding the protein of interest is to use polymerase chain reaction methodology (PCR) as described in
section 14 of Sambrook et al., supra. This method requires the use of oligonucleotides that will hybridize to the gene of interest; thus, at least some of the DNA sequence for this gene must be known in order to generate the oligonucleotides. - After the gene has been isolated, it may be inserted into a suitable vector (preferably a plasmid) for amplification, as described generally in Sambrook et al., supra.
- While several types of vectors are available and may be used to practice this invention, plasmid vectors are the preferred vectors for use herein, as they may be constructed with relative ease, and can be readily amplified. Plasmid vectors generally contain a variety of components including promoters, signal sequences, phenotypic selection genes, origin of replication sites, and other necessary components as are known to those of ordinary skill in the art.
- Promoters most commonly used in prokaryotic vectors include the lac Z promoter system, the alkaline phosphatase pho A promoter, the bacteriophage λPL promoter (a temperature sensitive promoter), the tac promoter (a hybrid trp-lac promoter that is regulated by the lac repressor), the tryptophan promoter, and the bacteriophage T7 promoter. For general descriptions of promoters, see
section 17 of Sambrook et al. supra. While these are the most commonly used promoters, other suitable microbial promoters may be used as well. - Preferred promoters for practicing this invention are those that can be tightly regulated such that expression of the fusion gene can be controlled. It is believed that the problem that went unrecognized in the prior art was that display of multiple copies of the fusion protein on the surface of the phagemid particle lead to multipoint attachment of the phagemid with the target. It is believed this effect, referred to as the “chelate effect”, results in selection of false “high affinity” polypeptides when multiple copies of the fusion protein are displayed on the phagemid particle in close proximity to one another so that the target was “chelated”. When multipoint attachment occurs, the effective or apparent Kd may be as high as the product of the individual Kds for each copy of the displayed fusion protein. This effect may be the reason Cwirla and coworkers supra were unable to separate moderate affinity peptides from higher affinity peptides.
- It has been discovered that by tightly regulating expression of the fusion protein so that no more than a minor amount, i.e. fewer than about 1%, of the phagemid particles contain multiple copies of the fusion protein the “chelate effect” is overcome allowing proper selection of high affinity polypeptides. Thus, depending on the promoter, culturing conditions of the host are adjusted to maximize the number of phagemid particles containing a single copy of the fusion protein and minimize the number of phagemid particles containing multiple copies of the fusion protein.
- Preferred promoters used to practice this invention are the lac Z promoter and the pho A promoter. The lac Z promoter is regulated by the lac repressor protein lac i, and thus transcription of the fusion gene can be controlled by manipulation of the level of the lac repressor protein. By way of illustration, the phagemid containing the lac Z promoter is grown in a cell strain that contains a copy of the lac i repressor gene, a repressor for the lac Z promoter. Exemplary cell strains containing the lac i gene include JM 101 and XL1-blue. In the alternative, the host cell can be cotransfected with a plasmid containing both the repressor lac i and the lac Z promoter. Occasionally both of the above techniques are used simultaneously, that is, phagemid particles containing the lac Z promoter are grown in cell strains containing the lac i gene and the cell strains are cotransfected with a plasmid containing both the lac Z and lac i genes. Normally when one wishes to express a gene, to the transfected host above one would add an inducer such as isopropylthiogalactoside (IPTG). In the present invention however, this step is omitted to (a) minimize the expression of the gene III fusion protein thereby minimizing the copy number (i.e. the number of gene III fusions per phagemid number) and to (b) prevent poor or improper packaging of the phagemid caused by inducers such as IPTG even at low concentrations. Typically, when no inducer is added, the number of fusion proteins per phagemid particle is about 0.1 (number of bulk fusion proteins/number of phagemid particles). The most preferred promoter used to practice this invention is pho A. This promoter is believed to be regulated by the level of inorganic phosphate in the cell where the phosphate acts to down-regulate the activity of the promoter. Thus, by depleting cells of phosphate, the activity of the promoter can be increased. The desired result is achieved by growing cells in a phosphate enriched medium such as 2YT or LB thereby controlling the expression of the gene III fusion.
- One other useful component of vectors used to practice this invention is a signal sequence. This sequence is typically located immediately 5′ to the gene encoding the fusion protein, and will thus be transcribed at the amino terminus of the fusion protein. However, in certain cases, the signal sequence has been demonstrated to be located at positions other 5′ to the gene encoding the protein to be secreted. This sequence targets the protein to which it is attached across the inner membrane of the bacterial cell. The DNA encoding the signal sequence may be obtained as a restriction endonuclease fragment from any gene encoding a protein that has a signal sequence. Suitable prokaryotic signal sequences may be obtained from genes encoding, for example, LamB or OmpF (Wong et al., Gene, 68:193 [1983]), MalE, PhoA and other genes. A preferred prokaryotic signal sequence for practicing this invention is the E. coli heat-stable enterotoxin II (STII) signal sequence as described by Chang et al., Gene, 55: 189 [1987].
- Another useful component of the vectors used to practice this invention is phenotypic selection genes. Typical phenotypic selection genes are those encoding proteins that confer antibiotic resistance upon the host cell. By way of illustration, the ampicillin resistance gene (amp), and the tetracycline resistance gene (tet) are readily employed for this purpose.
- Construction of suitable vectors comprising the aforementioned components as well as the gene encoding the desired polypeptide (gene 1) are prepared using standard recombinant DNA procedures as described in Sambrook et al. supra. Isolated DNA fragments to be combined to form the vector are cleaved, tailored, and ligated together in a specific order and orientation to generate the desired vector.
- The DNA is cleaved using the appropriate restriction enzyme or enzymes in a suitable buffer. In general, about 0.2-1 μg of plasmid or DNA fragments is used with about 1-2 units of the appropriate restriction enzyme in about 20 μl of buffer solution. Appropriate buffers, DNA concentrations, and incubation times and temperatures are specified by the manufacturers of the restriction enzymes. Generally, incubation times of about one or two hours at 37° C. are adequate, although several enzymes require higher temperatures. After incubation, the enzymes and other contaminants are removed by extraction of the digestion solution with a mixture of phenol and chloroform, and the DNA is recovered from the aqueous fraction by precipitation with ethanol.
- To ligate the DNA fragments together to form a functional vector, the ends of the DNA fragments must be compatible with each other. In some cases, the ends will be directly compatible after endonuclease digestion. However, it may be necessary to first convert the sticky ends commonly produced by endonuclease digestion to blunt ends to make them compatible for ligation. To blunt the ends, the DNA is treated in a suitable buffer for at least 15 minutes at 15° C. with 10 units of the Klenow fragment of DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates. The DNA is then purified by phenol-chloroform extraction and ethanol precipitation.
- The cleaved DNA fragments may be size-separated and selected using DNA gel electrophoresis. The DNA may be electrophoresed through either an agarose or a polyacrylamide matrix. The selection of the matrix will depend on the size of the DNA fragments to be separated. After electrophoresis, the DNA is extracted from the matrix by electroelution, or, if low-melting agarose has been used as the matrix, by melting the agarose and extracting the DNA from it, as described in sections 6.30-6.33 of Sambrook et al., supra.
- The DNA fragments that are to be ligated together (previously digested with the appropriate restriction enzymes such that the ends of each fragment to be ligated are compatible) are put in solution in about equimolar amounts. The solution will also contain ATP, ligase buffer and a ligase such as T4 DNA ligase at about 10 units per 0.5 μg of DNA. If the DNA fragment is to be ligated into a vector, the vector is at first linearized by cutting with the appropriate restriction endonuclease(s). The linearized vector is then treated with alkaline phosphatase or calf intestinal phosphatase. The phosphatasing prevents self-ligation of the vector during the ligation step.
- After ligation, the vector with the foreign gene now inserted is transformed into a suitable host cell. Prokaryotes are the preferred host cells for this invention. Suitable prokaryotic host cells include E. coli strain JM101, E. coli K12 strain 294 (ATCC number 31,446), E. coli strain W3110 (ATCC number 27,325), E. coli X1776 (ATCC number 31,537), E. coli XL-1 Blue (stratagene), and E. coli B; however many other strains of E. coli, such as HB101, NM522, NM538, NM539, and many other species and genera of prokaryotes may be used as well. In addition to the E. coli strains listed above, bacilli such as Bacillus subtilis, other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species may all be used as hosts.
- Transformation of prokaryotic cells is readily accomplished using the calcium chloride method as described in section 1.82 of Sambrook et al., supra. Alternatively, electroporation (Neumann et al., EMBO J., 1:841 [1982]) may be used to transform these cells. The transformed cells are selected by growth on an antibiotic, commonly tetracycline (tet) or ampicillin (amp), to which they are rendered resistant due to the presence of tet and/or amp resistance genes on the vector.
- After selection of the transformed cells, these cells are grown in culture and the plasmid DNA (or other vector with the foreign gene inserted) is then isolated. Plasmid DNA can be isolated using methods known in the art. Two suitable methods are the small scale preparation of DNA and the large-scale preparation of DNA as described in sections 1.25-1.33 of Sambrook et al., supra. The isolated DNA can be purified by methods known in the art such as that described in section 1.40 of Sambrook et al., supra. This purified plasmid DNA is then analyzed by restriction mapping and/or DNA sequencing. DNA sequencing is generally performed by either the method of Messing et al. Nucleic Acids Res., 9:309 [1981] or by the method of Maxam et al. Meth. Enzymol., 65: 499 [1980].
- This invention contemplates fusing the gene enclosing the desired polypeptide (gene 1) to a second gene (gene 2) such that a fusion protein is generated during transcription.
Gene 2 is typically a coat protein gene of a phage, and preferably it is the phage M13 gene III coat protein, or a fragment thereof. Fusion ofgenes gene 2 into a particular site on a plasmid that containsgene 1, or by insertinggene 1 into a particular site on a plasmid that containsgene 2. - Insertion of a gene into a plasmid requires that the plasmid be cut at the precise location that the gene is to be inserted. Thus, there must be a restriction endonuclease site at this location (preferably a unique site such that the plasmid will only be cut at a single location during restriction endonuclease digestion). The plasmid is digested, phosphatased, and purified as described above. The gene is then inserted into this linearized plasmid by ligating the two DNAs together. Ligation can be accomplished if the ends of the plasmid are compatible with the ends of the gene to be inserted. If the restriction enzymes are used to cut the plasmid and isolate the gene to be inserted create blunt ends or compatible sticky ends, the DNAs can be ligated together directly using a ligase such as bacteriophage T4 DNA ligase and incubating the mixture at 16° C. for 1-4 hours in the presence of ATP and ligase buffer as described in section 1.68 of Sambrook et al., supra. If the ends are not compatible, they must first be made blunt by using the Klenow fragment of DNA polymerase I or bacteriophage T4 DNA polymerase, both of which require the four deoxyribonucleotide triphosphates to fill-in overhanging single-stranded ends of the digested DNA. Alternatively, the ends may be blunted using a nuclease such as nuclease S1 or mung-bean nuclease, both of which function by cutting back the overhanging single strands of DNA. The DNA is then religated using a ligase as described above. In some cases, it may not be possible to blunt the ends of the gene to be inserted, as the reading frame of the coding region will be altered. To overcome this problem, oligonucleotide linkers may be used. The linkers serve as a bridge to connect the plasmid to the gene to be inserted. These linkers can be made synthetically as double stranded or single stranded DNA using standard methods. The linkers have one end that is compatible with the ends of the gene to be inserted; the linkers are first ligated to this gene using ligation methods described above. The other end of the linkers is designed to be compatible with the plasmid for ligation. In designing the linkers, care must be taken to not destroy the reading frame of the gene to be inserted or the reading frame of the gene contained on the plasmid. In some cases, it may be necessary to design the linkers such that they code for part of an amino acid, or such that they code for one or more amino acids.
- Between
gene 1 andgene 2, DNA encoding a termination codon may be inserted, such termination codons are UAG (amber), UAA (ocher) and UGA (opel). (Microbiology, Davis et al. Harper & Row, New York, 1980,pages 237, 245-47 and 274). The termination codon expressed in a wild type host cell results in the synthesis of thegene 1 protein product without thegene 2 protein attached. However, growth in a suppressor host cell results in the synthesis of detectable quantities of fused protein. Such suppressor host cells contain a tRNA modified to insert an amino acid in the termination codon position of the mRNA thereby resulting in production of detectable amounts of the fusion protein. Such suppressor host cells are well known and described, such as E. coli suppressor strain (Bullock et al.,BioTechniques 5, 376-379 [1987]). Any acceptable method may be used to place such a termination codon into the mRNA encoding the fusion polypeptide. - The suppressible codon may be inserted between the first gene encoding a polypeptide, and a second gene encoding at least a portion of a phage coat protein. Alternatively, the suppressible termination codon may be inserted adjacent to the fusion site by replacing the last amino acid triplet in the polypeptide or the first amino acid in the phage coat protein. When the phagemid containing the suppressible codon is grown in a suppressor host cell, it results in the detectable production of a fusion polypeptide containing the polypeptide and the coat protein. When the phagemid is grown in a non-suppressor host cell, the polypeptide is synthesized substantially without fusion to the phage coat protein due to termination at the inserted suppressible triplet encoding UAG, UAA, or UGA. In the non-suppressor cell the polypeptide is synthesized and secreted from the host cell due to the absence of the fused phage coat protein which otherwise anchored it to the host cell.
-
Gene 1, encoding the desired polypeptide, may be altered at one or more selected codons. An alteration is defined as a substitution, deletion, or insertion of one or more codons in the gene encoding the polypeptide that results in a change in the amino acid sequence of the polypeptide as compared with the unaltered or native sequence of the same polypeptide. Preferably, the alterations will be by substitution of at least one amino acid with any other amino acid in one or more regions of the molecule. The alterations may be produced be a variety of methods known in the art. These methods include but are not limited to oligonucleotide-mediated mutagenesis and cassette mutagenesis. - A. Oligonucleotide-Mediated Mutagenesis
- Oligonucleotide-mediated mutagenesis is preferred method for preparing substitution, deletion, and insertion variants of
gene 1. This technique is well known in the art as described by Zoller et al. Nucleic Acids Res. 10: 6487-6504 [1987]. Briefly,gene 1 is altered by hybridizing an oligonucleotide encoding the desired mutation to a DNA template, where the template is the single-stranded form of the plasmid containing the unaltered or native DNA sequence ofgene 1. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template will thus incorporate the oligonucleotide primer, and will code for the selected alteration ingene 1. - Generally, oligonucleotides of at least 25 nucleotides in length are used. An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single-stranded DNA template molecule. The oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al. Proc. Nat'l. Acad. Sci. USA, 75: 5765 [1978].
- The DNA template can only be generated by those vectors that are either derived from bacteriophage M13 vectors (the commercially available M13mp18 and M13mp19 vectors are suitable), or those vectors that contain a single-stranded phage origin of replication as described by Viera et al. Meth. Enzymol., 153: 3 [1987]. Thus, the DNA that is to be mutated must be inserted into one of these vectors in order to generate single-stranded template. Production of the single-stranded template is described in sections 4.21-4.41 of Sambrook et al., supra.
- To alter the native DNA sequence, the oligonucleotide is hybridized to the single stranded template under suitable hybridization conditions. A DNA polymerizing enzyme, usually the Klenow fragment of DNA polymerase I, is then added to synthesize the complementary strand of the template using the oligonucleotide as a primer for synthesis. A heteroduplex molecule is thus formed such that one strand of DNA encodes the mutated form of
gene 1, and the other strand (the original template) encodes the native, unaltered sequence ofgene 1. This heteroduplex molecule is then transformed into a suitable host cell, usually a prokaryote such as E. Coli JM101. After growing the cells, they are plated onto agarose plates and screened using the oligonucleotide primer radiolabelled with 32-Phosphate to identify the bacterial colonies that contain the mutated DNA. - The method described immediately above may be modified such that a homoduplex molecule is created wherein both strands of the plasmid contain the mutation(s). The modifications are as follows: The single-stranded oligonucleotide is annealed to the single-stranded template as described above. A mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), and deoxyribothymidine (dTTP), is combined with a modified thio-deoxyribocytosine called dCTP-(aS) (which can be obtained from Amersham). This mixture is added to the template-oligonucleotide complex. Upon addition of DNA polymerase to this mixture, a strand of DNA identical to the template except for the mutated bases is generated. In addition, this new strand of DNA will contain dCTP-(aS) instead of dCTP, which serves to protect it from restriction endonuclease digestion. After the template strand of the double-stranded heteroduplex is nicked with an appropriate restriction enzyme, the template strand can be digested with ExolII nuclease or another appropriate nuclease past the region that contains the site(s) to be mutagenized. The reaction is then stopped to leave a molecule that is only partially single-stranded. A complete double-stranded DNA homoduplex is then formed using DNA polymerase in the presence of all four deoxyribonucleotide triphosphates, ATP, and DNA ligase. This homoduplex molecule can then be transformed into a suitable host cell such as E. coli JM101, as described above.
- Mutants with more than one amino acid to be substituted may be generated in one of several ways. If the amino acids are located close together in the polypeptide chain, they may be mutated simultaneously using one oligonucleotide that codes for all of the desired amino acid substitutions. If, however, the amino acids are located some distance from each other (separated by more than about ten amino acids), it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed.
- In the first method, a separate oligonucleotide is generated for each amino acid to be substituted. The oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions. The alternative method involves two or more rounds of mutagenesis to produce the desired mutant. The first round is as described for the single mutants: wild-type DNA is used for the template, an oligonucleotide encoding the first desired amino acid substitution(s) is annealed to this template, and the heteroduplex DNA molecule is then generated. The second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template. Thus, this template already contains one or more mutations. The oligonucleotide encoding the additional desired amino acid substitution(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis. This resultant DNA can be used as a template in a third round of mutagenesis, and so on.
- B. Cassette Mutagenesis
- This method is also a preferred method for preparing substitution, deletion, and insertion variants of
gene 1. The method is based on that described by Wells et al. Gene, 34:315 [1985]. The starting material is the plasmid (or other vector) comprisinggene 1, the gene to be mutated. The codon(s) ingene 1 to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations ingene 1. After the restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 3′ and 5′ ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated DNA sequence ofgene 1. - In an alternative embodiment, this invention contemplates production of variants of a desired protein containing one or more subunits. Each subunit is typically encoded by separate gene. Each gene encoding each subunit can be obtained by methods known in the art (see, for example, Section II). In some instances it may be necessary to obtain the gene encoding the various subunits using separate techniques selected from any of the methods described in Section II.
- When constructing a replicable expression vector where the protein of interest contains more than one subunit, all subunits can be regulated by the same promoter, typically located 5′ to the DNA encoding the subunits, or each may be regulated by separate promoter suitably oriented in the vector so that each promoter is operably linked to the DNA it is intended to regulate. Selection of promoters is carried out as described in Section III above.
- In constructing a replicable expression vector containing DNA encoding the protein of interest having multiple subunits, the reader is referred to
FIG. 10 where, by way of illustration, a vector is diagrammed showing DNA encoding each subunit of an antibody fragment. This figure shows that, generally, one of the subunits of the protein of interest will be fused to a phage coat protein such as M13 gene III. This gene fusion generally will contain its own signal sequence. A separate gene encodes the other subunit or subunits, and it is apparent that each subunit generally has its own signal sequence.FIG. 10 also shows that a single promoter can regulate the expression of both subunits. Alternatively, each subunit may be independently regulated by a different promoter. The protein of interest subunit-phage coat protein fusion construct can be made as described in Section IV above. - When constructing a family of variants of the desired multi-subunit protein, DNA encoding each subunit in the vector may mutated in one or more positions in each subunit. When multi-subunit antibody variants are constructed, preferred sites of mutagenesis correspond to codons encoding amino acid residues located in the complementarity-determining regions (CDR) of either the light chain, the heavy chain, or both chains. The CDRs are commonly referred to as the hypervariable regions. Methods for mutagenizing DNA encoding each subunit of the protein of interest are conducted essentially as described in Section V above.
- VII. Preparing a Target Molecule and Binding with Phagemid
- Target proteins, such as receptors, may be isolated from natural sources or prepared by recombinant methods by procedures known in the art. By way of illustration, glycoprotein hormone receptors may be prepared by the technique described by McFarland et al., Science 245:494-499 [1989], nonglycosylated forms expressed in E. coli are described by Fuh et al. J. Biol. Chem 265:3111-3115 [1990] Other receptors can be prepared by standard methods.
- The purified target protein may be attached to a suitable matrix such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic copolymers, hydroxylalkyl methacrylate gels, polyacrylic and polymethacrylic copolymers, nylon, neutral and ionic carriers, and the like. Attachment of the target protein to the matrix may be accomplished by methods described in Methods in Enzymology 44 [1976], or by other means known in the art.
- After attachment of the target protein to the matrix, the immobilized target is contacted with the library of phagemid particles under conditions suitable for binding of at least a portion of the phagemid particles with the immobilized target. Normally, the conditions, including pH, ionic strength, temperature and the like will mimic physiological conditions.
- Bound phagemid particles (“binders”) having high affinity for the immobilized target are separated from those having a low affinity (and thus do not bind to the target) by washing. Binders may be dissociated from the immobilized target by a variety of methods. These methods include competitive dissociation using the wild-type ligand, altering pH and/or ionic strength, and methods known in the art.
- Suitable host cells are infected with the binders and helper phage, and the host cells are cultured under conditions suitable for amplification of the phagemid particles. The phagemid particles are then collected and the selection process is repeated one or more times until binders having the desired affinity for the target molecule are selected.
- Optionally the library of phagemid particles may be sequentially contacted with more than one immobilized target to improve selectivity for a particular target. For example, it is often the case that a ligand such as hGH has more than one natural receptor. In the case of hGH, both the growth hormone receptor and the prolactin receptor bind the hGH ligand. It may be desirable to improve the selectivity of hGH for the growth hormone receptor over the prolactin receptor. This can be achieved by first contacting the library of phagemid particles with immobilized prolactin receptor, eluting those with a low affinity (i.e. lower than wild type hGH) for the prolactin receptor and then contacting the low affinity prolactin “binders” or non-binders with the immobilized growth hormone receptor, and selecting for high affinity growth hormone receptor binders. In this case an hGH mutant having a lower affinity for the prolactin receptor would have therapeutic utility even if the affinity for the growth hormone receptor were somewhat lower than that of wild type hGH. This same strategy may be employed to improve selectivity of a particular hormone or protein for its primary function receptor over its clearance receptor.
- In another embodiment of this invention, an improved substrate amino acid sequence can be obtained. These may be useful for making better “cut sites” for protein linkers, or for better protease substrates/inhibitors. In this embodiment, an immobilizable molecule (e.g. hGH-receptor, biotin-avidin, or one capable of covalent linkage with a matrix) is fused to gene III through a linker. The linker will preferably be from 3 to 10 amino acids in length and will act as a substrate for a protease. A phagemid will be constructed as described above where the DNA encoding the linker region is randomly mutated to produce a randomized library of phagemid particles with different amino acid sequences at the linking site. The library of phagemid particles are then immobilized on a matrix and exposed to a desired protease. Phagemid particles having preferred or better substrate amino acid sequences in the liner region for the desired protease will be eluted, first producing an enriched pool of phagemid particles encoding preferred linkers. These phagemid particles are then cycled several more times to produce an enriched pool of particles encoding consense sequence(s) (see examples XIII and XIV).
- The cloned gene for hGH has been expressed in a secreted form in Eschericha coli (Chang, C. N>, et al., [1987]
Gene 55, 189) and its DNA and amino acid sequence has been reported (Goeddel, et al. [1979] Nature 281, 544; Gray et al., [1985] Gene 39, 247). The present invention describes novel hGH variants produced using the phagemid selection methods. Human growth hormone variants containing substitutions atpositions - Therapeutic formulations of hGH for therapeutic administration are prepared for storage by mixing hGH having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A., Ed., (1980), in the form of lyophilized cake or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; divalent metal ions such as zinc, cobalt or copper; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG). Formulations of the present invention may additionally contain a pharmaceutically acceptable buffer, amino acid, bulking agent and/or non-ionic surfactant. These include, for example, buffers, chelating agents, antioxidants, preservatives, cosolvents, and the like; specific examples of these could include, trimethylamaine salts (“Tris buffer”), and disodium edetate. The phagemids of the present invention may be used to produce quantities of the hGH variants free of the phage protein. To express hGH variants free of the gene III portion of the fusion, pS0643 and derivatives can simply be grown in a non-suppressor strain such as 16C9. In this case, the amber codon (TAG) leads to termination of translation, which yields free hormone, without the need for an independent DNA construction. The hGH variant is secreted from the host and may be isolated from the culture medium.
- One or more of the eight hGH amino acids F10, M14, H18, H21, R167, D171, T175 and I179 may be replaced by any amino acid other than the one found in that position in naturally occurring hGH as indicated. Therefore, 1, 2, 3, 4, 5, 6, 7, or all 8 of the indicated amino acids, F10, M14, H18, H21, R167, D171, T175 and I179, may be replaced by any of the other 19 amino acids out of the 20 amino acids listed below. In a preferred embodiment, all eight listed amino acids are replaced by another amino acid. The most preferred eight amino acids to be substituted are indicated in Table XIV in Example XII.
-
-
- Ala (A)
- Arg (R)
- Asn (N)
- Asp (D)
- Cys (C)
- Gln (Q)
- Glu (E)
- Gly (G)
- His (H)
- Ile (I)
- Leu (L)
- Lys (K)
- Met (M)
- Phe (F)
- Pro (P)
- Ser (S)
- Thr (T)
- Trp (W)
- Tyr (Y)
- Val (V)
The one letter hGH variant nomenclature first gives the hGH amino acid deleted, forexample glutamate 179; then the amino acid inserted; for example, serine; resulting in (E1795S).
- Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and illustrative examples, make and utilize the present invention to the fullest extent. The following working examples therefore specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way of the remainder of the disclosure.
- The plasmid phGH-M13gIII (
FIG. 1 ), was constructed from M13KO77 and the hGH producing plasmid, pBO473 (Cunningham, B. C., et al., Science, 243:1330-1336, [1989]). Asynthetic oligonucleotide 5′-AGC-TGT-GGC-TTC-GGG-CCC-TTA-GCA-TTT-AAT-GCG-GTA-3′ (SEQ ID NO:2) was used to introduce a unique ApaI restriction site (underlined) into pBO473 after the final Phe191 codon of hGH. Theoligonucleotide 5′-TTC-ACA-AAC-GAA-GGG-CCC-CTA-ATT-AAA-GCC-AGA-3′ (SEQ ID NO:3) was used to introduce a unique ApaI restriction site (underlined), and a Glu197-to-amber stop codon (bold lettering) into M13KO7 gene III. Theoligonucleotide 5′-CAA-TAA-TAA-CGG-GCT-AGC-CAA-AAG-AAC-TGG-3′ (SEQ ID NO:4) introduces a unique NheI site (underlined) after the 3′ end of the gene III coding sequence. The resulting 650 base pair (bp) ApaI-NheI fragment from the doubly mutated M13KO7 gene III was cloned into the large ApaI-NheI fragment of pBO473 to create the plasmid, pSO132. This fuses the carboxyl terminus of hGH (Phe191) to the Pro198 residue of the gene III protein with the insertion of a glycine residue encoded from the ApaI site and places the fusion protein under control of the E. coli alkaline phosphatase (phoA) promoter and stII secretion signal sequence (Chang, C. N., et al., Gene, 55:189-196, [1987]). For inducible expression of the fusion protein in rich media, we replaced the phoA promoter with the lac promoter and operator. A 138 bp EcoRI-XbaI fragment containing the lac promoter, operator, and Cap binding site was produced by PCR of plasmid pUC119 using theoligonucleotides 5′-CACGACAGAATTCCCGACTGGAAA-3′ (SEQ ID NO:5) and 5′-CTGTT TCTAGAGTGAAATTGTTA-3′ (SEQ ID NO:6) that flank the desired lac sequences and introduce the EcoRI and XbaI restriction sites (underlined). This lac fragment was gel purified and ligated into the large EcoRI-XbaI fragment of pSO132 to create the plasmid, phGH-M13gIII. The sequences of all tailored DNA junctions were verified by the dideoxy sequence method (Sanger, F., et al. Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467, [1977]). The R64A variant hGH phagemid was constructed as follows: the NsiI-BglII mutated fragment of hGH (Cunningham et al. supra) encoding the Arg64 to Ala substitution (R64A) (Cunningham, B. C., Wells, J. A., Science, 244:1081-1085, [1989]) was cloned between the corresponding restriction sites in the phGH-M13gIII plasmid (FIG. 1 ) to replace the wild-type hGH sequence. The R64A hGH phagemid particles were propagated and titered as described below for the wild-type hGH-phagemid. - Plasmids were transformed into a male strain of E. coli (JM101) and selected on carbenicillin plates. A single transformant was grown in 2 ml 2YT medium for 4 h at 37° C. and infected with 50 μl of M13KO7 helper phage. The infected culture was diluted into 30 ml 2YT, grown overnight, and phagemid particles were harvested by precipitation with polyethylene glycol (Vierra, J., Messing, J., Methods in Enzymology, 153:3-11, [1987]). Typical phagemid particle titers ranged from 2 to 5×1011 cfu/ml. The particles were purified to homogeneity by CsCl density centrifugation (Day, L. A. J. Mol. Biol., 39:265-277, [1969]) to remove any fusion protein not attached to virions.
- Rabbit polyclonal antibodies to hGH were purified with protein A, and coated onto microtiter plates (Nunc) at a concentration of 2 μg/ml in 50 mM sodium carbonate buffer (pH 10) at 4° C. for 16-20 hours. After washing in PBS containing 0.05
% Tween 20, hGH or hGH-phagemid particles were serially diluted from 2.0-0.002 nM in buffer A (50 mM Tris (pH 7.5), 50 mM NaCl, 2 mM EDTA, 5 mg/ml bovine serum albumin, and 0.05% Tween 20). After 2 hours at room temperature (rt), the plates were washed well and the indicated Mab (Cunningham et al. supra) was added at 1 μg/ml in buffer A for 2 hours at rt. Following washing, horseradish peroxidase conjugated goat anti-mouse IgG antibody was bound at rt for 1 hour. After a final wash, the peroxidase activity was assayed with the substrate, o-phenylenediamine. - Oxirane polyacrylamide beads (Sigma) were conjugated to the purified extracellular domain of the hGH receptor (hGHbp) (Fuh, G., et al., J. Biol. Chem., 265:3111-3115 [1990]) containing an extra cysteine residue introduced by site-directed mutagenesis at
position 237 that does not affect binding of hGH (J. Wells, unpublished). The hGHbp was conjugated as recommended by the supplier to a level of 1.7 pmol hGHbp/mg dry oxirane bead, as measured by binding of [125I] hGH to the resin. Subsequently, any unreacted oxirane groups were blocked with BSA and Tris. As a control for non-specific binding of phagemid particles, BSA was similarly coupled to the beads. Buffer for adsorption and washing contained 10 mM Tris.HCl (pH 7.5), 1 mM EDTA, 50 mM NaCl, 1 mg/ml BSA, and 0.02% Tween 20. Elution buffers contained wash buffer plus 200 nM hGH or 0.2 M glycine (pH 2.1). Parental phage M13KO7 was mixed with hGH phagemid particles at a ratio of nearly 3000:1 (original mixture) and tumbled for 8-12 h with a 5 μl aliquot (0.2 mg of acrylamide beads) of either absorbent in a 50 μl volume at room temperature. The beads were pelleted by centrifugation and the supernate carefully removed. The beads were resuspended in 200 μl wash buffer and tumbled at room temperature for 4 hours (wash 1). After a second wash (wash 2), the beads were eluted twice with 200 nM hGH for 6-10 hours each (eluate 1, eluate 2). The final elution was with a glycine buffer (pH 2.1) for 4 hours to remove remaining hGH phagemid particles (eluate 3). Each fraction was diluted appropriately in 2YT media, mixed with fresh JM101, incubated at 37° C. for 5 minutes, and plated with 3 ml of 2YT soft agar on LB or LB carbenicillin plates. - The gene III protein is composed of 410 residues divided into two domains that are separated by a flexible linker sequence (Armstrong, J., et al., FEBS Lett., 135:167-172, [1981]). The amino-terminal domain is required for attachment to the pili of E. coli, while the carboxyl-terminal domain is imbedded in the phage coat and required for proper phage assembly (Crissman, J. W., Smith, G. P., Virology, 132:445-455, [1984]). The signal sequence and amino-terminal domain of gene III was replaced with the stII signal and entire hGH gene (Chang et al. supra) by fusion to residue 198 in the carboxyl-terminal domain of gene III (
FIG. 1 ). The hGH-gene III fusion was placed under control of the lac promoter/operator in a plasmid (phGH-M13gIII;FIG. 1 ) containing the pBR322 β-lactamase gene and Col E1 replication origin, and the phage f1 intergenic region. The vector can be easily maintained as a small plasmid vector by selection on carbenicillin, which avoids relying on a functional gene III fusion for propagation. Alternatively, the plasmid can be efficiently packaged into virions (called phagemid particles) by infection with helper phage such as M13KO7 (Viera et al. supra) which avoids problems of phage assembly. Phagemid infectivity titers based upon transduction to carbenicillin resistance in this system varied from 2-5×1011 colony forming units (cfu)/ml. The titer of the M13KO7 helper phage in these phagemid stocks is ˜1010 plaque forming units (pfu)/ml. - With this system we confirmed previous studies (Parmley, Smith supra) that homogeneous expression of large proteins fused to gene III is deleterious to phage production (data not shown). For example, induction of the lac promoter in phGH-M13gIII by addition of IPTG produced low phagemid titers. Moreover, phagemid particles produced by co-infection with M13KO7 containing an amber mutation in gene III gave very low phagemid titers (<1010 cfu/ml). We believed that multiple copies of the gene III fusion attached to the phagemid surface could lead to multiple point attachment (the “chelate effect”) of the fusion phage to the immobilized target protein. Therefore to control the fusion protein copy number we limited transcription of the hGH-gene III fusion by culturing the plasmid in E. coli JM101 (lacIQ) which contains a constitutively high level of the lac repressor protein. The E. coli JM101 cultures containing phGH-M13gIII were best propagated and infected with M13KO7 in the absence of the lac operon inducer (IPTG); however, this system is flexible so that co-expression of other gene III fusion proteins can be balanced. We estimate that about 10% of the phagemid particles contain one copy of the hGH gene III fusion protein from the ratio of the amount of hGH per virion (based on hGH immuno-reactive material in CsCl gradient purified phagemid). Therefore, the titer of fusion phage displaying the hGH gene III fusion is about 2-5×1010/ml. This number is much greater than the titer of E. coli (˜108 to 109/ml) in the culture from which they are derived. Thus, on average every E. coli cell produces 10-100 copies of phage decorated with an hGH gene III fusion protein.
- Immunoblot analysis (
FIG. 2 ) of the hGH-gene III phagemid show that hGH cross-reactive material comigrates with phagemid particles in agarose gels. This indicates that the hGH is tightly associated with phagemid particles. The hGH-gene III fusion protein from the phagemid particles runs as a single immuno-stained band showing that there is little degradation of the hGH when it is attached to gene III. Wild-type gene III protein is clearly present because about 25% of the phagemid particles are infectious. This is comparable to specific infectivity estimates made for wild-type M13 phage that are similarly purified (by CsCl density gradients) and concentrations estimated by UV absorbance (Smith, G. P. supra and Parmley, Smith supra) Thus, both wild-type gene III and the hGH-gene III fusion proteins are displayed in the phage pool. - It was important to confirm that the tertiary structure of the displayed hGH was maintained in order to have confidence that results from binding selections will translate to the native protein. We used monoclonal antibodies (Mabs) to hGH to evaluate the structural integrity of the displayed hGH gene III fusion protein (Table I).
-
TABLE I Binding of Eight Different Monoclonal Antibodies (Mab's) to hGH and hGH Phagemid Particles* IC50 (nM) Mab hGH hGH- phagemid 1 0.4 0.4 2 0.04 0.04 3 0.2 0.2 4 0.1 0.1 5 0.2 >2.0 6 0.07 0.2 7 0.1 0.1 8 0.1 0.1 *Values given represent the concentration (nM) of hGH or hGH-phagemid particles to give half-maximal binding to the particular Mab. Standard errors in these measurements are typically at or below ±30% of the reported value. See Materials and Methods for further details. - The epitopes on hGH for these Mabs have been mapped (Cunningham et al. supra) and binding for 7 of 8 Mabs requires that hGH be properly folded. The IC50 values for all Mabs were equivalent to wild-type hGH except for
Mab Mabs - Previous workers (Parmley, Smith supra; Scott, Smith supra; Cwirla et al. supra; and Devlin et al. supra) have fractionated phage by panning with streptavidin coated polystyrene petri dishes or microtiter plates. However, chromatographic systems would allow more efficient fractionation of phagemid particles displaying mutant proteins with different binding affinities. We chose non-porous oxirane beads (Sigma) to avoid trapping of phagemid particles in the chromatographic resin. Furthermore, these beads have a small particle size (1 μm) to maximize the surface area to mass ratio. The extracellular domain of the hGH receptor (hGHbp) (Fuh et al., supra) containing a free cysteino residue was efficiently coupled to these beads and phagemid particles showed very low non-specific binding to beads coupled only to bovine serum albumin (Table II).
-
TABLE II Specific Binding of Hormone Phage to hGHbp-coated Beads Provides an Enrichment for hGH-phage over M13KO7 Phage* Sample Absorbent‡ Total pfu Total cfu Ratio (cfu/pfu) Enrichment§ Original mixture† 8.3 × 1011 2.9 × 108 3.5 × 10−4 (1) Supernatant BSA 7.4 × 1011 2.8 × 108 3.8 × 10−4 1.1 hGHbp 7.6 × 1011 3.3 × 108 4.3 × 10−4 1.2 Wash 1BSA 1.1 × 1010 6.0 × 106 5.5 × 10−4 1.6 hGHbp 1.9 × 1010 1.7 × 107 8.9 × 10−4 2.5 Wash 2BSA 5.9 × 107 2.8 × 104 4.7 × 10−4 1.3 hGHbp 4.9 × 107 2.7 × 106 5.5 × 10−2 1.6 × 102 Eluate 1 (hGH) BSA 1.1 × 106 1.9 × 103 1.7 × 10−3 4.9 hGHbp 1.2 × 106 2.1 × 106 1.8 5.1 × 103 Eluate 2 (hGH) BSA 5.9 × 105 1.2 × 103 2.0 × 10−3 5.7 hGHbp 5.5 × 105 1.3 × 106 2.4 6.9 × 103 Eluate 3 (pH 2.1) BSA 4.6 × 105 2.0 × 103 4.3 × 10−3 12.3 hGHbp 3.8 × 105 4.0 × 106 10.5 3.0 × 104 *The titers of M13KO7 and hGH-phagemid particles in each fraction was determined by multiplying the number of plaque forming units (pfu) or carbenicillin resistant colony forming units (cfu) by the dilution factor, respectively. See Example IV for details. †The ratio of M13KO7 to hGH-phagemid particles was adjusted to 3000:1 in the original mixture. ‡Absorbents were conjugated with BSA or hGHbp. §Enrichments are calculated by dividing the cfu/pfu ratio after each step by cfu/pfu ratio in the original mixture. - In a typical enrichment experiment (Table II), one part of hGH phagemid was mixed with >3,000 parts M13KO7 phage. After one cycle of binding and elution, 106 phage were recovered and the ratio of phagemid to M13KO7 phage was 2 to 1. Thus, a single binding selection step gave >5000-fold enrichment. Additional elutions with free hGH or acid treatment to remove remaining phagemids produced even greater enrichments. The enrichments are comparable to those obtained by Smith and coworkers using batch elution from coated polystyrene plates (Smith, G. P. supra and Parmely, Smith supra) however much smaller volumes are used on the beads (200 μl vs. 6 ml). There was almost no enrichment for the hGH phagemid over M13KO7 when we used beads linked only to BSA. The slight enrichment observed for control beads (˜10-fold for pH 2.1 elution; Table 2) may result from trace contaminants of bovine growth hormone binding protein present in the BSA linked to the bead. Nevertheless these data show the enrichments for the hGH phage depend upon the presence of the hGHbp on the bead suggesting binding occurs by specific interaction between hGH and the hGHbp.
- We evaluated the enrichment for wild-type hGH over a weaker binding variant of the hGH on fusion phagemids to further demonstrate enrichment specificity, and to link the reduction in binding affinity for the purified hormones to enrichment factors after panning fusion phagemids. A fusion phagemid was constructed with an hGH mutant in which Arg64 was substituted with Ala (R64A). The R64A variant hormone is about 20-fold reduced in receptor binding affinity compared to hGH (Kd values of 7.1 nM and 0.34 nM, respectively [Cunningham, Wells, supra]). The titers of the R64A hGH-gene III fusion phagemid were comparable to those of wild-type hGH phagemid. After one round of binding and elution (Table III) the wild-type hGH phagemid was enriched from a mixture of the two phagemids plus M13KO7 by 8-fold relative to the phagemid R64A, and ˜104 relative to M13KO7 helper phage.
-
TABLE III hGHbp-coated Beads Select for hGH Phagemids Over a Weaker Binding hGH Variant Phagemid Control beads hGHbp beads WT phagemid enrichment WT phagemid enrichment Sample total phagemid for WT/R64A total phagemid for WT/ R64A Original mixture 8/20 (1) 8/20 (1) Supernatant ND — 4/10 1.0 Elution 1 (hGH) 7/20 0.8 17/20 8.5‡ Elution 2 (pH 2.1) 11/20 1.8 21/27 5.2 *The parent M13KO7 phage, wild-type hGH phagemid and R64A phagemid particles were mixed at a ratio of 104:0.4:0.6. Binding selections were carried out using beads linked with BSA (control beads) or with the hGHbp (hGHbp beads) as described in Table II and the Materials and Methods After each step, plasmid DNA was isolated (Birnboim, H. C., Doly, J., Nucleic Acids Res., 7: 1513-1523, [1979]) from carbenicillin resistant colonies and analyzed by restriction analysis to determine if it contained the wild-type hGH or the R64A hGH gene III fusion. †The enrichment for wild-type hGH phagemid over R64A mutant was calculated from the ratio of hGH phagemid present after each step to that present in the original mixture (8/20), divided by the corresponding ratio for R64A phagemids. WT = wild-type; ND = not determined. ‡The enrichment for phagemid over total M13KO7 parental phage was ~104 after this step. - By displaying a mixture of wild-type gene III and the gene III fusion protein on phagemid particles one can assemble and propagate virions that display a large and proper folded protein as a fusion to gene III. The copy number of the gene III fusion protein can be effectively controlled to avoid “chelate effects” yet maintained at high enough levels in the phagemid pool to permit panning of large epitope libraries (>1010). We have shown that hGH (a 22 kD protein) can be displayed in its native folded form. Binding selections performed on receptor affinity beads eluted with free hGH, efficiently enriched for wild-type hGH phagemids over a mutant hGH phagemid shown to have reduced receptor binding affinity. Thus, it is possible to sort phagemid particles whose binding constants are down in the nanomolar range.
- Protein-protein and antibody-antigen interactions are dominated by discontinuous epitopes (Janin, J., et al., J. Mol. Biol., 204:155-164, [1988]; Argos, P., Prot. Eng., 2:101-113, [1988]; Barlow, D. J., et al., Nature, 322:747-748, [1987]; and Davies, D. R., et al., J. Biol. Chem., 263:10541-10544, [1988]); that is the residues directly involved in binding are close in tertiary structure but separated by residues not involved in binding. The screening system presented here should allow one to analyze more conveniently protein-receptor interactions and isolate discontinuous epitopes in proteins with new and high affinity binding properties.
- A mutant of the hGH-gene III fusion protein was constructed using the method of Kunkel, et al. Meth. Enzymol. 154, 367-382 [1987]. Template DNA was prepared by growing the plasmid pS0132 (containing the natural hGH gene fused to the carboxy-terminal half of M13 gene III, under control of the alkaline phosphatase promoter) in CJ236 cells with M13-K07 phage added as helper. Single-stranded, uracil-containing DNA was prepared for mutagenesis to introduce (1) a mutation in hGH which would greatly reduce binding to the hGH binding protein (hGHbp); and (2) a unique restriction site (KpnI) which could be used for assaying for—and selecting against—parental background phage. Oligonucleotide-directed mutagenesis was carried out using T7 DNA polymerase and the following oligodeoxy-nucleotide (SEQ ID NO:7):
-
hGH codon: Gly Thr 178 179 5′-G ACA TTC CTG GGT ACC GTG CAG T-3′ < KpnI > - This oligo introduces the KpnI site as shown, along with mutations (R178G, I179T) in hGH. These mutations are predicted to reduce binding of hGH to hGHbp by more than 30-fold. Clones from the mutagenesis were screened by KpnI digestion and confirmed by dideoxy DNA sequencing. The resulting construct, to be used as a template for random mutagenesis, was designated pHO415.
- Random Mutagenesis within Helix-4 of hGH
-
Codons -
hGH codon: 172 174 5′-GC TTC AGG AAG GAC ATG GAC NNS GTC NNS ACA-- Ile 176 178 179 NNS CTG NNS ATC GTG CAG TGC CGC TCT GTG G-3′
As shown, this oligo pool revertscodon 179 to wild-type (Ile), destroys the unique KpnI site of pH0415, and introduces random codons (NNS, where N=A, G, C, or T and S=G or C) atpositions - The mutagenesis products were extracted twice with phenol:chloroform (50:50) and ethanol precipitated with an excess of carrier tRNA to avoid adding salt that would confound the subsequent electroporation step. Approximately 50 ng (15 fmols) of DNA was electroporated into WJM101 cells (2.8×1010 cells/mL) in 45 μL total volume in a 0.2 cm cuvette at a voltage setting of 2.49 kV with a single pulse (time constant=4.7 msec.).
- The cells were allowed to recover 1 hour at 37° C. with shaking, then mixed with 25 mL 2YT medium, 100 μg/mL carbenicillin, and M13-K07 (multiplicity of infection=1000). Plating of serial dilutions from this culture onto carbenicillin-containing media indicated that 8.2×106 electrotransformants were obtained. After 10′ at 23° C., the culture was incubated overnight (15 hours) at 37° C. with shaking.
- After overnight incubation, the cells were pelleted, and double-stranded DNA (dsDNA), designated pLIB1, was prepared by the alkaline lysis method. The supernatant was spun again to remove any remaining cells, and the phage, designated phage pool φ1, were PEG-precipitated and resuspended in 1 mL STE buffer (10 mM Tris, pH 7.6, 1 mM EDTA, 50 mM NaCl). Phage titers were measured as colony-forming units (CFU) for the recombinant phagemid containing hGH-g3p gene III fusion (hGH-g3) plasmid, and plaque-forming units (PFU) for the M13-K07 helper phage.
- Binding Selection Using Immobilized hGHbp
- 1. BINDING: An aliquot of phage pool φ1 (6×109 CFU, 6×107 PFU) was diluted 4.5-fold in buffer A (Phosphate-buffered saline, 0.5% BSA, 0.05% Tween-20, 0.01% thimerosal) and mixed with a 5 μL suspension of oxirane-polyacrylamide beads coupled to the hGHbp containing a Ser237 Cys mutation (350 fmols) in a 1.5 mL silated polypropylene tube. As a control, an equivalent aliquot of phage were mixed in a separate tube with beads that had been coated with BSA only. The phage were allowed to bind to the beads by incubating 3 hours at room temperature (23° C.) with slow rotation (approximately 7 RPM). Subsequent steps were carried out with a constant volume of 200 μL and at room temperature.
- 2. WASH: The beads were spun 15 sec., and the supernatant was removed (Sup. 1). To remove phage/phagemid not specifically bound, the beads were washed twice by resuspending in buffer A, then pelleting. A final wash consisted of rotating the beads in buffer A for 2 hours.
- 3. hGH ELUTION: Phage/phagemid binding weakly to the beads were removed by stepwise elution with hGH. In the first step, the beads were rotated with buffer A containing 2 nM hGH. After 17 hours, the beads were pelleted and resuspended in buffer A containing 20 nM hGH and rotated for 3 hours, then pelleted. In the final hGH wash, the beads were suspended in buffer A containing 200 nM hGH and rotated for 3 hours then pelleted.
- 4. GLYCINE ELUTION: To remove the tightest-binding phagemid (i.e. those still bound after the hGH washes), beads were suspended in Glycine buffer (1 M Glycine, pH 2.0 with HCl), rotated 2 hours and pelleted. The supernatant (fraction “G”; 200 μL) was neutralized by adding 30 μL of 1 M Tris base.
- Fraction G eluted from the hGHbp-beads (1×106 CFU, 5×104 PFU) was not substantially enriched for phagemid over K07 helper phage. We believe this resulted from the fact that K07 phage packaged during propagation of the recombinant phagemid display the hGH-g3p fusion.
- However, when compared with fraction G eluted from the BSA-coated control beads, the hGHbp-beads yielded 14 times as many CFU's. This reflects the enrichment of tight-binding hGH-displaying phagemid over nonspecifically-binding phagemid.
- 5. PROPAGATION: An aliquot (4.3×105 CFU) of fraction G eluted from the hGHbp-beads was used to infect log-phase WJM101 cells. Transductions were carried out by mixing 100 μL fraction G with 1 mL WJM101 cells, incubating 20 min. at 37° C., then adding K07 (multiplicity of infection=1000). Cultures (25 mL 2YT plus carbenicillin) were grown as described above and the second pool of phage (
Library 1G, for first glycine elution) were prepared as described above. - Phage from
library 1G (FIG. 3 ) were selected for binding to hGHbp beads as described above. Fraction G eluted from hGHbp beads contained 30 times as many CFU's as fraction G eluted from BSA-beads in this selection. Again, an aliquot of fraction G was propagated in WJM101 cells to yieldlibrary 1G2 (indicating that this library had been twice selected by glycine elution). Double-stranded DNA (pLIB 1G2) was also prepared from this culture. - KpnI Assay and Restriction-Selection of dsDNA
- To reduce the level of background (KpnI+) template, an aliquot (about 0.5 μg) of
pLIB 1G2 was digested with KpnI and electroporated into WJM101 cells. These cells were grown in the presence of K07 (multiplicity of infection=100) as described for the initial library, and a new phage pool,pLIB 3, was prepared (FIG. 3 ). - In addition, an aliquot (about 0.5 μg) of dsDNA from the initial library (pLIB1) was digested with KpnI and electroporated directly into WJM101 cells. Transformants were allowed to recover as above, infected with M13-K07, and grown overnight to obtain a new library of phage, designated phage Library 2 (
FIG. 3 ). - Phagemid binding, elution, and propagation were carried out in successive rounds for phagemid derived from both
pLIB 2 and pLIB 3 (FIG. 3 ) as described above, except that (1) an excess (10-fold over CFU) of purified K07 phage (not displaying hGH) was added in the bead-binding cocktail, and (2) the hGH stepwise elutions were replaced with brief washings of buffer A alone. Also, in some cases, XL1-Blue cells were used for phagemid propagation. - An additional digestion of dsDNA with KpnI was carried out on
pLIB 2G3 and onpLIB 3G5 before the final round of bead-binding selection (FIG. 3 ). - Four independently isolated clones from
LIB 4G4 and four independently isolated clones fromLIB 5G6 were sequenced by dideoxy sequencing. All eight of these clones had identical DNA sequences (SEQ ID NO:9): -
hGH codon: 172 174 176 178 5′-AAG GTC TCC ACA TAC CTG AGG ATC-3′
Thus, all these encode the same mutant of hGH: (E174S, F176Y).Residue 172 in these clones is Lys as in wild-type. The codon selected for 172 is also identical to wild-type hGH. This is not surprising since AAG is the only lysine-codon possible from a degenerate “NNS” codon set. Residue 178-Arg is also the same as wild-type, but here, the codon selected from the library was AAG instead of CGC as is found in wild-type hGH, even though the latter codon is also possible using the “NNS” codon set. - The multiplicity of infection of K07 infection is an important parameter in the propagation of recombinant phagemids. The K07 multiplicity of infection must be high enough to insure that virtually all cells transformed or transfected with phagemid are able to package new phagemid particles. Furthermore, the concentration of wild-type gene III in each cell should be kept high to reduce the possibility of multiple hGH-gene III fusion molecules being displayed on each phagemid particle, thereby reducing chelate effects in binding. However, if the K07 multiplicity of infection is too high, the packaging of K07 will compete with that of recombinant phagemid. We find that acceptable phagemid yields, with only 1-10% background K07 phage, are obtained when the K07 multiplicity of infection is 100.
-
TABLE IV Enrichment hGHbp/BSA Phage Pool moi (K07) CFU/PFU beads Fraction Kpnl LIB 1 1000 ND 14 0.44 LIB 1G1000 ND 30 0.57 LIB 3100 ND 1.7 0.26 LIB 3G10 ND 8.5 0.18 LIB 3G100 460 220 0.13 LIB 5100 ND 15 ND LIB 2 100 ND 1.7 <0.05 LIB 2G10 ND 4.1 <0.10 LIB 2G100 1000 27 0.18 LIB 4100 170 38 ND - Phage pools are labeled as shown (
FIG. 3 ). The multiplicity of infection (moi) refers to the multiplicity of K07 infection (PFU/cells) in the propagation of phagemid. The enrichment of CFU over PFU is shown in those cases where purified K07 was added in the binding step. The ratio of CFU eluting from hGHbp-beads over CFU eluting from BSA-beads is shown. The fraction of KpnI-containing template (i.e., pH0415) remaining in the pool was determined by digesting dsDNA with KpnI plus EcoRI, running the products on a 1% agarose gel, and laser-scanning a negative of the ethidium bromide-stained DNA. - Receptor-Binding Affinity of the Hormone hGH(E174S, F176Y)
- The fact that a single clone was isolated from two different pathways of selection (
FIG. 3 ) suggested that the double mutant (E174S, F176Y) binds strongly to hGHbp. To determine the affinity of this mutant of hGH for hGHbp, we constructed this mutant of hGH by site-directed mutagenesis, using a plasmid (pBO720) which contains the wild-type hGH gene as template and the following oligonucleotide which changescodons 174 and 176 (SEQ ID NO:10) -
hGH codon: 172 174 176 178 Lys Ser Tyr Arg 5′-ATG GAC AAG GTG TCG ACA TAC CTG CGC ATC GTG-3′
The resulting construct, pH0458B, was transformed into E. coli strain 16C9 for expression of the mutant hormone. Scatchard analysis of competitive binding of hGH(E174S, F176Y) versus 125I-hGH to hGHbp indicated that the (E174S, F176Y) mutant has a binding affinity at least 5.0-fold tighter than that of wild-type hGH. - Human growth hormone variants were produced by the method of the present invention using the phagemid described in
FIG. 9 . - We designed a vector for cassette mutagenesis (Wells et al., Gene 34, 315-323 [1985]) and expression of the hGH-gene III fusion protein with the objectives of (1) improving the linkage between hGH and the gene III moiety to more favorably display the hGH moiety on the phage (2) limiting expression of the fusion protein to obtain essentially “monovalent display,” (3) allowing for restriction nuclease selection against the starting vector, (4) eliminating expression of fusion protein from the starting vector, and (5) achieving facile expression of the corresponding free hormone from a given hGH-gene III fusion mutant.
- Plasmid pS0643 was constructed by oligonucleotide-directed mutagenesis (Kunkel et al., Methods Enzymol. 154, 367-382 [1987]) of pS0132, which contains pBR322 and f1 origins of replication and expresses an hGH-gene III fusion protein (hGH residues 1-191, followed by a single Gly residue, fused to Pro-198 of gene III) under the control of the E. coli phoA promoter (Bass et al.,
Proteins 8, 309-314 [1990])(FIG. 9 ). Mutagenesis was carried out with theoligonucleotide 5′-GGC-AGC-TGT-GGC-TTC-TAG-AGT-GGC-GGC-GGC-TCT-GGT-3′ (SEQ ID NO:1), which introduces a XbaI site (underlined) and an amber stop codon (TAG) following Phe-191 of hGH. In the resulting construct, pS0643, a portion of gene III was deleted, and two silent mutations (underlined) occurred, yielding the following junction between hGH and gene III (SEQ ID NOS: 11 AND 12): -
---hGH --------------> gene III -------- 187 188 189 190 191 am* 249 250 251 252 253 254 Gly Ser Cys Gly Phe Glu Ser Gly Gly Gly Ser Gly GGC AGC TGT GGA TTC TAG AGT GGC GGT GGC TCT GGT - This shortens the total size of the fusion protein from 401 residues in pS0132 to 350 residues in pS0643. Experiments using monoclonal antibodies against hGH have demonstrated that the hGH portion of the new fusion protein, assembled on a phage particle, is more accessible than was the previous, longer fusion.
- For propagation of hormone-displaying phage, pS0643 and derivatives can be grown in a amber-suppressor strain of E. coli, such as JM101 or XL1-Blue (Bullock et al.,
BioTechniques 5, 376-379 [1987]). Shown above is substitution of Glu at the amber codon which occurs in supE suppressor strains. Suppression with other amino acids is also possible in various available strains of E. coli well known and publically available. - To express hGH (or mutants) free of the gene III portion of the fusion, pS0643 and derivatives can simply be grown in a non-suppressor strain such as 16C9. In this case, the amber codon (TAG) leads to termination of translation, which yields free hormone, without the need for an independent DNA construction.
- To create sites for cassette mutagenesis, pS0643 was mutated with the oligonucleotides (1) 5′-CGG-ACT-GGG-CAG-ATA-TTC-AAG-CAG-ACC-3′ (SEQ ID NO:13), which destroys the unique BglII site of pS0643; (2) 5′-CTC-AAG-AAC-TAC-GGG-TTA-CCC-TGA-CTG-CTT-CAG-GAA-GG-3′ (SEQ ID NO:14), which inserts a unique BstEII site, a single-base frameshift, and a non-amber stop codon (TGA); and (3) 5′-CGC-ATC-GTG-CAG-TGC-AGA-TCT-GTG-GAG-GGC-3′ (SEQ ID NO:15), which introduces a new BglII site, to yield the starting vector, pH0509. The addition of a frameshift along with a TGA stop codon insures that no geneIII-fusion can be produced from the starting vector. The BstEII-BglII segment is cut out of pH0509 and replaced with a DNA cassette, mutated at the codons of interest. Other restriction sites for cassette mutagenesis at other locations in hGH have also been introduced into the hormone-phage vector.
- Cassette Mutagenesis within
Helix 4 of hGH -
Codons helix 4; and they are each substituted by at least one amino acid among known evolutionary variants of hGH. - We chose to substitute NNS(N=A/G/C/T; S=G/C) at each of the target residues. The choice of the NNS degenerate sequence yields 32 possible codons (including at least one codon for each amino acid) at 4 sites, for a total of (32)4=1,048,576 possible nucleotide sequences, or (20)4=160,000 possible polypeptide sequences. Only one stop codon, amber (TAG), is allowed by this choice of codons, and this codon is suppressible as Glu in supE strains of E. coli.
- Two degenerate oligonucleotides, with NNS at
codons - The vector was prepared by digesting pH0509 with BstEII followed by BglII. The products were run on a 1% agarose gel and the large fragment excised, phenol-extracted, and ethanol precipitated. This fragment was treated with calf intestinal phosphatase (Boehringer), then phenol:chloroform extracted, ethanol precipitated, and resuspended for ligation with the mutagenic cassette.
- Following ligation, the reaction products were again digested with BstEII, then phenol:chloroform extracted, ethanol precipitated and resuspended in water. (A BstEII recognition site (GGTNACC) is created within cassettes which contain a G at
position 3 ofcodon 172 and an ACC (Thr) codon at 174. However, treatment with BstEII at this step should not select against any of the possible mutagenic cassettes, because virtually all cassettes will be heteroduplexes, which cannot be cleaved by the enzyme). Approximately 150 ng (45 fmols) of DNA was electroporated into XL1-Blue cells (1.8×109 cells in 0.045 mL) in a 0.2 cm cuvette at a voltage setting of 2.49 kV with a single pulse (time constant=4.7 msec.). - The cells were allowed to recover 1 hour at 37° C. in S.O.C media with shaking, then mixed with 25 mL 2YT medium, 100 mg/mL carbenicillin, and M13-K07 (moi=100). After 10′ at 23° C., the culture was incubated overnight (15 hours) at 37° C. with shaking. Plating of serial dilutions from this culture onto carbenicillin-containing media indicated that 3.9×107 electrotransformants were obtained.
- After overnight incubation, the cells were pelleted, and double-stranded DNA (dsDNA), designated pH0529E (the initial library), was prepared by the alkaline lysis method. The supernatant was spun again to remove any remaining cells, and the phage, designated phage pool φH0529E (the initial library of phage), were PEG-precipitated and resuspended in 1 mL STE buffer (10 mM Tris, pH 7.6, 1 mM EDTA, 50 mM NaCl). Phage titers were measured as colony-forming units (CFU) for the recombinant phagemid containing hGH-g3p. Approximately 4.5×1013 CFU were obtained from the starting library.
- From the pool of electrotransformants, 58 clones were sequenced in the region of the BstEII-BglII cassette. Of these, 17% corresponded to the starting vector, 17% contained at least one frame shift, and 7% contained a non-silent (non-terminating) mutation outside the four target codons. We conclude that 41% of the clones were defective by one of the above measures, leaving a total functional pool of 2.0×107 initial transformants. This number still exceeds the possible number of DNA sequences by nearly 20-fold. Therefore, we are confident of having all possible sequences represented in the starting library.
- We examined the sequences of non-selected phage to evaluate the degree of codon bias in the mutagenesis (Table V). The results indicated that, although some codons (and amino acids) are under- or over-represented relative to the random expectation, the library is extremely diverse, with no evidence of large-scale “sibling” degeneracy (Table VI).
-
TABLE V Codon distribution (per 188 codons) of non-selected hormone phage. Residue Number expected Number found Found/Expected Leu 17.6 18 1.0 Ser 17.6 26 1.5 Arg 17.6 10 0.57 Pro 11.8 16 1.4 Thr 11.8 14 1.2 Ala 11.8 13 1.1 Gly 11.8 16 1.4 Val 11.8 4 0.3 Ile 5.9 2 0.3 Met 5.9 1 0.2 Tyr 5.9 1 0.2 His 5.9 2 0.3 Trp 5.9 2 0.3 Phe 5.9 5 0.9 Cys 5.9 5 0.9 Gln 5.9 7 1.2 Asn 5.9 14 2.4 Lys 5.9 11 1.9 Asp 5.9 9 1.5 Glu 5.9 6 1.0 amber* 5.9 6 1.0 Clones were sequenced from the starting library (pH0529E). All codons were tabulated, including those from clones which contained spurious mutations and/or frameshifts. *Note: the amber stop codon (TAG) is suppressed as Glu in XL1-Blue cells. Highlighted codons were over/under-represented by 50% or more. -
TABLE VI Kε NT (SEQ ID NO: 46) TWGS (SEQ ID NO: 47) Pε ER (SEQ ID NO: 48) LPPS (SEQ ID NO: 49) SLDP (SEQ ID NO: 50) QQSN (SEQ ID NO: 51) GSKT (SEQ ID NO: 52) TPVT (SEQ ID NO: 53) RSRA (SEQ ID NO: 54) LCGL (SEQ ID NO: 55) TGRL (SEQ ID NO: 56) AKAS (SEQ ID NO: 57) GNDD (SEQ ID NO: 58) KTEQ (SEQ ID NO: 59) NNCR (SEQ ID NO: 60) FPCL (SEQ ID NO: 61) NSDF (SEQ ID NO: 62) HRPS (SEQ ID NO: 63) LSLE (SEQ ID NO: 64) NGSK (SEQ ID NO: 65) LTTE (SEQ ID NO: 66) PSGG (SEQ ID NO: 67) LWFP (SEQ ID NO: 68) PAGS (SEQ ID NO: 69) GRAK (SEQ ID NO: 70) GTNG (SEQ ID NO: 71) CVLQ (SEQ ID NO: 72) EASL (SEQ ID NO: 73) SSKE (SEQ ID NO: 74) ALLL (SEQ ID NO: 75) PSHP (SEQ ID NO: 76) SYAP (SEQ ID NO: 77) ASNG (SEQ ID NO: 78) EANN (SEQ ID NO: 79) KNAK (SEQ ID NO: 80) SRGK (SEQ ID NO: 81) GLDG (SEQ ID NO: 82) NDPI (SEQ ID NO: 83) Non-selected (pHO529E) clones with an open reading frame. The notation, e.g. TWGS, denotes the hGH mutant 172T/174W/176G/178S. Amber (TAG) codons, translated as Glu in XL1 -Blue cells are shown as {tilde over (□)}
Preparation of Immobilized hGHbp and hPRLbp - Immobilized hGHbp (“hGHbp-beads”) was prepared as described (Bass et al.,
Proteins 8, 309-314 [1990]), except that wild-type hGHbp (Fuh et al., J. Biol. Chem. 265, 3111-3115 [1990]) was used. Competitive binding experiments with [125I] hGH indicated that 58 fmols of functional hGHbp were coupled per μL of bead suspension. - Immobilized hPRLbp (“hPRLbp-beads”) was prepared as above, using the 211-residue extracellular domain of the prolactin receptor (Cunningham et al.,
Science 250, 1709-1712 [1990]). Competitive binding experiments with [125I] hGH in the presence of 50 μM zinc indicated that 2.1 fmols of functional hPRLbp were coupled per μL of bead suspension. - “Blank beads” were prepared by treating the oxirane-acrylamide beads with 0.6 M ethanolamine (pH 9.2) for 15 hours at 4° C.
- Binding Selection Using Immobilized hGHbp and hPRLbp
- Binding of hormone-phage to beads was carried out in one of the following buffers: Buffer A (PBS, 0.5% BSA, 0.05
% Tween 20, 0.01% thimerosal) for selections using hGHbp and blank beads; Buffer B (50 mM tris pH 7.5, 10 mM MgCl2, 0.5% BSA, 0.05% Tween % Tween 20, 0.01% thimerosal, 10 mM EDTA) for selections using hPRLbp in the absence of zinc (+EDTA). Binding selections were carried out according to each of the following paths: (1) binding to blank beads, (2) binding to hGHbp-beads, (3) binding to hPRLbp-beads (+Zn2+), (4) binding to hPRLbp-beads (+EDTA), (5) pre-adsorbing twice with hGHbp beads then binding the non-adsorbed fraction to hPRLbp-beads (“−hGHbp, +hPRLbp” selection), or (6) pre-adsorbing twice with hPRLbp-beads then binding the non-adsorbed fraction to hGHbp-beads (“−hPRLbp, +hGHbp” selection). The latter two procedures are expected to enrich for mutants binding hPRLbp but not hGHbp, or for mutants binding hGHbp but not hPRLbp, respectively. Binding and elution of phage was carried out in each cycle as follows: - 1. BINDING: An aliquot of hormone phage (typically 109-1010 CFU) was mixed with an equal amount of non-hormone phage (pCAT), diluted into the appropriate buffer (A, B, or C), and mixed with a 10 mL suspension of hGHbp, hPRLbp or blank beads in a total volume of 200 mL in a 1.5 mL polypropylene tube. The phage were allowed to bind to the beads by incubating 1 hour at room temperature (23° C.) with slow rotation (approximately 7 RPM). Subsequent steps were carried out with a constant volume of 200 μL and at room temperature.
- 2. WASHES: The beads were spun 15 sec., and the supernatant was removed. To reduce the number of phage not specifically bound, the beads were washed 5 times by resuspending briefly in the appropriate buffer, then pelleting.
- 3. hGH ELUTION: Phage binding weakly to the beads were removed by elution with hGH. The beads were rotated with the appropriate buffer containing 400 nM hGH for 15-17 hours. The supernatant was saved as the “hGH elution” and the beads. The beads were washed by resuspending briefly n buffer and pelleting.
- 4. GLYCINE ELUTION: To remove the tightest-binding phage (i.e. those still bound after the hGH wash), beads were suspended in Glycine buffer (Buffer A plus 0.2 M Glycine, pH 2.0 with HCl), rotated 1 hour and pelleted. The supernatant (“Glycine elution”; 200 μL) was neutralized by adding 30 mL of 1 M Tris base and stored at 4° C.
- 5. PROPAGATION: Aliquots from the hGH elutions and from the Glycine elutions from each set of beads under each set of conditions were used to infect separate cultures of log-phase XL1-Blue cells. Transductions were carried out by mixing phage with 1 mL XL1-Blue cells, incubating 20 min. at 37° C., then adding K07 (moi=100). Cultures (25 mL 2YT plus carbenicillin) were grown as described above and the next pool of phage was prepared as described above.
- Phage binding, elution, and propagation were carried out in successive rounds, according to the cycle described above. For example, the phage amplified from the hGH elution from hGHbp-beads were again selected on hGHbp-beads and eluted with hGH, then used to infect a new culture of XL1-Blue cells. Three to five rounds of selection and propagation were carried out for each of the selection procedures described above.
- From the hGH and Glycine elution steps of each cycle, an aliquot of phage was used to inoculate XL1-Blue cells, which were plated on LB media containing carbenicillin and tetracycline to obtain independent clones from each phage pool. Single-stranded DNA was prepared from isolated colony and sequenced in the region of the mutagenic cassette. The results of DNA sequencing are summarized in terms of the deduced amino acid sequences in
FIGS. 5 , 6, 7, and 8. - To determine the binding affinity of some of the selected hGH mutants for the hGHbp, we transformed DNA from sequenced clones into E. coli strain 16C9. As described above, this is a non-suppressor strain which terminates translation of protein after the final Phe-191 residue of hGH. Single-stranded DNA was used for these transformations, but double-stranded DNA or even whole phage can be easily electroporated into a non-suppressor strain for expression of free hormone.
- Mutants of hGH were prepared from osmotically shocked cells by ammonium sulfate precipitation as described for hGH (Olson et al., Nature 293, 408-411 [1981]), and protein concentrations were measured by laser densitomoetry of Coomassie-stained SDS-polyacrylamide gel electrophoresis gels, using hGH as standard (Cunningham and Wells, Science 244, 1081-1085 [1989]).
- The binding affinity of each mutant was determined by displacement of 125I hGH as described (Spencer et al., J. Biol. Chem. 263, 7862-7867 [1988]; Fuh et al., J. Biol. Chem. 265, 3111-3115 [1990]), using an anti-receptor monoclonal antibody (Mab263).
- The results for a number of hGH mutants, selected by different pathways (
FIG. 6 ) are shown in Table VII. Many of these mutants have a tighter binding affinity for hGHbp than wild-type hGH. The most improved mutant, KSYR (SEQ ID NO:84), has a binding affinity 5.6 times greater than that of wild-type hGH. The weakest selected mutant, among those assayed was only about 10-fold lower in binding affinity than hGH. - Binding assays may be carried out for mutants selected for hPRLbp-binding.
-
TABLE VII Competitive binding to hGHbp Mutant Kd (nM) Kd(mut)/Kd(hGH) Pool KSYR (6) (SEQ ID NO: 84) 0.06 + 0.01 0.18 1G, 3G RSFR (SEQ ID NO: 85) 0.10 + 0.05 0.30 3G RAYR (SEQ ID NO: 86) 0.13 + 0.04 0.37 3* KTYK (2) (SEQ ID NO: 87) 0.16 + 0.04 0.47 H, 3G RSYR (3) (SEQ ID NO: 88) 0.20 + 0.07 0.58 1G, 3H, 3G KAYR (3) (SEQ ID NO: 89) 0.22 + 0.03 0.66 3G RFFR (2) (SEQ ID NO: 90) 0.26 + 0.05 0.76 3H KQYR (SEQ ID NO: 91) 0.33 + 0.03 1.0 3G KEFR = wt (9) 0.34 + 0.05 1.0 3H, 3G, 3* RTYH (SEQ ID NO: 92) 0.68 + 0.17 2.0 3H QRYR (SEQ ID NO: 93) 0.83 + 0.14 2.5 3* KKYK (SEQ ID NO: 94) 1.1 + 0.4 3.2 3* RSFS (2) (SEQ ID NO: 95) 1.1 + 0.2 3.3 3G, * KSNR (SEQ ID NO: 96) 3.1 + 0.4 9.2 3* The selected pool in which each mutant was found is indicated as 1G (first glycine selection), 3G (third glycine selection), 3H (third hGH selection), 3* (third selection, not binding to hPRLbp, but binding to hGHbp). The number of times each mutant occurred among all sequenced clones is shown ( ). - At some residues, substitution of a particular amino acid has essentially the same effect independent of surrounding residues. For example, substitution of F176Y in the background of 172R/174S reduces binding affinity by 2.0-fold (RSFR (SEQ ID NO:85) vs. RSYR (SEQ ID NO:88)). Similarly, in the background of 172K/174A the binding affinity of the F176Y mutant (KAYR (SEQ ID NO:89)) is 2.9-fold weaker than the corresponding 176F mutant (KAFR; Cunningham and Wells, 1989).
- On the other hand, the binding constants determined for several selected mutants of hGH demonstrate non-additive effects of some amino acid substitutions at
residues - Such non-additive effects on binding for substitutions at proximal residues illustrate the utility of protein-phage binding selection as a means of selecting optimized mutants from a library randomized at several positions. In the absence of detailed structural information, without such a selection process, many combinations of substitutions might be tried before finding the optimum mutant.
- Using the methods described in Example VIII, we targeted another region of hGH involved in binding to the hGHbp and/or hPRLbp,
helix 1residues - We chose to use the “amber” hGH-g3 construct (called phGHam-g3p) because it appears to make the target protein, hGH, more accessible for binding. This is supported by data from comparative ELISA assays of monoclonal antibody binding. Phage produced from both pS0132 (S. Bass, R. Greene, J. A. Wells,
Proteins 8, 309 (1990)) and phGHam-g3 were tested with three antibodies (Medix 2, 1B5.G2, and 5B7.C10) that are known to have binding determinants near the carboxyl-terminus of hGH [B. C. Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science 243, 1330 (1989); B. C. Cunningham and J. A. Wells, Science 244, 1081 (1989); L. Jin and J. Wells, unpublished results], and one antibody (Medix 1) that recognizes determinants inhelices 1 and 3 ([B. C. Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science 243, 1330 (1989); B. C. Cunningham and J. A. Wells, Science 244, 1081 (1989)]). Phagemid particles from phGHam-g3 reacted much more strongly withantibodies Medix 2, 1B5.G2, and 5B7.C10 than did phagemid particles from pS0132. In particular, binding of pS0132 particles was reduced by >2000-fold for bothMedix 2 and 5B7.C10 and reduced by >25-fold for 1B5.G2 compared to binding toMedix 1. On the other hand, binding of phGHam-g3 phage was weaker by only about 1.5-fold, 1.2-fold, and 2.3-fold for theMedix 2, 1B5.G2, and 5B7.C10 antibodies, respectively, compared with binding toMEDIX 1. - We mutated residues in
helix 1 that were previously identified by alanine-scanning mutagenesis [B. C. Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science 243, 1330 (1989); B. C. Cunningham and J. A. Wells, Science 244, 1081 (1989), 15, 16) to modulate the binding of the extracellular domains of the hGH and/or hPRL receptors (called hGHbp and hPRLbp, respectively). Cassette mutagenesis was carried out essentially as described [J. A. Wells, M. Vasser, D. B. Powers, Gene 34, 315 (1985)]. This library was constructed by cassette mutagenesis that fully mutated four residues at a time (see Example VIII) which utilized a mutated version of phGHam-g3 into which unique KpnI (at hGH codon 27) and XhoI (at hGH codon 6) restriction sites (underlined below) had been inserted by mutagenesis [T. A. Kunkel, J. D. Roberts, R. A. Zakour, Methods Enzymol. 154, 367-382] with theoligonucleotides 5′-GCC TTT GAC AGG TAC CAG GAG TTT G-3′ (SEQ ID NO:18) and 5′-CCA ACT ATA CCA CTC TCG AGG TCT ATT CGA TAA C-3′ (SEQ ID NO:19), respectively. The later oligo also introduced a +1 frameshift (italicized) to terminate translation from the starting vector and minimize wild-type background in the phagemid library. This strating vector was designated pH0508B. Thehelix 1 library, whichmutated hGH residues complementary oligonucleotides 5′-pTCG AGG CTC NNS GAC AAC GCG NNS CTG CGT GCT NNS CGT CTT NNS CAG CTG GCC TTT GAC ACG TAC-3′ (SEQ ID NO:20) and 5′-pGT GTC AAA GGC CAG CTG SNN AAG ACG SNN AGC ACG CAG SNN CGC GTT GTC SNN GAG CC-3′ (SEQ ID NO:21). The KpnI site was destroyed in the junction of the ligation product so that restriction enzyme digestion could be used for analysis of non-mutated background. - The library contained at least 107 independent transformants so that if the library were absolutely random (106 different combinations of codons) we would have an average of about 10 copies of each possible mutated hGH gene. Restriction analysis using KpnI indicated that at least 80% of
helix 1 library constructs contained the inserted cassette. - Binding enrichments of hGH-phage from the libraries was carried out using hGHbp immobilized on oxirane-polyacrylamide beads (Sigma Chemical Co.) as described (Example VIII). Four residues in helix 1 (F10, M14, H18, and H21) were similarly mutated and after 4 and 6 cycles a non-wild-type consensus developed (Table VIII).
Position 10 on the hydrophobic face ofhelix 1 tended to be hydrophobic whereaspositions 21 and 18 on the hydrophillic face tended were dominated by Asn; no obvious consensus was evident for position 14 (Table IX). - The binding constants for these mutants of hGH to hGHbp was determined by expressing the free hormone variants in the non-suppressor E. coli strain 16C9, purifying the protein, and assaying by competitive displacement of labelled wt-hGH from hGHbp (see Example VIII). As indicated, several mutants bind tighter to hGHbp than does wt-hGH.
-
TABLE VIII Selection of hGH helix 1 mutantsGly elution F10 M14 H18 H21 4 Cycles H G N N A W D N (2) Y T V N I N I N L N S H F S F G 6 Cycles H G N N (6) F S F L Consensus: H G N N Variants of hGH (randomly mutated at residues F10, M14, H18, H21) expressed on phagemid particles were selected by binding to hGHbp-beads and eluting with hGH (0.4 mM) buffer followed by glycine (0.2 M, pH 2) buffer(see Example VIII). -
TABLE IX Consensus sequences from the selected helix 1 libraryObserved frequency is fraction of all clones sequenced with the indicated amino acid. The nominal frequency is calculated on the basis of NNS 32 codon degeneracy. The maximal enrichment factor varies from 11 to 32 depending upon the nominal frequency value for a given residue. Values of [Kd(Ala mut)/Kd(wt hGH)] for single alanine mutations were taken from B. C. Cunningham and J. A. Wells, Science 244, 1081 (1989); B. C. Cunningham, D. J. Henner, J. A. Wells, Science 247, 1461 (1990); B. C. Cunningham and J. A. Wells, Proc. Natl. Acad. Sci. USA 88, 3407 (1991). Wild type residue Selected residue Frequency observed nominal Enrich- ment F10 5.9 H 0.50 0.031 17 F 0.14 0.031 5 A 0.14 0.062 2 M14 2.2 G 0.50 0.062 8 W 0.14 0.031 5 N 0.14 0.031 5 S 0.14 0.093 2 H18 1.6 N 0.50 0.031 17 D 0.14 0.031 5 F 0.14 0.031 5 H21 0.33 N 0.79 0.031 26 H 0.07 0.031 2 -
TABLE X Binding of purified hGH helix 1 mutants to hGHbp Sequence position 10 14 18 21 P Kd (nM\f(Kd mut) Kd(Wt hGH)) H G N N 0.50 0.14 ± 0.04 0.42 A W D N 0.14 0.10 ± 0.03 0.30 wt= F M H H 0 0.34 ± 0.05 (1) F S F L 0.07 0.68 ± 0.19 2.0 Y T V N 0.07 0.75 ± 0.19 2.2 L N S H 0.07 0.82 ± 0.20 2.4 I N I N 0.07 1.2 ± 0.31 3.4 Competition binding experiments were performed using [125I] hGH (wild-type), hGHbp (containing the extracellular receptor domain, residues 1-238), and Mab263 [B. C. Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science 243, 1330 (1989)];. The number P indicates the fractional occurrence of each mutant among all the clones sequenced after one or more rounds of selection. - Design of Mutant Proteins with Improved Binding Properties by Iterative Selection Using Hormone-Phage
- Our experience with recruiting non-binding homologs of hGH evolutionary variants suggests that many individual amino acid substitutions can be combined to yield cumulatively improved mutants of hGH with respect to binding a particular receptor [B. C. Cunningham, D. J. Henner, J. A. Wells, Science 247, 1461 (1990); B. C. Cunningham and J. A. Wells, Proc. Natl. Acad. Sci. USA 88, 3407 (1991); H. B. Lowman, B. C. Cunningham, J. A. Wells, J. Biol. Chem. 266, in press (1991)].
- The helix 4b library was constructed in an attempt to further improve the
helix 4 double mutant (E174S/F176Y) selected from the helix 4a library that we found bound tighter to the hGH receptor (see Example VIII). With the E174S/F176Y hGH mutant as the background starting hormone, residues were mutated that surroundedpositions - Cassette mutagenesis was carried out essentially as described [J. A. Wells, M. Vasser, D. B. Powers, Gene 34, 315 (1985)]. The helix 4b library, which
mutated residues complementary oligonucleotides 5′-pG TTA CTC TAC TGC TTC NNS AAG GAC ATG NNS AAG GTC AGC NNS TAC CTG CGC NNS GTG CAG TGC A-3′ (SEQ ID NO:22) and 5′-pGA TCT GCA CTG CAC SNN GCG CAG GTA SNN GCT GAC CTT SNN CAT GTC CTT SNN GAA GCA GTA GA-3′ (SEQ ID NO:23). The BstEII site was eliminated in the ligated cassette. From the helix 4b library, 15 unselected clones were sequenced. Of these, none lacked a cassette insert, 20% were frame-shifted, and 7% had a non-silent mutation. - Results of hGHbp Enrichment
- Binding enrichments of hGH-phage from the libraries was carried out using hGHbp immobilized on oxirane-polyacrylamide beads (Sigma Chemical Co.) as described (Example VIII). After 6 cycles of binding a reasonably clear consensus developed (Table XI). Interestingly, all positions tended to contain polar residues, notably Ser, Thr and Asn (XII).
- The binding constants for some of these mutants of hGH to hGHbp was determined by expressing the free hormone variants in the non-suppressor E. coli strain 16C9, purifying the protein, and assaying by competitive displacement of labelled wt-hGH from hGHbp (see Example VIII). As indicated, the binding affinities of several helix-4b mutants for hGHbp were tighter than that of wt-hGH Table XIII).
- Finally, we have begun to analyze the binding affinity of several of the tighter hGHbp binding mutants for their ability to bind to the hPRLbp. The E174S/F176Y mutant binds 200-fold weaker to the hPRLbp than hGH. The E174T/F176Y/R178K and R167N/D171S/E174S/F176Y/I179T mutants each bind >500-fold weaker to the hPRLbp than hGH. Thus, it is possible to use the produce new receptor selective mutants of hGH by phage display technology.
- Of the 12 residues mutated in three hGH-phagemid libraries (Examples VIII, IX, X), 4 showed a strong, although not exclusive, conservation of the wild-type residues (K172, T175, F176, and R178). Not surprisingly, these were residues that when converted to Ala caused the largest disruptions (4- to 60-fold) in binding affinity to the hGHbp. There was a class of 4 other residues (F10, M14, D171, and I179) where Ala substitutions caused weaker effects on binding (2- to 7-fold) and these positions exhibited little wild-type consensus. Finally the other 4 residues (H18, H21, R167, and E174), that promote binding to the hPRLbp but not the hGHbp, did not exhibit any consensus for the wild-type hGH sequence by selection on hGHbp-beads. In fact two residues (E174 and H21), where Ala substitutions enhance binding affinity to the hGHbp by 2- to 4-fold [B. C. Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science 243, 1330 (1989); B. C. Cunningham and J. A. Wells, Science 244, 1081 (1989); B. C. Cunningham, D. J. Henner, J. A. Wells, Science 247, 1461 (1990); B. C. Cunningham and J. A. Wells, Proc. Natl. Acad. Sci. USA 88, 3407 (1991)]. Thus, the alanine-scanning mutagenesis data correlates reasonably well with the flexibility to substitute each position. In fact, the reduction in binding affinity caused by alanine substitutions [B. C. Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science 243, 1330 (1989); B. C. Cunningham and J. A. Wells, Science 244, 1081 (1989)], B. C. Cunningham, D. J. Henner, J. A. Wells, Science 247, 1461 (1990); B. C. Cunningham and J. A. Wells, Proc. Natl. Acad. Sci. USA 88, 3407 (1991)] is a reasonable predictor of the percentage that the wild-type residue is found in the phagemid pool after 3-6 rounds of selection. The alanine-scanning information is useful for targeting side-chains that modulate binding, and the phage selection is appropriate for optimizing them and defining the flexibility of each site (and/or combinations of sites) for substitution. The combination of scanning mutational methods [B. C. Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science 243, 1330 (1989); B. C. Cunningham and J. A. Wells, Science 244, 1081 (1989)] and phage display is a powerful approach to designing receptor-ligand interfaces and studying molecular evolution in vitro.
- In cases where combined mutations in hGH have additive effects on binding affinity to receptor, mutations learned through hormone-phagemid enrichment to improve binding can be combined by simple cutting and ligation of restriction fragments or mutagenesis to yield cumulatively optimized mutants of hGH.
- On the other hand, mutations in one region of hGH which optimize receptor binding may be structurally or functionally incompatible with mutations in an overlapping or another region of the molecule. In these cases, hormone phagemid enrichment can be carried out by one of several variations on the iterative enrichment approach (1) random DNA libraries can be generated in each of two (or perhaps more) regions of the molecule by cassette or another mutagenesis method. Thereafter, a combined library can be created by ligation of restriction fragments from the two DNA libraries; (2) an hGH variant, optimized for binding by mutation in one region of the molecule, can be randomly mutated in a second region of the molecule as in the helix-4b library example; (3) two or more random libraries can be partially selected for improved binding by hormone-phagemid enrichment; after this “roughing-in” of the optimized binding site, the still-partially-diverse libraries can be recombined by ligation of restriction fragments to generate a single library, partially diverse in two or more regions of the molecules, which in turn can be further selected for optimized binding using hormone-phagemid enrichment.
-
TABLE XI Mutant phagemids of hGH selected from helix 4b library after 4 and 6 cycles of enrichment. R167 D171 T175 1179 4 Cycles N S T T K S T T S N T T D S T T D S T T+ D S A T D S A N T D T T N D T N A N T N A S T T 6 Cycles N S T T (2) N N T T N S T Q D S S T E S T I K S T L Consensus: N S T T D N Selection of hGH helix4b mutants (randomly mutated at residues One mutant (+) contained the spurious mutation R178H. -
TABLE XII Consensus sequences from the selected library. Observed frequency is fraction of all clones sequenced with the indicated amino acid. The nominal frequency is calculated on the basis of NNS 32 codon degeneracy. The maximal enrichment factor varies from 11 to 16 to 32 depending upon the nominal frequency value for a given residue. Values of [Kd(Ala mut)/Kd(wt hGH)] for single alanine mutations were taken from refs. below; for position 175 we only have a value for theT175S mutant [B. C. Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science 243, 1330 (1989); B. C. Cunningham and J. A. Wells, Science 244, 1081 (1989); B. C. Cunningham, D. J. Henner, J. A. Wells, Science 247, 1461 (1990); B. C. Cunningham and J. A. Wells, Proc. Natl. Acad. Sci. USA 88, 3407 (1991).]. Wild type residue Selected residue Frequency observed nominal Enrich- ment R167 0.75 N 0.35 0.031 11 D 0.24 0.031 8 K 0.12 0.031 4 A 0.12 0.062 2 D171 7.1 S 0.76 0.093 8 N 0.18 0.031 6 D 0.12 0.031 4 T175 3.5 T 0.88 0.062 14 A 0.12 0.031 4 I179 2.7 T 0.71 0.062 11 N 0.18 0.031 6 -
TABLE XIII Binding of purified hGH mutants to hGHbp. Sequence position * * * * Kd(Ala mut) 167 171 175 179 F Kd (nM) Kd(wt hGH) N S T T 0.18 0.04 ± 0.02 0.12 E S T I 0.06 0.04 ± 0.02 0.12 K S T L 0.06 0.05 ± 0.03 0.16 N N T T 0.06 0.06 ± 0.03 0.17 R D T I 0 0.06 ± 0.01 (0.18) N S T Q 0.06 0.26 ± 0.11 0.77 Competition binding experiments were performed using [125I]hGH (wild-type), hGHbp (containing the extracellular receptor domain, residues 1-238), and Mab263 (11). The number P indicates the fractional occurrence of each mutant among all the clones sequenced after one or more rounds of selection. Note that the helix 4b mutations (*) are in the background of hGH(E174S/F176Y). In the list of helix 4b mutants,, the E174S/F176Y mutant (*), with wt residues at 167, 171, 175, 179, is shown in bold. - Plasmid pDH 188 contains the DNA encoding the Fab portion of a humanized IgG antibody, called 4D5, that recognizes the HER-2 receptor. This plasmid is contained in E. coli strain SR 101, and has been deposited with the ATCC in Rockville, Md.
- Briefly, the plasmid was prepared as follows: the starting plasmid was pS0132, containing the alkaline phosphatase promoter as described above. The DNA encoding human growth hormone was excised and, after a series of manipulations to make the ends of the plasmid compatible for ligation, the DNA encoding 4D5 was inserted. The 4D5 DNA contains two genes. The first gene encodes the variable and constant regions of the light chain, and contains at its 5′ end the DNA encoding the st II signal sequence. The second gene contains four portions: first, at its 5′ end is the DNA encoding the st II signal sequence. This is followed by the DNA encoding the variable domain of the heavy chain, which is followed by the DNA encoding the first domain of the heavy chain constant region, which in turn is followed by the DNA encoding the M13 gene III. The salient features of this construct are shown in
FIG. 10 . The sequence of the DNA encoding 4D5 is shown inFIG. 11 . - E. coli Transformation and Phage Production.
- Both polyethylene glycol (PEG) and electroporation were used to transform plasmids into SR101 cells. (PEG competent cells were prepared and transformed according to the method of Chung and Miller (Nucleic Acids Res. 16:3580 [1988]). Cells that were competent for electroporation were prepared, and subsequently transformed via electroporation according to the method of Zabarovsky and Winberg (Nucleic Acids Res. 18:5912 [1990]). After placing the cells in 1 ml of the SOC media (described in Sambrook et al., supra), they were grown for 1 hour at 37° C. with shaking. At this time, the concentration of the cells was determined using light scattering at OD600. A titered KO7 phage stock was added to achieve an multiplicity of infection (MOI) of 100, and the phage were allowed to adhere to the cells for 20 minutes at room temperature. This mixture was then diluted into 25 mls of 2YT broth (described in Sambrook et al., supra) and incubated with shaking at 37° C. overnight. The next day, cells were pelleted by centrifugation at 5000×g for 10 minutes, the supernatant was collected, and the phage particles were precipitated with 0.5 M NaCl and 4% PEG (final concentration) at room temperature for 10 minutes. Phage particles were pelleted by centrifugation at 10,000×g for 10 minutes, resuspended in 1 ml of TEN (10 mM Tris, pH 7.6, 1 mM EDTA, and 150 mM NaCl), and stored at 4° C.
- Aliquots of 0.5 ml from a solution of 0.1 mg/ml of the extra-cellular domain of the HER-2 antigen (ECD) or a solution of 0.5 mg/ml of BSA (control antigen) in 0.1 M sodium bicarbonate, pH 8.5 were used to coat one well of a Falcon 12 well tissue culture plate. Once the solution was applied to the wells, the plates were incubated at 4° C. on a rocking platform overnight. The plates were then blocked by removing the initial solution, applying 0.5 ml of blocking buffer (30 mg/ml BSA in 0.1 M sodium bicarbonate), and incubating at room temperature for one hour. Finally, the blocking buffer was removed, 1 ml of buffer A (PBS, 0.5% BSA, and 0.05% Tween-20) was added, and the plates were stored up to 10 days at 4° C. before being used for phage selection.
- Approximately 109 phage particles were mixed with a 100-fold excess of KO7 helper phage and 1 ml of buffer A. This mixture was divided into two 0.5 ml aliquots; one of which was applied to ECD coated wells, and the other was applied to BSA coated wells. The plates were incubated at room temperature while shaking for one to three hours, and were then washed three times over a period of 30 minutes with 1 ml aliquots of buffer A. Elution of the phage from the plates was done at room temperature by one of two methods: 1) an initial overnight incubation of 0.025 mg/ml purified Mu4D5 antibody (murine) followed by a 30 minute incubation with 0.4 ml of the acid elution buffer (0.2 M glycine, pH 2.1, 0.5% BSA, and 0.05% Tween-20), or 2) an incubation with the acid elution buffer alone. Eluates were then neutralized with 1 M Tris base, and a 0.5 ml aliquot of TEN was added. These samples were then propagated, titered, and stored at 4° C.
- Aliquots of eluted phage were added to 0.4 ml of 2YT broth and mixed with approximately 108 mid-log phase male E. coli strain SR101. Phage were allowed to adhere to the cells for 20 minutes at room temperature and then added to 5 ml of 2YT broth that contained 50 μg/ml of carbenicillin and 5 μg/ml of tetracycline. These cells were grown at 37° C. for 4 to 8 hours until they reached mid-log phase. The OD600 was determined, and the cells were superinfected with KO7 helper phage for phage production. Once phage particles were obtained, they were titered in order to determine the number of colony forming units (cfu). This was done by taking aliquots of serial dilutions of a given phage stock, allowing them to infect mid-log phase SR101, and plating on LB plates containing 50 υg/ml carbenicillin.
- The affinity of h4D5 Fab fragments and Fab phage for the ECD antigen was determined using a competitive receptor binding RIA (Burt, D. R., Receptor Binding in Drug Research. O'Brien, R. A. (Ed.). pp. 3-29, Dekker, New York [1986]). The ECD antigen was labeled with 125-Iodine using the sequential chloramine-T method (De Larco, J. E. et al., J. Cell. Physiol. 109:143-152 [1981]) which produced a radioactive tracer with a specific activity of 14 μCi/μg and incorporation of 0.47 moles of Iodine per mole of receptor. A series of 0.2 ml solutions containing 0.5 ng (by ELISA) of Fab or Fab phage, 50 nCi of 125I ECD tracer, and a range of unlabeled ECD amounts (6.4 ng to 3277 ng) were prepared and incubated at room temperature overnight. The labeled ECD-Fab or ECD-Fab phage complex was separated from the unbound labeled antigen by forming an aggregate complex induced by the addition of an anti-human IgG (Fitzgerald 40-GH23) and 6% PEG 8000. The complex was pelleted by centrifugation (15,000×g for 20 minutes) and the amount of labeled ECD (in cpm) was determined by a gamma counter. The dissociation constant (Kd) was calculated by employing a modified version of the program LIGAND (Munson, P. and Rothbard, D., Anal. Biochem. 107:220-239 [1980]) which utilizes Scatchard analysis (Scatchard, G., Ann. N.Y. Acad. Sci. 51:660-672 [1949]). The Kd values are shown in
FIG. 13 . - Murine 4D5 antibody was labeled with 125-I to a specific activity of 40-50 μCi/μg using the Iodogen procedure. Solutions containing a constant amount of labeled antibody and increasing amounts of unlabeled variant Fab were prepared and added to near confluent cultures of SK-BR-3 cells grown in 96-well microtiter dishes (final concentration of labeled antibody was 0.1 nM). After an overnight incubation at 4° C., the supernatant was removed, the cells were washed and the cell associated radioactivity was determined in a gamma counter. Kd values were determined by analyzing the data using a modified version of the program LIGAND (Munson, P. and Rothbard, D., supra)
- This deposit of plasmid pDH188 ATCC no. 68663 was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture for 30 years from the date of deposit. The organisms will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc. and ATCC, which assures permanent and unrestricted availability of the progeny of the cultures to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC §122 and the Commissioner's rules pursuant thereto (including 37 CFR §1.14 with particular reference to 886 OG 638).
- The assignee of the present application has agreed that if the cultures on deposit should die or be lost or destroyed when cultivated under suitable conditions, they will be promptly replaced on notification with a viable specimen of the same culture. Availability of the deposited cultures is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.
- The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the cultures deposited, since the deposited embodiments are intended as separate illustrations of certain aspects of the invention and any cultures that are functionally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.
- While the invention has necessarily been described in conjunction with preferred embodiments, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and alterations to the subject matter set forth herein, without departing from the spirit and scope thereof. Hence, the invention can be practiced in ways other than those specifically described herein. It is therefore intended that the protection granted by Letters Patent hereon be limited only by the appended claims and equivalents thereof.
- According to additivity principles [J. A. Wells, Biochemistry 29, 8509 (1990)], mutations in different parts of a protein, if they are not mutually interacting, are expected to combine to produce additive changes in the free energy of binding to another molecule (changes are additive in terms of ΔΔGbinding, or multiplicative in terms of Kd=exp[−ΔG/RT]). Thus a mutation producing a 2-fold increase in binding affinity, when combined with a second mutation causing a 3-fold increase, would be predicted to yield a double mutant with a 6-fold increased affinity over the starting variant.
- To test whether multiple mutations obtained from hGH-phage selections would produce cumulatively favorable effects on hGHbp (hGH-binding protein; the extracellular domain of the hGH receptor) binding, we combined mutations found in the three tightest-binding variants of hGH from the helix-1 library (Example IX: F10A/M14W/H18D/H21N, F10H/M14G/H18N/H21N, and F10F/M14S/H18F/H21L) with those found in the three tightest binding variants found in the helix-4b library (Example X: R167N/D171S/T175/I179T, R167E/D171S/T175/I179, and R167N/D171N/T175/I179T).
- hGH-phagemid double-stranded DNA (dsDNA) from each of the one-helix variants was isolated and digested with the restriction enzymes EcoRI and BstXI. The large fragment from each helix-4b variant was then isolated and ligated with the small fragment from each helix-1 variant to yield the new two-helix variants shown in Table XIII. All of these variants also contained the mutations E174S/F176Y obtained in earlier hGH-phage binding selections (see Example X for details).
- Although additivity principles appear to hold for a number of combinations of mutations, some combinations (e.g. E174S with F176Y) are clearly non-additive (see examples VIII and X). In order to identify with certainty the tightest binding variant with, for example, 4 mutations in helix-1 and 4 mutations in helix-4, one would ideally mutate all 8 residues at once and then sort the pool for the globally tightest binding variant. However, such a pool would consist of 1.1×1012 DNA sequences (utilizing NNS codon degeneracy) encoding 2.6×1010 different polypeptides. Obtaining a random phagemid library large enough to assure representation of all variants (perhaps 1013 transformants) is not practical using current transformation technology.
- We have addressed this difficulty first by utilizing successive rounds of mutagenesis, taking the tightest binding variant from one library, then mutating other residues to further improve binding (Example X). In a second method, we have utilized the principle of additivity to combine the best mutations from two independently sorted libraries to create multiple mutants with improved binding (described above). Here, we further searched through the possible combinations of mutations at
positions helix 1 library (independently sorted for 0, 2, or 4 cycles; Example IX) and the pool from the helix-4b library (independently sorted for 0, 2, or 4 cycles; Example X) and sorted the combined variant pool for hGHbp binding. Since some amount of sequence diversity exists in each of these pools, the resulting combinatorial library can explore more sequence combinations than what we might construct manually (e.g. Table XIII). - hGH-phagemid double-stranded DNA (dsDNA) from each of the one-helix library pools (selected for 0, 2, or 4 rounds) was isolated and digested with the restriction enzymes AccI and BstXI. The large fragment from each helix-1 variant pool was then isolated and ligated with the small fragment from each helix-4b variant pool to yield the three combinatorial libraries pH0707A (unselected
helix 1 and helix 4b pools, as described in examples IX and X), pH0707B (twice-selected helix-1 pool with twice-selected helix-4b pool), and pH0707C (4-times selected helix-1 pool with 4-times selected helix-4b pool). Duplicate ligations were also set up with less DNA and designated as pH0707D, pH0707E, and pH0707F, corresponding to the 0-, 2-, and 4-round starting libraries respectively. All of these variant pools also contained the mutations E174S/F176Y obtained in earlier hGH-phage binding selections (see Example X for details). - Sorting Combinatorial Libraries of hGH-Phage Variants
- The ligation products pH0707A-F were processed and electro-transformed into XL1-Blue cells as described (Example VIII). Based on colony-forming units (CFU), the number of transformants obtained from each pool was as follows: 2.4×106 from pH0707A, 1.8×106 from pH0707B, 1.6×106 from pH0707C, 8×105 from pH0707D, 3×105 from pH0707E, and 4×105 from pH0707F. hGH-phagemid particles were prepared and selected for hGHbp-binding over 2 to 7 cycles as described in Example VIII.
- Rapid Sorting of hGH-Phagemid Libraries
- In addition to sorting phagemid libraries for tight-binding protein variants, as measured by equilibrium binding affinity, it is of interest to sort for variants which are altered in either the on-rate (kon) or the off-rate (koff) of binding to a receptor or other molecule. From thermodynamics, these rates are related to the equilibrium dissociation constant, Kd=(koff/kon). We envision that certain variants of a particular protein have similar Kd's for binding while having very different kon's and koff's. Conversely, changes in Kd from one variant to another may be due to effects on kon, effects on koff, or both. The pharmacological properties of a protein may be dependent on binding affinity or on kon or koff, depending on the detailed mechanism of action. Here, we sought to identify hGH variants with higher on-rates to investigate the effects of changes in kon. We envision that the selection could alternatively be weighted toward koff by increasing the binding time and increasing the wash time and/or concentration with cognate ligand (hGH).
- From time-course analysis of wild-type hGH-phagemid binding to immobilized hGHbp, it appears that, of the total hGH-phagemid particles that can be eluted in the
final pH 2 wash (see Example VIII for the complete binding and elution protocol), less than 10% are bound after 1 minute of incubation, while greater than 90% are bound after 15 minutes of incubation. - For “rapid-binding selection,” phagemid particles from the pH0707B pool (twice-selected for
helices - The binding constants for some of these mutants of hGH to hGHbp was determined by expressing the free hormone variants in the non-suppressor E. coli strain 16C9 or 34B8, purifying the protein, and assaying by competitive displacement of labelled wt-hGH from hGHbp (see Example VIII) in a radio-immunoprecipitation assay. In Table XIII below, all the variants have glutamate174 replaced by serine174 and phenylalanine176 replaced by tyrosine176 (E174S and F1176Y) plus the additional substitutions as indicated at hGH amino acid positions 10, 14, 18, 21, 167, 171, 175 and 179.
-
TABLE XIII-A hGH variants from addition of helix-1 and helix-4b mutations wild- type residue Helix 1 Helix 4Variant F10 M14 H18 H21 R167 D171 T175 I179 H0650AD H G N N N S T T H0650AE H G N N E S T I H0650AF H G N N N N T T H0650BD A W D N N S T T H0650BE A W D N E S T I H06508F A W D N N N T T H0650CD F S F L N S T T H0650CD F S F L E S T I H0650CD F S F L N N T T - In Table XIV below, hGH variants were selected from combinatorial libraries by the phagemid binding selection process. All hGH variants in Table XIV contain two background mutations (E174S/F176Y). hGH-phagemid pools from the libraries pH0707A (Part A), pH0707B and pH0707E (Part B), or pH0707C (Part C) were sorted for 2 to 7 cycles for binding to hGHbp. The number P indicates the fractional occurrence of each variant type among the set of clones sequenced from each pool.
-
TABLE XIV hGH variants from hormone-phagemid binding selection of combinatorial libraries. wild-type residue: Helix 1 Helix 4 P Variant F10 M14 H18 H21 R167 D171 T175 I179 Part A: 4 cycles: 0.60 H0714A.1 H G N N N S T N 0.40 H0714A.4 A N D A N N T N* Part B: 2 cycles: 0.13 H0712B.1 F S F G H S T T 0.13 H0712B.2 H Q T S A D N S 0.13 H0712B.4 H G N N N A T T 0.13 H07128.5 F S F L S D T T 0.13 H07128.6 A S T N R D T I 0.13 H0712B.7 Q Y N N H S T T 0.13 H0712B.8 W G S S R D T I 0.13 H0712E.1 F L S S K N T V 0.13 H0712E.2 W N N S H S T T 0.13 H0712E.3 A N A S N S T T 0.13 H0712E.4 P S D N R D T I 0.13 H0712E.5 H G N N N N T S 0.13 H0712E.6 F S T G R D T I 0.13 H0712E.7 M T S N Q S T T 0.13 H0712E.8 F S F L T S T S 4 cycles: 0.17 H0714B.1 A W D N R D T I 0.17 H0714B.2 A W D N H S T N 0.17 H0714B.3 M Q M N N S T T 0.17 H0714B.4 H Y D H R D T T 0.17 H0714B.5 L N S H R D T I 0.17 H0714B.6 L N S H T S T T 7 cycles: 0.57 H0717B.1 A W D N N A T T 0.14 H0717B.2 F S T G R D T I 0.14 H0717B.6 A W D N R D T I 0.14 H0717B.7 I Q E H N S T T 0.50 H0717E.1 F S L A N S T V Part C: 4 cycles: 0.67 H0714C.2 F S F L K D T T * = also contained the mutations L15R, K168R. - In Table XV below, hGH variants were selected from combinatorial libraries by the phagemid binding selection process. All hGH variants in Table XV contain two background mutations (E174S/F176Y). The number P is the fractional occurrence of a given variant among all clones sequenced after 4 cycles of rapid-binding selection.
-
TABLE XV hGH variants from RAPID hGHbp binding selection of an hGH- phagemid combinatorial library wild-type residue: Helix 1Helix 4 P Variant F10 M14 H18 H21 R167 D171 T175 I179 0.14 H07BF4.2 W G S S R D T I 0.57 H07BF4.3 M A D N N S T T 0.14 H07BF4.6 A W D N S S V T‡ 0.14 H076F4.7 H D T S R D T I ‡= also contained the mutation Y176F (wild-type hGH also contains F176). - In table XVI below, binding constants were measured by competitive displacement of 125I-labelled hormone H0650BD or labelled hGH using hGHbp (1-238) and either Mab5 or Mab263. The variant H0650BD appears bind more than 30-fold tighter than wild-type hGH.
-
TABLE XVI Equilibrium binding constants of selected hGH variants. hGH Kd(variant) Kd(variant) Variant Kd(H0650BD) Kd(hGH) Kd (pM) hGH 32 -1- 340 ± 50 H0650BD -1- 0.031 10 ± 3 H0650BF 1.5 0.045 15 ± 5 H0714B.6 3.4 0.099 34 ± 19 H0712B.7 7.4 0.22 74 ± 30 H0712E.2 16 0.48 60 ± 70 - As described in Example I, the plasmid pS0132 contains the gene for hGH fused to the residue Pro198 of the gene III protein with the insertion of an extra glycine residue. This plasmid may be used to produce hGH-phage particles in which the hGH-gene III fusion product is displayed monovalently on the phage surface (Example IV). The fusion protein comprises the entire hGH protein fused to the carboxy terminal domain of gene III via a flexible linker sequence.
- To investigate the feasibility of using phage display technology to select favourable substrate sequences for a given proteolytic enzyme, a genetically engineered variant of subtilisin BPN′ was used. (Carter, P. et al., Proteins: Structure, function and genetics 6:240-248 (1989)). This variant (hereafter referred to as A64SAL subtilisin) contains the following mutations: Ser24Cys, His64Ala, Glu156Ser, Gly169Ala and Tyr217Leu. Since this enzyme lacks the essential catalytic residue His64, its substrate specificity is greatly restricted so that certain histidine-containing substrates are preferentially hyrdrolysed (Carter et al., Science 237:394-399 (1987)).
- Construction of a hGH-Substrate-Phage Vector
- The sequence of the linker region in pS0132 was mutated to create a substrate sequence for A64SAL subtilisin, using the
oligonucleotide 5′-TTC-GGG-CCC-TTC-GCT-GCT-CAC-TAT-ACG-CGT-CAG-TCG-ACT-GAC-CTG-CCT-3′ (SEQ ID NO:27). This resulted in the introduction of the protein sequence Phe-Gly-Pro-Phe-Ala-Ala-His-Tyr-Thr-Arg-Gln-Ser-Thr-Asp (SEQ ID NO:107) in the linker region between hGH and the carboxy terminal domain of gene III, where the first Phe residue in the above sequence is Phe191 of hGH. The sequence Ala-Ala-His-Tyr-Thr-Agr-Gln (SEQ ID NO:97) is known to be a good substrate for A64SAL subtilisin (Carter et al (1989), supra). The resulting plasmid was designated pS0640. - Selective Enrichment of hGH-Substrate-Phage
- Phagemid particles derived from pS0132 and pS0640 were constructed as described in Example I. In initial experiments, a 10 μl aliquot of each phage pool was separately mixed with 30 μl of oxirane beads (prepared as described in Example II) in 100 μl of buffer comprising 20 mM Tris-HCl pH 8.6 and 2.5M NaCl. The binding and washing steps were performed as described in example VII. The beads were then resuspended in 400 μl of the same buffer, with or without 50 nM of A64SAL subtilisin. Following incubation for 10 minutes, the supernatants were collected and the phage titres (cfu) measured. Table XVII shows that approximately 10 times more substrate-containing phagemid particles (pS0640) were eluted in the presence of enzyme than in the absence of enzyme, or than in the case of the non-substrate phagemids (pS0132) in the presence or absence of enzyme. Increasing the enzyme, phagemid or bead concentrations did not improve this ratio.
- In an attempt to decrease the non-specific elution of immobilised phagemids, a tight-binding variant of hGH was introduced in place of the wild-type hGH gene in pS0132 and pS0640. The hGH variant used was as described in example XI (pH0650bd) and contains the mutations Phe10Ala, Met14Trp, His18Asp, His21Asn, Arg167Asn, Asp171Ser, Glu174Ser, Phe176Tyr and Ile179Thr. This resulted in the construction of two new phagemids: pDM0390 (containing tight-binding hGH and no substrate sequence) and pDM0411 (containing tight-binding hGH and the substrate sequence Ala-Ala-His-Tyr-Thr-Agr-Gln). The binding washing and elution protocol was also changed as follows:
- (i) Binding: COSTAR 12-well tissue culture plates were coated for 16 hours with 0.5 ml/well 2 ug/ml hGHbp in sodium carbonate buffer pH 10.0. The plates were then incubated with 1 ml/well of blocking buffer (phosphate buffered saline (PBS) containing 0.1% w/v bovine serum albumen) for 2 hours and washed in an assay buffer containing 10 mM Tris-HCl pH 7.5, 1 mM EDTA and 100 mM NaCl. Phagemids were again prepared as described in Example I: the phage pool was diluted 1:4 in the above assay buffer and 0.5 ml of phage incubated per well for 2 hours.
- (ii) Washing: The plates were washed thoroughly with PBS+0.05
% Tween 20 and incubated for 30 minuted with 1 ml of this wash buffer. This washing step was repeated three times. - (iii) Elution: The plates were incubated for 10 minutes in an elution buffer consisting of 20 mM Tris-HCl pH 8.6+100 mM NaCl, then the phage were eluted with 0.5 ml of the above buffer with or without 500 nM of A64SAL subtilisin.
- Table XVII shows that there was a dramatic increase in the ratio of specifically eluted substrate-phagemid particles compared to the method previously described for pS0640 and pS0132. It is likely that this is due to the fact that the tight-binding hGH mutant has a significantly slower off-rate for binding to hGH binding protein compared to wild-type hGH.
-
TABLE XVII Specific elution of substrate-phagemids by A64SAL subtilisin phagemid +50 nM A64SAL no enzyme (i) Wild-type hGH gene: binding to hGHbp-oxirane beads pS0640 (substrate) 9 × 106 cfu/10 μl 1.5 × 106 cfu/10 μl pS0132 (non-sub- 6 × 105 cfu/10 μl 3 × 105 cfu/10 μl strate) (ii) pH0650bd mutant hGH gene: binding to hGHbp-coated plates pDM0411 (substrate) 1.7 × 105 cfu/10 μl 2 × 103 cfu/10 μl pDM0390 (non-sub- 2 × 103 cfu/10 μl 1 × 103 cfu/10 μl strate) Colony forming units (cfu) were estimated by plating out 10 μl of 10-fold dilutions of phage on 10 μl spots of XL-1 blue cells, on LB agar plates containing 50 μg/ml carbenicillinl - We sought to employ the selective enrichment procedure described in Example XIII to identify good substrate sequences from a library of random substrate sequences.
- We designed a vector suitable for introduction of randomised substrate cassettes. and subsequent expression of a library of substrate sequences. The starting point was the vector pS0643, described in Example VIII. Site-directed mutagenesis was carried out using the
oligonucleotide 5′-AGC-TGT-GGC-TTC-GGG-CCC-GCC-GCC-GCG-TCG-ACT-GGC-GGT-GGC-TCT-3′ (SEQ ID NO:28), which introduces ApaI (GGGCCC) and SalI (GTCGAC) restriction sites between hGH and Gene III. This new construct was designated pDM0253 (The actual sequence of pDM0253 is 5′-AGC-TGT-GGC-TTC-GGG-CCC-GCC-CCC-GCG-TCG-ACT-GGC-GGT-GGC-TCT-3′ (SEQ ID NO:29), where the underlined base substitution is due to a spurious error in the mutagenic oligonucleotide). In addition, the tight-binding hGH variant described in example was introduced by exchanging a fragment from pDM0411 (example XIII). The resulting library vector was designated pDM0454. - To introduce a library cassette, pDM0454 was digested with ApaI followed by SalI, then precipitated with 13% PEG 8000+10 mM MgCl2, washed twice in 70% ethanol and resuspended This efficiently precipitates the vector but leaves the small Apa-Sal fragment in solution (Paithankar, K. R. and Prasad, K. S. N., Nucleic Acids Research 19:1346). The product was run on a 1% agarose gel and the ApaI-SalI digested vector excised, purified using a Bandprep kit (Pharmacia) and resuspended for ligation with the mutagenic cassette.
- The cassette to be inserted contained a DNA sequence similar to that in the linker region of pS0640 and pDM0411, but with the codons for the histidine and tyrosine residues in the substrate sequence replaced by randomised codons. We chose to substitute NNS(N=G/A/T/C; S=G/C) at each of the randomised positions as described in example VIII. The oligonucleotides used in the mutagenic cassettes were: 5′-C-TTC-GCT-GCT-NNS-NNS-ACC-CGG-CAA-3′ (coding strand) (SEQ ID NO:30) and 5′-T-CGA-TTG-CCG-GGT-SNN-SNN-AGC-AGC-GAA-GGG-CC-3′ (non-coding strand) (SEQ ID NO:31). This cassette also destroys the SalI site, so that digestion with SalI may be used to reduce the vector background. The oligonucleotides were not phosphorylated before insertion into the Apa-Sal cassette site, as it was feared that subsequent oligomerisation of a small population of the cassettes may lead to spurious results with multiple cassette inserts. Following annealing and ligation, the reaction products were phenol:chloroform extracted, ethanol precipitated and resuspended in water. Initially, no digestion with SalI to reduce the background vector was performed. Approximately 200 ng was electroporated into XL-1 blue cells and a phagemid library was prepared as described in example VIII.
- Selection of Highly Cleavable Substrates from the Substrate Library
- The selection procedure used was identical to that described for pDM0411 and pDM0390 in example XIII. After each round of selection, the eluted phage were propagated by transducing a fresh culture of XL-1 blue cells and propagating a new phagemid library as described for hGH-phage in example VIII. The progress of the selection procedure was monitored by measuring eluted phage titres and by sequencing individual clones after each round of selection.
- Table A shows the successive phage titres for elution in the presence and absence of enzyme after 1, 2 and 3 rounds of selection.
- Clearly, the ratio of specifically eluted phage:non-specifically eluted phage (ie phage eluted with enzyme:phage eluted without enzyme) increases dramatically from
round 1 toround 3, suggesting that the population of good substrates is increasing with each round of selection. - Sequencing of 10 isolates from the starting library showed them all to consist of the wild-type pDM0464 sequence. This is attributed to the fact that after digestion with ApaI, the SalI site is very close to the end of the DNA fragment, thus leading to low efficiency of digestion. Nevertheless, there are only 400 possible sequences in the library, so this population should still be well represented.
- Tables B1 and B2 shows the sequences of isolates obtained after
round 2 andround 3 of selection. After 2 rounds of selection, there is clearly a high incidence of histidine residues. This is exactly what is expected: as described in example XIII, A64SAL subtilisin requires a histidine residue in the substrate as it employs a substrate-assisted catalytic mechanism. After 3 rounds of selection, each of the 10 clones sequenced has a histidine in the randomised cassette. Note, however, that 2 of the sequences are of pDM0411, which was not present in the starting library and is therefore a contaminant. -
TABLE A Titration of initial phage pools and eluted phage from 3 rounds of selective enrichment ROUND 1 Starting library: 3 × 1012 cfu/ml LIBRARY: +500 nM A64SAL 4 × 103 cfu/10 μl no enzyme 3 × 103 cfu/10 μl pDM0411: +500 nM A64SAL 2 × 106 cfu/10 μl (control) no enzyme 8 × 103 cfu/10 μl ROUND 2 Round 1 library:7 × 1012 cfu/ml LIBRARY: +500 nM A64SAL 3 × 104 cfu/10 μl no enzyme 6 × 103 cfu/10 μl pDM0411: +500 nM A64SAL 3 × 106 cfu/10 μl (control) no enzyme 1.6 × 104 cfu/10 μl ROUND 3 Round 2 library:7 × 1011 cfu/ml LIBRARY: +500 nM A64SAL 1 × 105 cfu/10 μl no enzyme <103 cfu/10 μl pDM0411: +500 nM A64SAL 5 × 106 cfu/10 μl (control) no enzyme 3 × 104 cfu/10 μl Colony forming units (cfu) were estimated by plating out 10 μl of 10-fold dilutions of phage on 10 μl spots of XL-1 blue cells, on LB agar plates containing 50 μg/ml carbenicillin -
TABLE B1 Sequences of eluted phage after 2 rounds of selective enrichment. No. of Sequence occurrences After round 2: * * A A H Y T R Q (SEQ ID NO: 97) 2 . . . GCT GCT CAC TAC ACC CGG CAA . . . (SEQ ID NO: 32) A A H M T R Q (SEQ ID NO: 98) 1 . . . GCT GCT CAC ATG ACC CGG CAA . . . (SEQ ID NO: 33) A A L H T R Q (SEQ ID NO: 99) 1 . . . GCT GCT CTC CAC ACC CGG CAA . . . (SEQ ID NO: 34) A A L H T R Q (SEQ ID NO: 99) 1 . . . GCT GCT CTG CAC ACC CGG CAA . . . (SEQ ID NO: 35) A A H T R Q (SEQ ID NO: 100) 1 # . . . GCT GCT CAC ACC CGG CAA . . . (SEQ ID NO: 36) A A ? H T R Q (SEQ ID NO: 101) 1 ## . . . GCT GCT ??? CAC ACC CGG CAA (SEQ ID NO: 37) . . . wild- type pDM0454 3 #- spurious deletion of 1 codon within the cassette ##- ambiguous sequence All protein sequences should be of the form AA**TRQ, where * represents a randomised codon. In the table below, the randomised codons and amino acids are underlined and in bold. -
TABLE B2 Sequences of eluted Dhaqe after 3 rounds of selective enrichment. No. of Sequence occurrences After round 3: * * A A H Y T R Q (SEQ ID NO: 97) 2# . . . GCT GCT CAC TAT ACG CGT CAG . . . (SEQ ID NO: 38) A A L H T R Q (SEQ ID NO: 99) 2 . . . GCT GCT CTC CAC ACC CGG CAA . . . (SEQ ID NO: 34) A A Q H T R Q (SED ID NO: 102) 1 . . . GCT GCT CAG CAC ACC CGG CAA . . . (SEQ ID NO: 39) A A T H T R Q (SEQ ID NO: 103) 1 . . . GCT GCT ACG CAC ACC CGG CAA . . . (SEQ ID NO: 40) A A H S R Q (SEQ ID NO: 104) 1 . . . GCT GCT CAC TCC CGG CAA . . . (SEQ ID NO: 41) A A H H T R Q (SEQ ID NO: 105) 1## . . . GCT GCT CAT CAT ACC CGG CAA . . . (SEQ ID NO: 42) A A H F R Q (SEQ ID NO: 106) 1 . . . GCT GCT CAC TTC CGG CAA . . . (SEQ ID NO: 43) A A H T R Q (SEQ ID NO: 100) 1 . . . GCT GCT CAC ACC CGG CAA . . . (SEQ ID NO: 36) #- contaminating sequence from pDM0411 ##- contains the “illegal” codon CAT - T should not appear in the 3rd position of a codon. All protein sequences should be of the form AA**TRQ, where * represents a randomised codon. In the table below, the randomised codons and amino acids are underlined and in bold.
Claims (52)
1. A method for selecting novel binding polypeptides comprising:
(a) constructing a replicable expression vector comprising a transcription regulatory element operably linked to a gene fusion encoding a fusion protein wherein the gene fusion comprises a first gene encoding a polypeptide, and a second gene encoding at least a portion of a phage coat protein;
(b) mutating the vector at one or more selected positions within the first gene thereby forming a family of related plasmids;
(c) transforming suitable host cells with the plasmids;
(d) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein;
(e) culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface of the particle;
(f) contacting the phagemid particles with a target molecule so that at least a portion of the phagemid particles bind to the target molecule; and
(g) separating the phagemid particles that bind from those that do not.
2. The method of claim 1 further comprising infecting a suitable host cells with the phagemid particles that bind and repeating steps (d) through (g).
3. The method of claim 2 wherein the steps are repeated one or more times.
4. The method of claim 1 wherein the expression vector further comprises a secretory signal sequence.
5. The method of claim 1 wherein the transcription regulatory element is a promoter system selected from the group; lac Z, pho A, tryptophan, tac, λPL, bacteriophage T7, and combinations thereof.
6. The method of claim 1 wherein the first gene encodes a mammalian protein.
7. The method of claim 6 wherein the protein is selected from the group; growth hormone, human growth hormone (hGH), des-N methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin A-chain, insulin B-chain, proinsulin, relaxin A-chain, relaxin B-chain, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), leutinizing hormone (LH), glycoprotein hormone receptors, calcitonin, glucagon, factor VIII, an antibody, lung surfactant, urokinase, streptokinase, human tissue-type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hemopoietic growth factor, tumor necrosis factor-alpha and -beta, enkephalinase, human serum albumin, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, β-lactamase, tissue factor protein, inhibin, activin, vascular endothelial growth factor, integrin receptors, thrombopoietin, protein A or D, rheumatoid factors, NGF-β, platelet-growth factor, transforming growth factor; TGF-alpha and TGF-beta, insulin-like growth factor-I and -II, insulin-like growth factor binding proteins, CD-4, DNase, latency associated peptide, erythropoietin, HER2 ligands, osteoinductive factors, interferon-alpha, -beta, and -gamma, colony stimulating factors (CSFs), M-CSF, GM-CSF, and G-CSF, interleukins (ILs), IL-1, IL-2, IL-3, IL-4, superoxide dismutase; decay accelerating factor, viral antigen, HIV envelope proteins GP120 and GP140, atrial natriuretic peptides A, B, or C, or immuno globulins, and fragments of the above-listed proteins.
8. The method of claim 7 wherein the protein is a human protein.
9. The method of claim 8 wherein the protein comprises more than about 100 amino acid residues.
10. The method of claim 1 wherein the protein comprises a plurality of rigid secondary structures displaying amino acids capable of interacting with the target, and the mutations are primarily produced at positions corresponding to codons encoding the amino acids.
12. The method of claim 10 wherein the mutations are produced at more than one codon.
13. The method of claim 12 wherein the mutations are produced on more than one rigid secondary structure.
14. The method of claim 1 wherein the helper phage is selected from the group M13KO7, M13R408, M13-VCS, and Phi X 174.
15. The method of claim 14 wherein the helper phage is M13KO7 and the coat protein is the M13 phage gene III coat protein.
16. The method of claim 15 wherein the host is E. coli.
17. The method of claim 16 wherein the plasmid is under tight control of the transcription regulatory element.
18. The method of claim 17 wherein the amount is less than about 1%.
19. The method of claim 18 wherein the amount is less than 20% the amount of phagemid particles displaying a single copy of the fusion protein.
20. The method of claim 19 wherein the amount is less than 10%.
21. The method of claim 1 further comprising in step (a), inserting a DNA triplet, encoding an mRNA suppressible terminator codon between said first gene encoding a polypeptide, and said second gene encoding at least a portion of a phage coat protein.
22. The method of claim 21 wherein said mRNA suppressible terminator codon is selected from the following: UAG (amber), UAA (ocher) and UGA (opel).
23. The method of claim 22 wherein said suppressible mutation results in the detectable production of a fusion polypeptide containing sadi polypeptide and said coat protein when said expression vector is grown in a suppressor host cell; and, when grown in a non-suppressor host cell said polypeptide is synthesized substantially without fusion to said phage coat protein.
24. A human growth hormone variant wherein hGH amino acids 172, 174, 176 and 178 respectively are as a group sequentially selected from one of the following: (1) R,S,F,R; (2) R,A,Y,R; (3) K,T,Y,K; (4) R,S,Y,R; (5) K,A,Y,R; (6) R,F,F,R; (7) K,Q,Y,R; (8) R,T,Y,H; (9) Q,R,Y,R; (10) K,K,Y,K; (11) R,S,F,S; and (12) K,S,N,R.
25. A phagemid comprising a replicable expression vector comprising a transcription regulatory element operably linked to a gene fusion encoding a fusion protein wherein the gene fusion comprises a first gene encoding a polypeptide, and a second gene encoding at least a portion of a phage coat protein, wherein a DNA triplet codon encoding an mRNA suppressible terminator codon selected from UAG, UAA and UGA is inserted between the fused ends of the first and second genes, or is substituted for an amino acid encoding triplet codon adjacent to the gene fusion junction.
26. The phagemid of claim 25 wherein said first gene encodes a mammalian protein.
27. The phagemid of claim 26 wherein the protein is selected from the group: growth hormone, human growth hormone (hGH), des-N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin A-chain, insulin B-chain, proinsulin, relaxin A-chain, relaxin B-chain, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), leutinizing hormone (LH), glycoprotein hormone receptors, calcitonin, glucagon, factor VIII, an antibody, lung surfactant, urokinase, streptokinase, human tissue-type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hemopoietic growth factor, tumor necrosis factor-alpha and -beta, enkephalinase, human serum albumin, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, β-lactamase, tissue factor protein, inhibin, activin, vascular endothelial growth factor, integrin receptors, thrombopoietin, protein A or D, rheumatoid factors, NGF-β platelet-growth factor, transforming growth factor; TGF-alpha and TGF-beta, insulin-like growth-I and -II, insulin-like growth factor binding proteins, CD-4, DNase, latency associated peptide, erythropoietin, osteoinductive factors, interferon-alpha, -beta, and -gamma, colony stimulating factors (CSFs), M-CSF, GM-CSF, and G-CSF, interleukins (ILs), IL-1, IL-2, IL-3, IL-4, superoxide dismutase; decay accelerating factor, viral antigen, HIV envelope proteins GP120 and GP140, atrial natriuretic peptides A, B or C immuno globulins, and fragments of the above-listed proteins.
28. The phagemid of claim 27 wherein said protein is a human protein.
29. The phagemid of claim 28 wherein the protein comprises more than about 100 amino acid residues.
30. The phagemid of claim 25 wherein said protein comprises a plurality of rigid secondary structures displaying amino acids capable of interacting with the target.
31. The phagemid of claim 30 wherein said rigid secondary structures comprises structures selected from the group; α-(3.613) helix, 310 helix, π-(4.416) helix, parallel and anti-parallel β-pleated sheets, reverse turns, and non-ordered structures.
32. The phagemid of claim 25 wherein the helper phage is selected from the group M13KO7, M13R408, M13-VCS, and Phi X 174.
33. The phagemid of claim 32 wherein the helper phage is M13KO7 and the coat protein is the M13 phage gene III coat protein.
34. The phagemid of claim 33 wherein the host is the E. coli wild type or suppressor type.
35. The phagemid of claim 34 wherein the plasmid is under tight control of the transcription regulatory element.
36. The phagemid of claim 35 wherein the number of phagemid particles displaying more than one copy of the fusion protein on the surface of the particles is less than 1%.
37. The phagemid of claim 36 wherein said number of phagemid particles is less than about 10%.
38. The phagemid of claim 37 wherein the number of phagemid particles is less than about 20%.
39. A human growth variant wherein hGH amino acids 10, 14, 18, and 21 respectively are as a group sequentially selected from one of the following:
(1) H,G,N,N; (2) A,W,D,N; (3) F,S,F,L; (4) Y,T,V,N and (5) I,N,I,N.
40. A human growth variant wherein hGH amino acids 174 is serine and 176 is tyrosine and hGH amino acids 167, 171, 175 and 179 respectively are as a group sequentially selected from one of the following: (1) N,S,T,T; (2) E,S,T,I; (3) K,S,T,L; (4) N,N,T,T; (5) R,D,I,I; and (6) N,S,T,Q.
41. A method for selecting novel binding polypeptides comprising
(a) constructing a replicable expression vector comprising a transcription regulatory element operably linked to DNA encoding a protein of interest containing one or more subunits, wherein the DNA encoding at least one of the subunits is fused to the DNA encoding at least a portion of a phage coat protein;
(b) mutating the DNA encoding the protein of interest at one or more selected positions thereby forming a family of related vectors;
(c) transforming suitable host cells with the vectors;
(d) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein;
(e) culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface of the particle;
(f) contacting the phagemid particles with a target molecule so that at least a portion of the phagemid particles bind to the target molecule; and
(g) separating the phagemid particles that bind from those that do not.
42. The method of claim 41 wherein the expression vector further comprises a secretory signal sequence operably linked to the DNA encoding each subunit of the protein of interest.
43. The method of claim 42 wherein the protein of interest is a mammalian protein.
44. The method of claim 43 wherein the protein of interest is selected from the group; insulin, relaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), leutinizing hormone (LH), glycoprotein hormone receptors, monoclonal and polyclonal antibodies, lung surfactant, integrin receptors, insulin-like growth factor-I and -II, and fragments of the above-listed proteins.
45. The method of claim 44 wherein the protein of interest is a humanized antibody.
46. The method of claim 45 wherein the protein of interest is a humanized Fab fragment capable of binding to the HER-2 receptor (human epidermal growth factor receptor-2).
47. A human growth hormone (hGH) variant wherein hGH amino acid glutamate174 is replaced by serine174 and phenylalanine176 is replaced by tyrosine176 and one or more of the eight naturally occurring hGH amino acids F10, M14, H18, H21, R167, D171, T175 and I179 are replaced by another natural amino acid.
48. The hGH variant of claim 47 wherein the eight naturally occurring hGH amino acids F10, M14, H18, H21, R167, D171, T175 and I179 respectively are as a group replaced with a corresponding amino acid sequentially selected from one of the following groups:
49. The method of claim 48 wherein said human growth hormone variant (11) further contains leucine15 replaced by arginine15 and lysine168 replaced by arginine168.
50. The method of claim 48 wherein said human growth hormone variant
(40) further contains phenylalanine176
51. A method for selecting novel binding polypeptides comprising:
(a) constructing a replicable expression vector comprising a transcription regulatory element operably linked to a gene fusion encoding a fusion protein wherein the gene fusion comprises a first gene encoding a polypeptide operable connected to a linking amino acid sequence, and a second gene encoding at least a portion of a phage coat protein;
(b) mutating the vector at one or more selected positions within the amino acid linking sequence of the first gene thereby forming a family of related plasmids;
(c) transforming suitable host cells with the plasmids;
(d) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein;
(e) culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface of the particle;
(f) contacting the phagemid particles with a target molecule so that at least a portion of the phagemid particles bind to the target molecule; and
(g) contacting the bound phagemid particles with a protease capable of hydrolysing the linking a amino acid sequence of at least a portion of the bound phagemid particles, and
(h) isolating the hydrolyzed phagemid particles.
52. The method of claim 51 further comprising infecting suitable host cells with the hydrolyzed phagemid particles and repeating steps (d) through (h).
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US08/923,854 Expired - Fee Related US6040136A (en) | 1990-12-03 | 1997-09-03 | Enrichment method for variant proteins with altered binding properties |
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Families Citing this family (2177)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5534617A (en) * | 1988-10-28 | 1996-07-09 | Genentech, Inc. | Human growth hormone variants having greater affinity for human growth hormone receptor at site 1 |
US5688666A (en) * | 1988-10-28 | 1997-11-18 | Genentech, Inc. | Growth hormone variants with altered binding properties |
US5350836A (en) * | 1989-10-12 | 1994-09-27 | Ohio University | Growth hormone antagonists |
US5723286A (en) * | 1990-06-20 | 1998-03-03 | Affymax Technologies N.V. | Peptide library and screening systems |
GB9206318D0 (en) * | 1992-03-24 | 1992-05-06 | Cambridge Antibody Tech | Binding substances |
US6916605B1 (en) | 1990-07-10 | 2005-07-12 | Medical Research Council | Methods for producing members of specific binding pairs |
GB9015198D0 (en) | 1990-07-10 | 1990-08-29 | Brien Caroline J O | Binding substance |
US7063943B1 (en) | 1990-07-10 | 2006-06-20 | Cambridge Antibody Technology | Methods for producing members of specific binding pairs |
US6172197B1 (en) | 1991-07-10 | 2001-01-09 | Medical Research Council | Methods for producing members of specific binding pairs |
DE69129154T2 (en) * | 1990-12-03 | 1998-08-20 | Genentech Inc | METHOD FOR ENRICHING PROTEIN VARIANTS WITH CHANGED BINDING PROPERTIES |
IE921169A1 (en) | 1991-04-10 | 1992-10-21 | Scripps Research Inst | Heterodimeric receptor libraries using phagemids |
JP3417558B2 (en) | 1991-05-10 | 2003-06-16 | ジェネンテク,インコーポレイテッド | Choice of ligand agonists and antagonists |
US5858657A (en) * | 1992-05-15 | 1999-01-12 | Medical Research Council | Methods for producing members of specific binding pairs |
US5962255A (en) * | 1992-03-24 | 1999-10-05 | Cambridge Antibody Technology Limited | Methods for producing recombinant vectors |
US6225447B1 (en) | 1991-05-15 | 2001-05-01 | Cambridge Antibody Technology Ltd. | Methods for producing members of specific binding pairs |
US6492160B1 (en) | 1991-05-15 | 2002-12-10 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
DE69230142T2 (en) * | 1991-05-15 | 2000-03-09 | Cambridge Antibody Tech | METHOD FOR PRODUCING SPECIFIC BINDING PAIRS |
US6800738B1 (en) * | 1991-06-14 | 2004-10-05 | Genentech, Inc. | Method for making humanized antibodies |
WO1992022653A1 (en) | 1991-06-14 | 1992-12-23 | Genentech, Inc. | Method for making humanized antibodies |
DE4122599C2 (en) | 1991-07-08 | 1993-11-11 | Deutsches Krebsforsch | Phagemid for screening antibodies |
US5733731A (en) * | 1991-10-16 | 1998-03-31 | Affymax Technologies N.V. | Peptide library and screening method |
US5270170A (en) * | 1991-10-16 | 1993-12-14 | Affymax Technologies N.V. | Peptide library and screening method |
ATE463573T1 (en) | 1991-12-02 | 2010-04-15 | Medimmune Ltd | PRODUCTION OF AUTOANTIBODIES ON PHAGE SURFACES BASED ON ANTIBODIES SEGMENT LIBRARIES |
US5733743A (en) * | 1992-03-24 | 1998-03-31 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
US6297026B1 (en) * | 1993-07-26 | 2001-10-02 | Cor Therapeutics Inc. | Nucleic acids encoding the C140 receptor |
WO1995018856A1 (en) | 1993-12-30 | 1995-07-13 | President And Fellows Of Harvard College | Vertebrate embryonic pattern-inducing hedgehog-like proteins |
US5837458A (en) * | 1994-02-17 | 1998-11-17 | Maxygen, Inc. | Methods and compositions for cellular and metabolic engineering |
US6335160B1 (en) | 1995-02-17 | 2002-01-01 | Maxygen, Inc. | Methods and compositions for polypeptide engineering |
US6406855B1 (en) | 1994-02-17 | 2002-06-18 | Maxygen, Inc. | Methods and compositions for polypeptide engineering |
US6576236B1 (en) | 1994-07-01 | 2003-06-10 | Dana Farber Cancer Institute | Methods for stimulating T cell responses by manipulating a common cytokine receptor γ chain |
US7597886B2 (en) * | 1994-11-07 | 2009-10-06 | Human Genome Sciences, Inc. | Tumor necrosis factor-gamma |
US7820798B2 (en) * | 1994-11-07 | 2010-10-26 | Human Genome Sciences, Inc. | Tumor necrosis factor-gamma |
US6335319B1 (en) * | 1994-11-15 | 2002-01-01 | Metabolic Pharmaceuticals, Inc. | Treatment of obesity |
US7429646B1 (en) | 1995-06-05 | 2008-09-30 | Human Genome Sciences, Inc. | Antibodies to human tumor necrosis factor receptor-like 2 |
US6475806B1 (en) | 1995-06-07 | 2002-11-05 | Praecis Pharmaceuticals, Inc. | Anchor libraries and identification of peptide binding sequences |
DE122006000003I2 (en) * | 1995-09-21 | 2011-01-13 | Genentech Inc | VARIANTS OF HUMAN GROWTH HORMONE |
US7368111B2 (en) | 1995-10-06 | 2008-05-06 | Cambridge Antibody Technology Limited | Human antibodies specific for TGFβ2 |
DK0859959T3 (en) * | 1995-11-10 | 2003-11-24 | Elan Corp Plc | Peptides that enhance tissue transport and methods for identifying and using them |
US6090382A (en) | 1996-02-09 | 2000-07-18 | Basf Aktiengesellschaft | Human antibodies that bind human TNFα |
US7888466B2 (en) | 1996-01-11 | 2011-02-15 | Human Genome Sciences, Inc. | Human G-protein chemokine receptor HSATU68 |
AU2531397A (en) * | 1996-03-21 | 1997-10-10 | President And Fellows Of Harvard College | Enantiomeric screening process, and compositions therefor |
US6777217B1 (en) | 1996-03-26 | 2004-08-17 | President And Fellows Of Harvard College | Histone deacetylases, and uses related thereto |
US6361938B1 (en) | 1996-11-08 | 2002-03-26 | Elan Corporation, Plc | Peptides which enhance transport across tissues and methods of identifying and using the same |
US20070185032A1 (en) * | 1996-12-11 | 2007-08-09 | Praecis Pharmaceuticals, Inc. | Pharmaceutical formulations for sustained drug delivery |
US6497874B1 (en) | 1997-02-05 | 2002-12-24 | Maardh Sven | Recombinant phages |
ATE505543T1 (en) | 1997-04-16 | 2011-04-15 | Millennium Pharm Inc | A COMPOSITION COMPRISING AN ANTIBODY THAT SELECTIVELY BINDS TO A CYSTEINE-RICH SECRETED PROTEIN (CRSP). |
US6306826B1 (en) | 1997-06-04 | 2001-10-23 | The Regents Of The University Of California | Treatment of heart failure with growth hormone |
US5994511A (en) | 1997-07-02 | 1999-11-30 | Genentech, Inc. | Anti-IgE antibodies and methods of improving polypeptides |
US6172213B1 (en) * | 1997-07-02 | 2001-01-09 | Genentech, Inc. | Anti-IgE antibodies and method of improving polypeptides |
US7378506B2 (en) | 1997-07-21 | 2008-05-27 | Ohio University | Synthetic genes for plant gums and other hydroxyproline-rich glycoproteins |
US6639050B1 (en) | 1997-07-21 | 2003-10-28 | Ohio University | Synthetic genes for plant gums and other hydroxyproline-rich glycoproteins |
WO1999006587A2 (en) | 1997-08-01 | 1999-02-11 | Morphosys Ag | Novel method and phage for the identification of nucleic acid sequences encoding members of a multimeric (poly)peptide complex |
GB9723955D0 (en) * | 1997-11-14 | 1998-01-07 | Generic Biolog Limited | Improvements in or relating to detection of molecules in samples |
GB2375765B (en) * | 1997-11-14 | 2003-02-26 | Kymed Gb Ltd | Tagged growth hormone molecules |
US7192589B2 (en) | 1998-09-16 | 2007-03-20 | Genentech, Inc. | Treatment of inflammatory disorders with STIgMA immunoadhesins |
JP3497133B2 (en) | 1997-11-21 | 2004-02-16 | ジェネンテック・インコーポレーテッド | A-33 related antigens and their pharmacological uses |
WO1999047538A1 (en) | 1998-03-19 | 1999-09-23 | Human Genome Sciences, Inc. | Cytokine receptor common gamma chain like |
US7163682B2 (en) | 1998-04-13 | 2007-01-16 | The Forsyth Institute | Glucan binding protein and glucosyltransferase immunogens |
US7056517B2 (en) | 1998-04-13 | 2006-06-06 | The Forsyth Institute | Glucosyltransferase immunogens |
JP2002522029A (en) * | 1998-07-27 | 2002-07-23 | ジェネンテック・インコーポレーテッド | Improved transformation efficiency in phage display by modification of coat protein |
US6387888B1 (en) | 1998-09-30 | 2002-05-14 | American Foundation For Biological Research, Inc. | Immunotherapy of cancer through expression of truncated tumor or tumor-associated antigen |
US6485972B1 (en) | 1998-10-15 | 2002-11-26 | President And Fellows Of Harvard College | WNT signalling in reproductive organs |
US6420110B1 (en) | 1998-10-19 | 2002-07-16 | Gpc Biotech, Inc. | Methods and reagents for isolating biologically active peptides |
DE69939484D1 (en) * | 1998-10-28 | 2008-10-16 | Cornell Res Foundation Inc | METHODS FOR REGULATING ANGIOGENESIS AND VASCULAR INTEGRITY BY TRK RECEPTOR LIGANDS BDNF, NT-3 AND NT-4 |
US6927024B2 (en) | 1998-11-30 | 2005-08-09 | Genentech, Inc. | PCR assay |
US6696063B1 (en) * | 1998-12-30 | 2004-02-24 | Applied Research Systems Ars Holding N.V. | Treatment of HIV-associated dysmorphia/dysmetabolic syndrome (HADDS) with or without lipodystrophy |
US20030194800A1 (en) * | 2001-08-31 | 2003-10-16 | Megede Jan Zur | Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides and uses thereof |
CA2358385C (en) * | 1998-12-31 | 2013-08-06 | Chiron Corporation | Polynucleotides encoding antigenic hiv type c polypeptides, polypeptides and uses thereof |
US7935805B1 (en) * | 1998-12-31 | 2011-05-03 | Novartis Vaccines & Diagnostics, Inc | Polynucleotides encoding antigenic HIV Type C polypeptides, polypeptides and uses thereof |
AU2221600A (en) * | 1998-12-31 | 2000-07-31 | Chiron Corporation | Improved expression of hiv polypeptides and production of virus-like particles |
WO2000039303A2 (en) | 1998-12-31 | 2000-07-06 | Chiron Corporation | Modified hiv env polypeptides |
CA2356109C (en) | 1999-01-06 | 2008-04-22 | Genentech, Inc. | Insulin-like growth factor (igf) i mutant variants |
IL143834A0 (en) * | 1999-01-06 | 2002-04-21 | Genentech Inc | Insulin-like growth factor (igf) i mutant variants |
AU3501700A (en) | 1999-02-26 | 2000-09-14 | Human Genome Sciences, Inc. | Human endokine alpha and methods of use |
EP2301947A3 (en) | 1999-02-26 | 2011-11-23 | Millennium Pharmaceuticals, Inc. | Secreted proteins and uses thereof |
IL139321A0 (en) | 1999-03-03 | 2001-11-25 | Biogen Inc | Methods and compositions for modulating lipid metabolism |
US7883704B2 (en) | 1999-03-25 | 2011-02-08 | Abbott Gmbh & Co. Kg | Methods for inhibiting the activity of the P40 subunit of human IL-12 |
US6914128B1 (en) | 1999-03-25 | 2005-07-05 | Abbott Gmbh & Co. Kg | Human antibodies that bind human IL-12 and methods for producing |
CA2669512A1 (en) | 1999-03-25 | 2000-09-28 | Abbott Gmbh & Co. Kg | Human antibodies that bind human il-12 and methods for producing |
WO2000058521A2 (en) * | 1999-03-31 | 2000-10-05 | Rosetta Inpharmatics, Inc. | Methods for the identification of reporter and target molecules using comprehensive gene expression profiles |
DE19915057A1 (en) | 1999-04-01 | 2000-10-19 | Forschungszentrum Borstel | Monoclonal antibodies to the human Mcm3 protein, process for their preparation and their use |
US6492497B1 (en) | 1999-04-30 | 2002-12-10 | Cambridge Antibody Technology Limited | Specific binding members for TGFbeta1 |
US7270969B2 (en) * | 1999-05-05 | 2007-09-18 | Phylogica Limited | Methods of constructing and screening diverse expression libraries |
US7803765B2 (en) * | 1999-05-05 | 2010-09-28 | Phylogica Limited | Methods of constructing biodiverse gene fragment libraries and biological modulators isolated therefrom |
CA2721199A1 (en) * | 1999-05-05 | 2000-11-16 | Phylogica Limited | Isolating biological modulators from biodiverse gene fragment libraries |
US6221597B1 (en) | 1999-05-21 | 2001-04-24 | Rosetta Inpharmatics, Inc. | Essential genes of yeast as targets for antifungal agents, herbicides, insecticides and anti-proliferative drugs |
US7396905B1 (en) | 1999-05-21 | 2008-07-08 | Mckeon Frank | Calcipressins: endogenous inhibitors of calcineurin, uses and reagents related thereto |
US6200803B1 (en) | 1999-05-21 | 2001-03-13 | Rosetta Inpharmatics, Inc. | Essential genes of yeast as targets for antifungal agents, herbicides, insecticides and anti-proliferative drugs |
US6197517B1 (en) | 1999-05-21 | 2001-03-06 | Rosetta Inpharmatics, Inc. | Essential genes of yeast as targets for antifungal agents, herbicides, insecticides and anti-proliferative drugs |
US20030166003A1 (en) * | 1999-06-14 | 2003-09-04 | Cochran Andrea G. | Structured peptide scaffold for displaying turn libraries on phage |
JP2003502304A (en) | 1999-06-14 | 2003-01-21 | ジェネンテック・インコーポレーテッド | Structured peptide scaffolds for displaying turn libraries on phage |
US7291714B1 (en) * | 1999-06-30 | 2007-11-06 | Millennium Pharmaceuticals, Inc. | Glycoprotein VI and uses thereof |
US20040001826A1 (en) | 1999-06-30 | 2004-01-01 | Millennium Pharmaceuticals, Inc. | Glycoprotein VI and uses thereof |
ES2327382T3 (en) | 1999-07-20 | 2009-10-29 | Morphosys Ag | METHODS FOR SUBMITTING (POLI) PEPTIDES / PROTEINS IN PARTICLES OF BACTERIOPHAGES THROUGH DISULFIDE LINKS. |
HU228477B1 (en) | 1999-08-23 | 2013-03-28 | Dana Farber Cancer Inst Inc | Pd-1, a receptor for b7-4, and uses therefor |
DE19943743C2 (en) * | 1999-09-03 | 2002-02-07 | Jerini Biotools Gmbh | Procedure for the identification of binding partners with position-specific arrays |
JP4931310B2 (en) | 1999-10-20 | 2012-05-16 | ジェネンテック, インコーポレイテッド | Regulation of T cell differentiation for the treatment of helper T cell diseases |
US20060073509A1 (en) * | 1999-11-18 | 2006-04-06 | Michael Kilpatrick | Method for detecting and quantitating multiple subcellular components |
US6951839B1 (en) | 1999-11-30 | 2005-10-04 | Curis, Inc. | Methods and compositions for regulating lymphocyte activity |
US8168178B2 (en) | 1999-11-30 | 2012-05-01 | Curis, Inc. | Methods and compositions for regulating lymphocyte activity |
JP2003516755A (en) * | 1999-12-15 | 2003-05-20 | ジェネンテック・インコーポレーテッド | Shotgun scanning, a combined method for mapping functional protein epitopes |
US20020004247A1 (en) * | 1999-12-23 | 2002-01-10 | Genentech, Inc. | Assay method |
WO2001049717A2 (en) | 1999-12-30 | 2001-07-12 | President And Fellows Of Harvard College | Methods and compositions relating to modulation of hepatocyte growth, plasma cell differentiation or t cell subset activity by modulation of xbp-1 activity |
JP2004500086A (en) | 2000-02-10 | 2004-01-08 | アボット・ラボラトリーズ | Antibodies that bind to human interleukin 18, and methods for their preparation and use |
US6632645B1 (en) | 2000-03-02 | 2003-10-14 | Promega Corporation | Thermophilic DNA polymerases from Thermoactinomyces vulgaris |
US6436677B1 (en) | 2000-03-02 | 2002-08-20 | Promega Corporation | Method of reverse transcription |
US20030129724A1 (en) | 2000-03-03 | 2003-07-10 | Grozinger Christina M. | Class II human histone deacetylases, and uses related thereto |
JP2003531588A (en) | 2000-04-11 | 2003-10-28 | ジェネンテック・インコーポレーテッド | Multivalent antibodies and their uses |
EP1832599A3 (en) | 2000-04-12 | 2007-11-21 | Human Genome Sciences, Inc. | Albumin fusion proteins |
US8288322B2 (en) | 2000-04-17 | 2012-10-16 | Dyax Corp. | Methods of constructing libraries comprising displayed and/or expressed members of a diverse family of peptides, polypeptides or proteins and the novel libraries |
CA2406236C (en) * | 2000-04-17 | 2013-02-19 | Dyax Corp. | Novel methods of constructing libraries of genetic packages that collectively display the members of a diverse family of peptides, polypeptides or proteins |
WO2001081375A2 (en) * | 2000-04-24 | 2001-11-01 | Yale University | Dna & protein binding miniature proteins |
US7495070B2 (en) * | 2000-04-24 | 2009-02-24 | Yale University | Protein binding miniature proteins |
AU2001263215A1 (en) | 2000-05-16 | 2001-11-26 | Genentech Inc. | Method for treating cartilage disorders |
EP1714661A3 (en) | 2000-05-19 | 2012-03-14 | The Center for Blood Research, INC. | Methods for diagnosing and treating hemostatic disorders by modulating p-selectin activity |
US6573370B1 (en) | 2000-05-19 | 2003-06-03 | Regents Of The University Of Michigan | PON3 and uses thereof |
US20030031675A1 (en) | 2000-06-06 | 2003-02-13 | Mikesell Glen E. | B7-related nucleic acids and polypeptides useful for immunomodulation |
JP2004502412A (en) | 2000-06-08 | 2004-01-29 | ザ センター フォー ブラッド リサーチ インコーポレーティッド | Methods and compositions for inhibiting immunoglobulin-mediated reperfusion injury |
CA2413160A1 (en) | 2000-06-15 | 2001-12-20 | Human Genome Sciences, Inc. | Human tumor necrosis factor delta and epsilon |
PT2281843T (en) | 2000-06-16 | 2017-01-02 | Human Genome Sciences Inc | Antibodies that immunospecifically bind to blys |
CZ20024214A3 (en) * | 2000-06-28 | 2003-04-16 | Bristol-Myers Squibb Company | Selective androgen receptor modulator, method of its identification, its construction, and use |
NZ522844A (en) | 2000-06-28 | 2005-02-25 | Brigham & Womens Hospital | PD-L2 molecules: novel PD-1 ligands and methods to identify compounds to modulate T cell activation |
CN102120773B (en) | 2000-06-29 | 2013-07-24 | Abbvie公司 | Dual specificity antibodies and methods of making and using |
US6951947B2 (en) * | 2000-07-13 | 2005-10-04 | The Scripps Research Institute | Labeled peptides, proteins and antibodies and processes and intermediates useful for their preparation |
US7176037B2 (en) * | 2000-07-13 | 2007-02-13 | The Scripps Research Institute | Labeled peptides, proteins and antibodies and processes and intermediates useful for their preparation |
DE60143986D1 (en) | 2000-07-14 | 2011-03-17 | Cropdesign Nv | INHIBITORS FOR CYCLIN DEPENDENT KINASE OF PLANTS |
US6878861B2 (en) | 2000-07-21 | 2005-04-12 | Washington State University Research Foundation | Acyl coenzyme A thioesterases |
AU2001279055A1 (en) | 2000-07-27 | 2002-02-13 | Genentech, Inc. | Apo-2L receptor agonist and CPT-11 synergism |
US6984522B2 (en) * | 2000-08-03 | 2006-01-10 | Regents Of The University Of Michigan | Isolation and use of solid tumor stem cells |
US8044259B2 (en) | 2000-08-03 | 2011-10-25 | The Regents Of The University Of Michigan | Determining the capability of a test compound to affect solid tumor stem cells |
US20080194022A1 (en) * | 2000-08-03 | 2008-08-14 | Clarke Michael F | Isolation and use of solid tumor stem cells |
US6902734B2 (en) | 2000-08-07 | 2005-06-07 | Centocor, Inc. | Anti-IL-12 antibodies and compositions thereof |
US7288390B2 (en) | 2000-08-07 | 2007-10-30 | Centocor, Inc. | Anti-dual integrin antibodies, compositions, methods and uses |
AU2001281113B2 (en) | 2000-08-07 | 2006-07-20 | Nektar Therapeutics | Inhaleable spray dried 4-helix bundle protein powders having minimized aggregation |
UA81743C2 (en) | 2000-08-07 | 2008-02-11 | Центокор, Инк. | HUMAN MONOCLONAL ANTIBODY WHICH SPECIFICALLY BINDS TUMOR NECROSIS FACTOR ALFA (TNFα), PHARMACEUTICAL MIXTURE CONTAINING THEREOF, AND METHOD FOR TREATING ARTHRITIS |
AU2001289079A1 (en) * | 2000-09-13 | 2002-03-26 | Praecis Pharmaceuticals Incorporated | Pharmaceutical compositions for sustained drug delivery |
GB0022978D0 (en) | 2000-09-19 | 2000-11-01 | Oxford Glycosciences Uk Ltd | Detection of peptides |
AU2002214545B2 (en) | 2000-09-26 | 2008-04-03 | Genentech, Inc. | IGE receptor antagonists |
US7393532B1 (en) | 2000-10-18 | 2008-07-01 | Genentech, Inc. | Modulation of T cell differentiation for the treatment of T helper cell mediated diseases |
US6673580B2 (en) * | 2000-10-27 | 2004-01-06 | Genentech, Inc. | Identification and modification of immunodominant epitopes in polypeptides |
US6841359B2 (en) | 2000-10-31 | 2005-01-11 | The General Hospital Corporation | Streptavidin-binding peptides and uses thereof |
CA2427820A1 (en) | 2000-11-01 | 2002-06-13 | Elusys Therapeutics, Inc. | Method of producing biospecific molecules by protein trans-splicing |
EP1356108A2 (en) | 2000-11-28 | 2003-10-29 | Wyeth | Expression analysis of fkbp nucleic acids and polypeptides useful in the diagnosis and treatment of prostate cancer |
TWI327599B (en) | 2000-11-28 | 2010-07-21 | Medimmune Llc | Methods of administering/dosing anti-rsv antibodies for prophylaxis and treatment |
AU3650302A (en) | 2000-11-28 | 2002-06-11 | Wyeth Corp | Expression analysis of kiaa nucleic acids and polypeptides useful in the diagnosis and treatment of prostate cancer |
US20040253242A1 (en) * | 2000-12-05 | 2004-12-16 | Bowdish Katherine S. | Rationally designed antibodies |
US7396917B2 (en) * | 2000-12-05 | 2008-07-08 | Alexion Pharmaceuticals, Inc. | Rationally designed antibodies |
PT1642910E (en) * | 2000-12-05 | 2012-03-22 | Alexion Pharma Inc | Rationally designed antibodies |
US9249229B2 (en) * | 2000-12-08 | 2016-02-02 | Alexion Pharmaceuticals, Inc. | Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof |
US20060057651A1 (en) | 2000-12-08 | 2006-03-16 | Bowdish Katherine S | Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof |
WO2002059280A2 (en) * | 2000-12-08 | 2002-08-01 | Alexion Pharmaceuticals, Inc. | Chronic lymphocytic leukemia cell line and its use for producing an antibody |
US7408041B2 (en) | 2000-12-08 | 2008-08-05 | Alexion Pharmaceuticals, Inc. | Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof |
US20040198661A1 (en) * | 2000-12-08 | 2004-10-07 | Bowdish Katherine S. | Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof |
AU2002248184C1 (en) | 2000-12-12 | 2018-01-04 | Board Of Regents, The University Of Texas System | Molecules with extended half-lives, compositions and uses thereof |
DE60144063D1 (en) | 2000-12-18 | 2011-03-31 | Dyax Corp | DIRECTED LIBRARIES GENETICALLY PACKAGED |
US8372954B2 (en) * | 2000-12-22 | 2013-02-12 | National Research Council Of Canada | Phage display libraries of human VH fragments |
AU2002231736A1 (en) | 2000-12-22 | 2002-07-08 | Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. | Use of repulsive guidance molecule (rgm) and its modulators |
MY143465A (en) | 2001-01-05 | 2011-05-13 | Pfizer | Antibodies to insulin-like growth factor i receptor |
EP1366058B1 (en) | 2001-02-09 | 2011-01-26 | Human Genome Sciences, Inc. | Human g-protein chemokine receptor (ccr5) hdgnr10 |
US7087726B2 (en) | 2001-02-22 | 2006-08-08 | Genentech, Inc. | Anti-interferon-α antibodies |
CN100582226C (en) | 2001-02-23 | 2010-01-20 | Dsmip资产有限公司 | Genes encoding proteolytic enzymes from aspargilli |
ES2360205T3 (en) | 2001-03-02 | 2011-06-01 | Agennix Ag | THREE HYBRID TEST SYSTEM. |
WO2002098370A2 (en) * | 2001-03-02 | 2002-12-12 | Medimmune, Inc. | Methods of administering/dosing cd2 antagonists for the prevention and treatment of autoimmune disorders or inflammatory disorders |
US8231878B2 (en) * | 2001-03-20 | 2012-07-31 | Cosmo Research & Development S.P.A. | Receptor trem (triggering receptor expressed on myeloid cells) and uses thereof |
US20090081199A1 (en) * | 2001-03-20 | 2009-03-26 | Bioxell S.P.A. | Novel receptor trem (triggering receptor expressed on myeloid cells) and uses thereof |
US8981061B2 (en) | 2001-03-20 | 2015-03-17 | Novo Nordisk A/S | Receptor TREM (triggering receptor expressed on myeloid cells) and uses thereof |
CA2342376C (en) * | 2001-03-20 | 2013-11-12 | Marco Colonna | A receptor trem (triggering receptor expressed on myeloid cells) and uses thereof |
WO2002076196A1 (en) | 2001-03-22 | 2002-10-03 | Abbott Gmbh & Co. Kg | Transgenic animals expressing antibodies specific for genes of interest and uses thereof |
NZ528265A (en) | 2001-04-02 | 2005-10-28 | Wyeth Corp | Screening of compounds which modulate PD-1 signalling by testing for compounds that modulate phosphorylation of SHP-2, ERK1 or ERK-2, and PKC-theta |
EP2228389B1 (en) | 2001-04-13 | 2015-07-08 | Human Genome Sciences, Inc. | Antibodies against vascular endothelial growth factor 2 |
EP2258718A1 (en) | 2001-04-16 | 2010-12-08 | Wyeth Holdings Corporation | Streptococcus pneumoniae open reading frames encoding polypeptide antigens and uses thereof |
US6914123B2 (en) | 2001-04-17 | 2005-07-05 | Genentech, Inc. | Hairpin peptides with a novel structural motif and methods relating thereto |
US7244853B2 (en) | 2001-05-09 | 2007-07-17 | President And Fellows Of Harvard College | Dioxanes and uses thereof |
US7064189B2 (en) | 2001-05-25 | 2006-06-20 | Human Genome Sciences, Inc. | Antibodies that immunospecifically bind to trail receptors |
AU2002322033A1 (en) * | 2001-06-04 | 2002-12-16 | Ikonisys Inc. | Method for detecting infectious agents using computer controlled automated image analysis |
US20030113714A1 (en) * | 2001-09-28 | 2003-06-19 | Belcher Angela M. | Biological control of nanoparticles |
US20030148380A1 (en) * | 2001-06-05 | 2003-08-07 | Belcher Angela M. | Molecular recognition of materials |
US20050164515A9 (en) * | 2001-06-05 | 2005-07-28 | Belcher Angela M. | Biological control of nanoparticle nucleation, shape and crystal phase |
US20070160576A1 (en) | 2001-06-05 | 2007-07-12 | Genentech, Inc. | IL-17A/F heterologous polypeptides and therapeutic uses thereof |
CA2817619A1 (en) | 2001-06-08 | 2002-12-08 | Abbott Laboratories (Bermuda) Ltd. | Methods of administering anti-tnf.alpha. antibodies |
US7803915B2 (en) * | 2001-06-20 | 2010-09-28 | Genentech, Inc. | Antibody compositions for the diagnosis and treatment of tumor |
DK2000545T3 (en) | 2001-06-20 | 2011-11-28 | Genentech Inc | Compositions and methods for diagnosis and treatment of lung tumor |
US20050107595A1 (en) * | 2001-06-20 | 2005-05-19 | Genentech, Inc. | Compositions and methods for the diagnosis and treatment of tumor |
MXPA03011890A (en) | 2001-06-22 | 2004-06-03 | Du Pont | Defensin polynucleotides and methods of use. |
US20030198621A1 (en) * | 2001-07-05 | 2003-10-23 | Megede Jan Zur | Polynucleotides encoding antigenic HIV type B and/or type C polypeptides, polypeptides and uses thereof |
CA2634992C (en) | 2001-07-05 | 2012-10-16 | Novartis Vaccines And Diagnostics, Inc. | Polynucleotides encoding antigenic hiv type c polypeptides, polypeptides and uses thereof |
DE10135039C1 (en) * | 2001-07-18 | 2003-03-13 | Nemod Immuntherapie Ag | Method for isolating large variances of specific molecules for a target molecule from phagemid gene libraries |
US6867189B2 (en) * | 2001-07-26 | 2005-03-15 | Genset S.A. | Use of adipsin/complement factor D in the treatment of metabolic related disorders |
KR100458083B1 (en) * | 2001-08-29 | 2004-11-18 | 주식회사 아이지세라피 | Method for the construction of phage display library using helper phage variants |
US20030170614A1 (en) * | 2001-08-31 | 2003-09-11 | Megede Jan Zur | Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides and uses thereof |
AU2002347404A1 (en) * | 2001-09-14 | 2003-04-01 | Cytos Biotechnology Ag | In vivo activation of antigen presenting cells for enhancement of immune responses induced by virus like particles |
US20040142325A1 (en) | 2001-09-14 | 2004-07-22 | Liat Mintz | Methods and systems for annotating biomolecular sequences |
CA2460120A1 (en) | 2001-09-18 | 2003-03-27 | Genentech, Inc. | Compositions and methods for the diagnosis and treatment of tumor |
US20030073104A1 (en) * | 2001-10-02 | 2003-04-17 | Belcher Angela M. | Nanoscaling ordering of hybrid materials using genetically engineered mesoscale virus |
US20050123925A1 (en) | 2002-11-15 | 2005-06-09 | Genentech, Inc. | Compositions and methods for the diagnosis and treatment of tumor |
AR039067A1 (en) | 2001-11-09 | 2005-02-09 | Pfizer Prod Inc | ANTIBODIES FOR CD40 |
US7858297B2 (en) | 2001-12-18 | 2010-12-28 | Centre National De La Recherche Scientifique Cnrs | Chemokine-binding protein and methods of use |
AU2002361385B2 (en) | 2001-12-18 | 2009-11-19 | Centre National De La Recherche Scientifique Cnrs | Novel death associated proteins of the THAP family and related Par4 pathways involved in apoptosis control |
EP1463807A4 (en) | 2001-12-19 | 2006-04-12 | Bristol Myers Squibb Co | Pichia pastoris formate dehydrogenase and uses therefor |
DK1463751T3 (en) | 2001-12-21 | 2013-08-26 | Human Genome Sciences Inc | Albumin Fusion Proteins. |
JP2005525095A (en) | 2002-01-02 | 2005-08-25 | ジェネンテック・インコーポレーテッド | Compositions and methods for tumor diagnosis and treatment |
AU2003207459A1 (en) * | 2002-01-03 | 2003-07-24 | The Scripps Research Institute | Cancer-associated epitope |
US20070166704A1 (en) * | 2002-01-18 | 2007-07-19 | Fei Huang | Identification of polynucleotides and polypeptide for predicting activity of compounds that interact with protein tyrosine kinases and/or protein tyrosine kinase pathways |
WO2003104428A2 (en) | 2002-01-21 | 2003-12-18 | Vaccinex, Inc. | Gene differentially expressed in breast and bladder cancer and encoded polypeptides |
US7094579B2 (en) | 2002-02-13 | 2006-08-22 | Xoma Technology Ltd. | Eukaryotic signal sequences for prokaryotic expression |
EP1489968B1 (en) * | 2002-03-01 | 2009-02-18 | Siemens Healthcare Diagnostics Inc. | Assays for cancer patient monitoring based on levels of analyte components of the plasminogen activator system in body fluid samples |
DE60329020D1 (en) | 2002-03-01 | 2009-10-08 | Siemens Healthcare Diagnostics | ASSAYS FOR THE MONITORING OF CANCER PATIENTS BASED ON THE MIRROR OF THE EXTRACELLULAR DOMAIN (ECD) ANALYSIS OF THE EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR), ALONE OR IN COMBINATION WITH OTHER ANALYTES, IN SAMPLES FROM BODY FLUIDS |
JP2005531511A (en) | 2002-03-07 | 2005-10-20 | ザ フォーシス インスティチュート | Immunogenicity of glucan binding proteins |
AU2003218456A1 (en) * | 2002-04-01 | 2003-10-20 | Human Genome Sciences, Inc. | Antibodies that specifically bind to gmad |
GB0207533D0 (en) | 2002-04-02 | 2002-05-08 | Oxford Glycosciences Uk Ltd | Protein |
US7745192B2 (en) | 2002-04-03 | 2010-06-29 | Venomics Pty Limited | Prothrombin activating protein |
JP2005535572A (en) | 2002-04-12 | 2005-11-24 | メディミューン,インコーポレーテッド | Recombinant anti-interleukin-9 antibody |
AU2003230874A1 (en) | 2002-04-16 | 2003-11-03 | Genentech, Inc. | Compositions and methods for the diagnosis and treatment of tumor |
US20030206898A1 (en) | 2002-04-26 | 2003-11-06 | Steven Fischkoff | Use of anti-TNFalpha antibodies and another drug |
DE60333629D1 (en) | 2002-05-21 | 2010-09-16 | Dsm Ip Assets Bv | NEW PHOSPHOLIPASES AND ITS USES |
EP2305710A3 (en) | 2002-06-03 | 2013-05-29 | Genentech, Inc. | Synthetic antibody phage libraries |
AU2003239966B9 (en) * | 2002-06-03 | 2010-08-26 | Genentech, Inc. | Synthetic antibody phage libraries |
WO2003106640A2 (en) * | 2002-06-14 | 2003-12-24 | Case Western Reserve University | Cell targeting methods and compositions |
US7425618B2 (en) | 2002-06-14 | 2008-09-16 | Medimmune, Inc. | Stabilized anti-respiratory syncytial virus (RSV) antibody formulations |
AU2003247582A1 (en) | 2002-06-20 | 2004-01-06 | Board Of Trustees Operating Michigan State University | Plastid division and related genes and proteins, and methods of use |
USRE47770E1 (en) | 2002-07-18 | 2019-12-17 | Merus N.V. | Recombinant production of mixtures of antibodies |
ES2442615T5 (en) | 2002-07-18 | 2023-03-16 | Merus Nv | Recombinant production of antibody mixtures |
PT1944322E (en) | 2002-07-19 | 2015-07-01 | Abbvie Biotechnology Ltd | Treatment of tnf alpha related disorders |
US7250551B2 (en) | 2002-07-24 | 2007-07-31 | President And Fellows Of Harvard College | Transgenic mice expressing inducible human p25 |
ES2346868T3 (en) | 2002-08-10 | 2010-10-21 | Yale University | NOGO RECEIVER ANTAGONISTS. |
US20040067532A1 (en) * | 2002-08-12 | 2004-04-08 | Genetastix Corporation | High throughput generation and affinity maturation of humanized antibody |
ES2381617T5 (en) | 2002-08-14 | 2016-02-24 | Macrogenics, Inc. | Specific antibodies against FcgammaRIIB and its procedures for use |
EP2338986B1 (en) | 2002-08-19 | 2014-10-29 | DSM IP Assets B.V. | Novel lipases and uses thereof |
DE10238846A1 (en) * | 2002-08-20 | 2004-03-04 | Nemod Immuntherapie Ag | Active fusion proteins and processes for their production |
US7361511B2 (en) | 2002-08-20 | 2008-04-22 | Millenium Pharmaceuticals, Inc. | Compositions, kits, and methods for identification, assessment, prevention, and therapy of cervical cancer |
US7455989B2 (en) | 2002-08-20 | 2008-11-25 | Yeda Research And Development Co. Ltd. | AKAP84 and its use for visualization of biological structures |
JP2006512901A (en) * | 2002-08-29 | 2006-04-20 | ジェネンテック・インコーポレーテッド | Achaete-Scute-like-2 polypeptide and its encoding nucleic acid and methods for tumor diagnosis and treatment |
JP2006502167A (en) | 2002-09-04 | 2006-01-19 | バイオポリマー エンジニアリング,インコーポレイテッド | Cancer treatment using whole glucan particles and antibodies |
AU2003263552A1 (en) * | 2002-09-09 | 2004-03-29 | Nautilus Biotech | Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules |
US20050064508A1 (en) * | 2003-09-22 | 2005-03-24 | Semzyme | Peptide mediated synthesis of metallic and magnetic materials |
WO2004032870A2 (en) | 2002-10-08 | 2004-04-22 | Rinat Neuroscience Corp. | Methods for treating post-surgical pain by admisnistering a nerve growth factor antagonist and compositions containing the same |
US7255860B2 (en) | 2002-10-08 | 2007-08-14 | Rinat Neuroscience Corp. | Methods for treating post-surgical pain by administering an anti-nerve growth factor antagonist antibody |
US20040146512A1 (en) * | 2002-10-09 | 2004-07-29 | Arnon Rosenthal | Methods of treating Alzheimer's disease using antibodies directed against amyloid beta peptide and compositions thereof |
SI2891666T1 (en) | 2002-10-16 | 2017-11-30 | Purdue Pharma L.P. | Antibodies that bind cell-associated CA 125/O722P and methods of use thereof |
WO2004050683A2 (en) * | 2002-12-02 | 2004-06-17 | Abgenix, Inc. | Antibodies directed to tumor necrosis factor and uses thereof |
US7056702B2 (en) * | 2002-12-16 | 2006-06-06 | Kimberly Clark Co | Detecting lipocalin |
WO2004058968A1 (en) * | 2002-12-23 | 2004-07-15 | Apalexo Biotechnologie Gmbh | Purification of recombinant filamental bacteriophages by means of affinity chromatography to increase the immunogenicity and efficacy of phagic vaccines |
NZ540879A (en) * | 2002-12-23 | 2008-08-29 | Rinat Neuroscience Corp | Methods for treating taxol-induced sensory neuropathy |
US7569364B2 (en) * | 2002-12-24 | 2009-08-04 | Pfizer Inc. | Anti-NGF antibodies and methods using same |
PT1575517E (en) | 2002-12-24 | 2012-05-28 | Rinat Neuroscience Corp | Anti-ngf antibodies and methods using same |
US9498530B2 (en) | 2002-12-24 | 2016-11-22 | Rinat Neuroscience Corp. | Methods for treating osteoarthritis pain by administering a nerve growth factor antagonist and compositions containing the same |
EP2290107A1 (en) | 2003-01-07 | 2011-03-02 | Dyax Corporation | Kunitz domain library |
WO2004065416A2 (en) * | 2003-01-16 | 2004-08-05 | Genentech, Inc. | Synthetic antibody phage libraries |
WO2004065551A2 (en) * | 2003-01-21 | 2004-08-05 | Bristol-Myers Squibb Company | Polynucleotide encoding a novel acyl coenzyme a, monoacylglycerol acyltransferase-3 (mgat3), and uses thereof |
CA2514564A1 (en) | 2003-01-31 | 2005-07-26 | Promega Corporation | Covalent tethering of functional groups to proteins |
DE10303974A1 (en) | 2003-01-31 | 2004-08-05 | Abbott Gmbh & Co. Kg | Amyloid β (1-42) oligomers, process for their preparation and their use |
US7429472B2 (en) * | 2003-01-31 | 2008-09-30 | Promega Corporation | Method of immobilizing a protein or molecule via a mutant dehalogenase that is bound to an immobilized dehalogenase substrate and linked directly or indirectly to the protein or molecule |
US7531169B2 (en) * | 2003-02-01 | 2009-05-12 | Tanox, Inc. | High affinity anti-human IgE antibodies |
US7655231B2 (en) * | 2003-02-19 | 2010-02-02 | Pfizer Inc. | Methods for treating pain by administering a nerve growth factor antagonist and an NSAID |
JP5356648B2 (en) | 2003-02-20 | 2013-12-04 | シアトル ジェネティックス, インコーポレイテッド | Anti-CD70 antibody-drug conjugates and their use for the treatment of cancer and immune disorders |
US20040180387A1 (en) | 2003-03-13 | 2004-09-16 | Fujirebio Diagnostics, Inc. | Detection of urinary mesothelin-/megakaryocyte potentiating factor-related peptides for assessment of ovarian cancer |
EP2248899B8 (en) * | 2003-03-19 | 2015-07-15 | Biogen MA Inc. | NOGO receptor binding protein |
US20070014786A1 (en) * | 2003-03-20 | 2007-01-18 | Rinat Neuroscience Corp. | Methods for treating taxol-induced gut disorder |
US7294701B2 (en) * | 2003-04-02 | 2007-11-13 | Technion Research & Development Foundation Ltd. | Antibody fragment capable of modulating multidrug resistance and compositions and kits and methods using same |
CA2521826C (en) | 2003-04-11 | 2013-08-06 | Jennifer L. Reed | Recombinant il-9 antibodies and uses thereof |
TWI353991B (en) | 2003-05-06 | 2011-12-11 | Syntonix Pharmaceuticals Inc | Immunoglobulin chimeric monomer-dimer hybrids |
US20050014932A1 (en) | 2003-05-15 | 2005-01-20 | Iogenetics, Llc | Targeted biocides |
ES2408582T3 (en) | 2003-05-30 | 2013-06-21 | Merus B.V. | Fab library for the preparation of a mixture of antibodies |
JP2007525466A (en) | 2003-05-30 | 2007-09-06 | ジェネンテック・インコーポレーテッド | Treatment with anti-VEGF antibody |
US20100069614A1 (en) | 2008-06-27 | 2010-03-18 | Merus B.V. | Antibody producing non-human mammals |
US9708410B2 (en) | 2003-05-30 | 2017-07-18 | Janssen Biotech, Inc. | Anti-tissue factor antibodies and compositions |
JP2007526447A (en) | 2003-06-06 | 2007-09-13 | ジェネンテック・インコーポレーテッド | Regulation of interaction between HGF β chain and C-MET |
EP1635763B1 (en) | 2003-06-09 | 2012-08-08 | Alnylam Pharmaceuticals Inc. | Method of treating neurodegenerative disease |
US20060019256A1 (en) * | 2003-06-09 | 2006-01-26 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing cancer |
PL1641822T3 (en) | 2003-07-08 | 2013-10-31 | Genentech Inc | Il-17 a/f heterologous polypeptides and therapeutic uses thereof |
EP1644408A1 (en) * | 2003-07-15 | 2006-04-12 | Barros Research Institute | Eimeria tenella antigen for immunotherapy of coccidiosis |
WO2005010163A2 (en) * | 2003-07-15 | 2005-02-03 | Barros Research Institute | Compositions and methods for immunotherapy of human immunotherapy of human immunodeficiency virus (hiv) |
US7727752B2 (en) | 2003-07-29 | 2010-06-01 | Life Technologies Corporation | Kinase and phosphatase assays |
US20050106667A1 (en) | 2003-08-01 | 2005-05-19 | Genentech, Inc | Binding polypeptides with restricted diversity sequences |
US7758859B2 (en) * | 2003-08-01 | 2010-07-20 | Genentech, Inc. | Anti-VEGF antibodies |
HN2004000285A (en) | 2003-08-04 | 2006-04-27 | Pfizer Prod Inc | ANTIBODIES DIRECTED TO c-MET |
AU2004263896A1 (en) | 2003-08-08 | 2005-02-17 | Genenews Inc. | Osteoarthritis biomarkers and uses thereof |
CA2536238C (en) | 2003-08-18 | 2015-04-07 | Medimmune, Inc. | Humanization of antibodies |
CA2537984A1 (en) * | 2003-09-05 | 2005-03-17 | The Scripps Research Institute | Detection of cholesterol ozonation products |
JP2007504244A (en) * | 2003-09-05 | 2007-03-01 | ザ スクリプス リサーチ インスティテュート | Cholesterol ozonation products for the treatment and prevention of atherosclerosis and / or cardiovascular disease |
AR045563A1 (en) | 2003-09-10 | 2005-11-02 | Warner Lambert Co | ANTIBODIES DIRECTED TO M-CSF |
KR20060120652A (en) | 2003-10-07 | 2006-11-27 | 밀레니엄 파머슈티컬스 인코퍼레이티드 | Nucleic acid molecules and proteins for the identification, assessment, prevention, and therapy of ovarian cancer |
IL158287A0 (en) | 2003-10-07 | 2004-05-12 | Yeda Res & Dev | Antibodies to nik, their preparation and use |
EP1677735B1 (en) | 2003-10-17 | 2014-07-23 | Joslin Diabetes Center, Inc. | Methods and compositions for modulating adipocyte function |
US7329725B1 (en) * | 2003-10-29 | 2008-02-12 | Nastech Pharmaceutical Company Inc. | Phage displayed Trp cage ligands |
GB0325836D0 (en) * | 2003-11-05 | 2003-12-10 | Celltech R&D Ltd | Biological products |
CA2545603A1 (en) | 2003-11-12 | 2005-05-26 | Biogen Idec Ma Inc. | Neonatal fc receptor (fcrn)-binding polypeptide variants, dimeric fc binding proteins and methods related thereto |
US20050100965A1 (en) | 2003-11-12 | 2005-05-12 | Tariq Ghayur | IL-18 binding proteins |
CA2747871C (en) | 2003-11-17 | 2018-04-10 | Genentech, Inc. | Compositions and methods for the treatment of tumor of hematopoietic origin |
ES2305879T3 (en) | 2003-11-21 | 2008-11-01 | Ucb Pharma, S.A. | METHOD FOR THE TREATMENT OF MULTIPLE SCLEROSIS BY INHIBITION OF ACTIVITY IL-17. |
JP5042631B2 (en) * | 2003-12-04 | 2012-10-03 | バクシネックス インコーポレーティッド | Method of killing tumor cells by targeting intracellular antigens exposed on apoptotic tumor cells |
CA2548282A1 (en) | 2003-12-11 | 2005-06-30 | Genentech, Inc. | Methods and compositions for inhibiting c-met dimerization and activation |
GB0329825D0 (en) * | 2003-12-23 | 2004-01-28 | Celltech R&D Ltd | Biological products |
CA2551097A1 (en) | 2003-12-23 | 2005-07-14 | Rinat Neuroscience Corp. | Agonist anti-trkc antibodies and methods using same |
US20050266425A1 (en) * | 2003-12-31 | 2005-12-01 | Vaccinex, Inc. | Methods for producing and identifying multispecific antibodies |
KR20120048644A (en) | 2004-01-09 | 2012-05-15 | 암젠 프레몬트 인코포레이티드 | Antibodies to madcam |
US20060148680A1 (en) | 2004-01-14 | 2006-07-06 | Kieliszewski Marcia J | Glycoproteins produced in plants and methods of their use |
JP5912211B2 (en) | 2004-01-20 | 2016-04-27 | メルス ビー.ヴィー. | Mixture of binding proteins |
EP1761561B1 (en) | 2004-01-20 | 2015-08-26 | KaloBios Pharmaceuticals, Inc. | Antibody specificity transfer using minimal essential binding determinants |
WO2005070468A2 (en) | 2004-01-21 | 2005-08-04 | Novo Nordisk A/S | Transglutaminase mediated conjugation of peptides |
EP1714151A4 (en) | 2004-01-21 | 2009-06-10 | Fujirebio America Inc | Detection of mesothelin-/megakaryocyte potentiating factor-related peptides in peritoneal fluid for assessment of the peritoneum and the peritoneal cavity |
AU2005211362B2 (en) | 2004-02-02 | 2008-03-13 | Ambrx, Inc. | Modified human interferon polypeptides and their uses |
WO2005074633A2 (en) * | 2004-02-03 | 2005-08-18 | The Regents Of The University Of Michigan | Compositions and methods for characterizing, regulating, diagnosing, and treating cancer |
DK1729795T3 (en) | 2004-02-09 | 2016-04-11 | Human Genome Sciences Inc | Albumin fusion proteins |
ATE452147T1 (en) | 2004-02-19 | 2010-01-15 | Genentech Inc | ANTIBODIES WITH CORRECTED CDR |
CA2560066A1 (en) | 2004-03-01 | 2005-09-15 | The Cbr Institute For Biomedical Research, Inc. | Natural igm antibodies and inhibitors thereof |
US7592188B2 (en) * | 2004-03-12 | 2009-09-22 | The Scripps Research Institute | Live cell biosensors |
KR20110027823A (en) | 2004-03-24 | 2011-03-16 | 트리패스 이미징, 인코포레이티드 | Methods and compositions for the detection of cervical disease |
US7973139B2 (en) * | 2004-03-26 | 2011-07-05 | Human Genome Sciences, Inc. | Antibodies against nogo receptor |
NZ550217A (en) | 2004-03-31 | 2009-11-27 | Genentech Inc | Humanized anti-TGF-beta antibodies |
CA2562024C (en) * | 2004-04-07 | 2014-05-27 | Rinat Neuroscience Corp. | Methods for treating bone cancer pain by administering a nerve growth factor antagonist |
US7794713B2 (en) | 2004-04-07 | 2010-09-14 | Lpath, Inc. | Compositions and methods for the treatment and prevention of hyperproliferative diseases |
US7785903B2 (en) * | 2004-04-09 | 2010-08-31 | Genentech, Inc. | Variable domain library and uses |
TWI439284B (en) | 2004-04-09 | 2014-06-01 | Abbvie Biotechnology Ltd | Multiple-variable dose regimen for treating tnfα-related disorders |
EP1745149A4 (en) | 2004-04-15 | 2008-08-06 | Univ Florida | Neural proteins as biomarkers for nervous system injury and other neural disorders |
EP1735336A2 (en) * | 2004-04-16 | 2006-12-27 | Genentech, Inc. | Omi pdz modulators |
WO2005110015A2 (en) | 2004-04-19 | 2005-11-24 | Ohio University | Cross-linkable glycoproteins and methods of making the same |
JP2008501638A (en) | 2004-04-23 | 2008-01-24 | ドイツ連邦共和国 | Method of treating T cell mediated pathology by in vivo depletion of ICOS positive cells |
EP1789453A2 (en) * | 2004-05-18 | 2007-05-30 | Genentech, Inc. | M13 virus major coat protein variants for c-terminal and bi-terminal display of a heterologous protein |
EP1602928A1 (en) * | 2004-06-01 | 2005-12-07 | Universiteit Maastricht | Process and kit for determining binding parameters of bioaffinity binding reactions |
WO2005118810A1 (en) | 2004-06-03 | 2005-12-15 | Athlomics Pty Ltd | Agents and methods for diagnosing stress |
US20110218118A1 (en) * | 2004-06-03 | 2011-09-08 | Phylogica Limited | Peptide modulators of cellular phenotype and bi-nucleic acid fragment library |
US7604947B2 (en) * | 2004-06-09 | 2009-10-20 | Cornell Research Foundation, Inc. | Detection and modulation of cancer stem cells |
NZ582684A (en) * | 2004-06-18 | 2011-05-27 | Ambrx Inc | Use of an antibody or binding fragment thereof comprising a non naturally encoded amino acid coupled to a linker |
GB0414054D0 (en) | 2004-06-23 | 2004-07-28 | Owen Mumford Ltd | Improvements relating to automatic injection devices |
AU2005258335B2 (en) * | 2004-06-24 | 2011-03-17 | Biogen Ma Inc. | Treatment of conditions involving demyelination |
US6986264B1 (en) * | 2004-07-15 | 2006-01-17 | Carrier Corporation | Economized dehumidification system |
EP2335727A3 (en) | 2004-07-16 | 2011-09-28 | Pfizer Products Inc. | Combination treatment for non-hematologic malignancies using an anti-IGF-1R antibody |
US8604185B2 (en) | 2004-07-20 | 2013-12-10 | Genentech, Inc. | Inhibitors of angiopoietin-like 4 protein, combinations, and their use |
CN101044164A (en) * | 2004-07-20 | 2007-09-26 | 健泰科生物技术公司 | Inhibitors of angiopoietin-like 4 protein, combinations, and their use |
EP1771573A4 (en) * | 2004-07-21 | 2009-02-18 | Ambrx Inc | Biosynthetic polypeptides utilizing non-naturally encoded amino acids |
US7342093B2 (en) | 2004-07-23 | 2008-03-11 | University Of Massachusetts | Compounds that inhibit Hsp90 protein-protein interactions with IAP proteins |
US7425436B2 (en) * | 2004-07-30 | 2008-09-16 | Promega Corporation | Covalent tethering of functional groups to proteins and substrates therefor |
US20070087400A1 (en) * | 2004-07-30 | 2007-04-19 | Aldis Darzins | Covalent tethering of functional groups to proteins and substrates therefor |
AP2007003890A0 (en) * | 2004-07-30 | 2007-02-28 | Rinat Neuroscience Corp | Antibodies directed against amy-loid-beta peptide and methods using same |
EP2329714A1 (en) | 2004-08-03 | 2011-06-08 | Biogen Idec MA Inc. | Influence of TAJ in the neuronal functions |
PT1784426E (en) | 2004-09-03 | 2012-03-06 | Genentech Inc | Humanized anti-beta7 antagonists and uses therefor |
US7700720B2 (en) | 2004-09-21 | 2010-04-20 | Medimmune, Llc | Antibodies against and methods for producing vaccines for respiratory syncytial virus |
WO2006047325A1 (en) | 2004-10-21 | 2006-05-04 | Genentech, Inc. | Method for treating intraocular neovascular diseases |
WO2006047417A2 (en) | 2004-10-21 | 2006-05-04 | University Of Florida Research Foundation, Inc. | Detection of cannabinoid receptor biomarkers and uses thereof |
JP2008518023A (en) | 2004-10-27 | 2008-05-29 | メディミューン,インコーポレーテッド | Regulation of antibody specificity by altering affinity for cognate antigens |
US7998930B2 (en) | 2004-11-04 | 2011-08-16 | Hanall Biopharma Co., Ltd. | Modified growth hormones |
GB0426146D0 (en) | 2004-11-29 | 2004-12-29 | Bioxell Spa | Therapeutic peptides and method |
EP1828250A2 (en) * | 2004-12-16 | 2007-09-05 | Genentech, Inc. | Methods for treating autoimmune disorders |
JP4990792B2 (en) * | 2004-12-22 | 2012-08-01 | アンブレツクス・インコーポレイテツド | Compositions of aminoacyl-tRNA synthetases and uses thereof |
WO2006071840A2 (en) * | 2004-12-22 | 2006-07-06 | Ambrx, Inc. | Formulations of human growth hormone comprising a non-naturally encoded amino acid |
MX2007007591A (en) * | 2004-12-22 | 2007-07-25 | Ambrx Inc | Methods for expression and purification of recombinant human growth hormone. |
JP2008525473A (en) | 2004-12-22 | 2008-07-17 | アンブレツクス・インコーポレイテツド | Modified human growth hormone |
RU2441071C2 (en) | 2004-12-22 | 2012-01-27 | Дженентек, Инк. | Method for producing soluble multipass transmembrane proteins |
EP1772465B1 (en) | 2005-01-05 | 2009-02-18 | f-star Biotechnologische Forschungs- und Entwicklungsges.m.b.H. | Synthetic immunoglobulin domains with binding properties engineered in regions of the molecule different from the complementarity determining regions |
CN101137673B (en) | 2005-01-05 | 2013-12-04 | 比奥根艾迪克Ma公司 | Cripto binding molecules |
GT200600031A (en) | 2005-01-28 | 2006-08-29 | ANTI-BETA ANTIBODY FORMULATION | |
US8029783B2 (en) | 2005-02-02 | 2011-10-04 | Genentech, Inc. | DR5 antibodies and articles of manufacture containing same |
CA2599589A1 (en) | 2005-02-07 | 2006-08-17 | Genenews,Inc. | Mild osteoarthritis biomarkers and uses thereof |
CA2597924C (en) | 2005-02-15 | 2018-10-02 | Duke University | Anti-cd19 antibodies and uses in oncology |
JP2008531730A (en) * | 2005-03-04 | 2008-08-14 | キュアーディーエム、インク. | Methods and pharmaceutical compositions for treating type I diabetes mellitus and other conditions |
US20090142338A1 (en) * | 2005-03-04 | 2009-06-04 | Curedm, Inc. | Methods and Compositions for Treating Type 1 and Type 2 Diabetes Mellitus and Related Conditions |
RU2007137489A (en) | 2005-03-10 | 2009-04-20 | Дженентек, Инк. (Us) | METHODS AND COMPOSITIONS FOR MODULATION OF VESSEL INTEGRITY |
WO2006102095A2 (en) | 2005-03-18 | 2006-09-28 | Medimmune, Inc. | Framework-shuffling of antibodies |
SG171690A1 (en) | 2005-03-22 | 2011-06-29 | Harvard College | Treatment of protein degradation disorders |
CA2602375C (en) | 2005-03-23 | 2018-07-24 | Genmab A/S | Antibodies against cd38 for treatment of multiple myeloma |
GB0506912D0 (en) | 2005-04-05 | 2005-05-11 | Celltech R&D Ltd | Biological products |
EP1868650B1 (en) | 2005-04-15 | 2018-10-03 | MacroGenics, Inc. | Covalent diabodies and uses thereof |
US20060269556A1 (en) * | 2005-04-18 | 2006-11-30 | Karl Nocka | Mast cell activation using siglec 6 antibodies |
JP5122441B2 (en) | 2005-04-19 | 2013-01-16 | シアトル ジェネティックス, インコーポレイテッド | Humanized anti-CD70 binding agents and uses thereof |
EA015534B1 (en) | 2005-04-25 | 2011-08-30 | Пфайзер Инк. | Antibodies to myostatin and methods of use thereof |
EP2444421A1 (en) | 2005-04-26 | 2012-04-25 | Pfizer Inc. | P-Cadherin antibodies |
AR054260A1 (en) * | 2005-04-26 | 2007-06-13 | Rinat Neuroscience Corp | METHODS OF TREATMENT OF DISEASES OF THE LOWER MOTOR NEURONE AND COMPOSITIONS USED IN THE SAME |
US7595380B2 (en) | 2005-04-27 | 2009-09-29 | Tripath Imaging, Inc. | Monoclonal antibodies and methods for their use in the detection of cervical disease |
MY148086A (en) * | 2005-04-29 | 2013-02-28 | Rinat Neuroscience Corp | Antibodies directed against amyloid-beta peptide and methods using same |
PE20061324A1 (en) | 2005-04-29 | 2007-01-15 | Centocor Inc | ANTI-IL-6 ANTIBODIES, COMPOSITIONS, METHODS AND USES |
CN102961746B (en) | 2005-05-16 | 2016-06-15 | 艾伯维生物技术有限公司 | The purposes of TNF α inhibitor for treatment of erosive polyarthritis |
EP1899364B1 (en) | 2005-05-17 | 2020-02-19 | University of Connecticut | Compositions and methods for immunomodulation in an organism |
EP1883417A2 (en) | 2005-05-25 | 2008-02-06 | Curedm Inc. | Peptides, derivatives and analogs thereof, and methods of using same |
LT2460831T (en) | 2005-05-27 | 2016-12-12 | Biogen Ma Inc. | Tweak binding antibodies |
JP2008541769A (en) * | 2005-06-03 | 2008-11-27 | アンブレツクス・インコーポレイテツド | Improved human interferon molecules and their use |
CA2610709A1 (en) | 2005-06-06 | 2006-12-14 | Genentech, Inc. | Transgenic models for different genes and their use for gene characterization |
WO2006135886A2 (en) * | 2005-06-13 | 2006-12-21 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing cancer |
AU2006261185B2 (en) | 2005-06-17 | 2012-02-16 | Janssen Alzheimer Immunotherapy | Methods of purifying anti A beta antibodies |
KR20080025174A (en) | 2005-06-23 | 2008-03-19 | 메디뮨 인코포레이티드 | Antibody formulations having optimized aggregation and fragmentation profiles |
EP2452694B1 (en) | 2005-06-30 | 2018-11-14 | Janssen Biotech, Inc. | Anti-IL-23 antibodies, compositions, methods and uses |
KR20130080058A (en) | 2005-06-30 | 2013-07-11 | 아보트 러보러터리즈 | Il-12/p40 binding proteins |
EP2476761A3 (en) | 2005-07-07 | 2012-10-17 | Athlomics Pty Ltd | Polynucleotide marker genes and their expression, for diagnosis of endotoxemia |
MX2008000253A (en) | 2005-07-08 | 2008-04-02 | Biogen Idec Inc | Sp35 antibodies and uses thereof. |
WO2007008604A2 (en) * | 2005-07-08 | 2007-01-18 | Bristol-Myers Squibb Company | Single nucleotide polymorphisms associated with dose-dependent edema and methods of use thereof |
CA2615615A1 (en) | 2005-07-22 | 2007-02-01 | Y's Therapeutics Co., Ltd. | Anti-cd26 antibodies and methods of use thereof |
CA2617637C (en) | 2005-08-02 | 2017-07-18 | Xbiotech Inc. | Diagnosis, treatment, and prevention of vascular disorders using il-1.alpha. autoantibodies |
US20080307549A1 (en) | 2005-08-03 | 2008-12-11 | Adelaide Research & Innovation Pty Ltd. | Polysaccharide Synthases |
WO2007021423A2 (en) | 2005-08-15 | 2007-02-22 | Genentech, Inc. | Gene disruptions, compositions and methods relating thereto |
CN103103238B (en) | 2005-08-18 | 2016-08-10 | Ambrx公司 | A kind of manufacture in cell has selected amino acid whose antibody or the method for antibody fragment polypeptide in specific location |
US7612181B2 (en) * | 2005-08-19 | 2009-11-03 | Abbott Laboratories | Dual variable domain immunoglobulin and uses thereof |
US20070202512A1 (en) * | 2005-08-19 | 2007-08-30 | Bristol-Myers Squibb Company | Human single nucleotide polymorphisms associated with dose-dependent weight gain and methods of use thereof |
US20070041905A1 (en) | 2005-08-19 | 2007-02-22 | Hoffman Rebecca S | Method of treating depression using a TNF-alpha antibody |
NZ612578A (en) | 2005-08-19 | 2014-11-28 | Abbvie Inc | Dual variable domain immunoglobin and uses thereof |
US20090215992A1 (en) * | 2005-08-19 | 2009-08-27 | Chengbin Wu | Dual variable domain immunoglobulin and uses thereof |
EP2500354A3 (en) | 2005-08-19 | 2012-10-24 | Abbott Laboratories | Dual variable domain immunoglobulin and uses thereof |
TWI541253B (en) | 2005-09-07 | 2016-07-11 | 艾默根佛蒙特有限公司 | Human monoclonal antibodies to activin receptor-like kinase-1 |
EP1926757B1 (en) * | 2005-09-14 | 2012-02-22 | UCB Pharma, S.A. | Antibody-comb polymer conjugate |
EP1928905B1 (en) | 2005-09-30 | 2015-04-15 | AbbVie Deutschland GmbH & Co KG | Binding domains of proteins of the repulsive guidance molecule (rgm) protein family and functional fragments thereof, and their use |
KR101176830B1 (en) | 2005-10-17 | 2012-08-27 | 주식회사 아이지세라피 | Novel method for phage display |
US8249814B2 (en) | 2005-10-21 | 2012-08-21 | Genenews Inc. | Method, computer readable medium, and system for determining a probability of colorectal cancer in a test subject |
US8597646B2 (en) | 2005-10-25 | 2013-12-03 | The Johns Hopkins University | Methods and compositons featuring TGF-beta antagonists for the treatment of marfan syndrome and associated disorders |
US7723112B2 (en) * | 2005-10-31 | 2010-05-25 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing cancer |
US7723477B2 (en) * | 2005-10-31 | 2010-05-25 | Oncomed Pharmaceuticals, Inc. | Compositions and methods for inhibiting Wnt-dependent solid tumor cell growth |
EP1961065A4 (en) | 2005-10-31 | 2009-11-11 | Oncomed Pharm Inc | Compositions and methods for diagnosing and treating cancer |
CN101663048A (en) | 2005-11-01 | 2010-03-03 | 艾博特生物技术有限公司 | Use the method and composition of biomarker diagnosing ankylosing spondylitis |
AU2006311828B2 (en) | 2005-11-04 | 2013-07-11 | Biogen Ma Inc. | Methods for promoting neurite outgrowth and survival of dopaminergic neurons |
EP1957531B1 (en) | 2005-11-07 | 2016-04-13 | Genentech, Inc. | Binding polypeptides with diversified and consensus vh/vl hypervariable sequences |
UA94922C2 (en) | 2005-11-07 | 2011-06-25 | Зе Скріпс Рісьорч Інстітьют | Method for controlling tissue factor signaling specificity in a mammalian subject, method for identifying an agent which specifically inhibits tissue factor signaling |
ATE500215T1 (en) * | 2005-11-08 | 2011-03-15 | Ambrx Inc | ACCELERATOR FOR THE MODIFICATION OF NON-NATURAL AMINO ACIDS AND NON-NATURAL AMINO ACID POLYPEPTIDES |
UA96139C2 (en) | 2005-11-08 | 2011-10-10 | Дженентек, Інк. | Anti-neuropilin-1 (nrp1) antibody |
US7807790B2 (en) | 2005-11-14 | 2010-10-05 | Metamol Theranostics, Llc | Peptide sequence that promotes tumor invasion |
ME00419B (en) | 2005-11-14 | 2011-10-10 | Rinat Neuroscience Corp | Antagonist antibodies directed against calcitonin gene-related peptide and methods using same |
PT2339014E (en) | 2005-11-16 | 2015-10-13 | Ambrx Inc | Methods and compositions comprising non-natural amino acids |
JP2009516514A (en) | 2005-11-21 | 2009-04-23 | ジェネンテック・インコーポレーテッド | Novel gene disruptions, compositions and methods related thereto |
DK1963369T3 (en) | 2005-11-28 | 2013-06-03 | Zymogenetics Inc | IL-21 Antagonists |
CN102898519B (en) | 2005-11-30 | 2015-10-28 | Abbvie公司 | Monoclonal antibody of anti-amyloid beta protein and uses thereof |
EP2289909B1 (en) | 2005-11-30 | 2014-10-29 | AbbVie Inc. | Screening method, process for purifying of non-diffusible a-beta oligomers, selective antibodies against said non-diffusible a-beta oligomers and a process for manufacturing of said antibodies |
CN101495869B (en) | 2005-11-30 | 2013-06-19 | 麻省理工学院 | Pathogen detection biosensor |
JP5808070B2 (en) * | 2005-12-02 | 2015-11-10 | ジェネンテック, インコーポレイテッド | Binding polypeptides and uses thereof |
CA2631961A1 (en) | 2005-12-02 | 2007-11-08 | Genentech, Inc. | Compositions and methods for the treatment of diseases and disorders associated with cytokine signaling relating to antibodies that bind to il-22 |
US20070237764A1 (en) * | 2005-12-02 | 2007-10-11 | Genentech, Inc. | Binding polypeptides with restricted diversity sequences |
WO2007064882A2 (en) | 2005-12-02 | 2007-06-07 | Biogen Idec Ma Inc. | Treatment of conditions involving demyelination |
UA96141C2 (en) | 2005-12-09 | 2011-10-10 | Юсиби Фарма, С.А. | Neutralising antibody having specificity for human il-6 |
CN101448512B (en) * | 2005-12-14 | 2015-11-25 | Ambrx公司 | Purposes containing alpha-non-natural amino acid and the compositions of polypeptide, the method relating to alpha-non-natural amino acid and polypeptide and alpha-non-natural amino acid and polypeptide |
CA2633887C (en) | 2005-12-15 | 2015-12-22 | Genentech, Inc. | Methods and compositions for targeting polyubiquitin |
US20070264687A1 (en) | 2005-12-15 | 2007-11-15 | Min-Yuan Chou | Recombinant triplex scaffold-based polypeptides |
US10183986B2 (en) | 2005-12-15 | 2019-01-22 | Industrial Technology Research Institute | Trimeric collagen scaffold antibodies |
US7632498B2 (en) | 2005-12-19 | 2009-12-15 | Tripath Imaging, Inc. | MCM6 and MCM7 monoclonal antibodies and methods for their use in the detection of cervical disease |
PT3219328T (en) | 2005-12-29 | 2020-08-28 | Janssen Biotech Inc | Human anti-il-23 antibodies, compositions, method and uses |
PT1973950E (en) | 2006-01-05 | 2014-12-29 | Genentech Inc | Anti-ephb4 antibodies and methods using the same |
BRPI0707126A2 (en) | 2006-01-12 | 2011-04-19 | Alexion Pharma Inc | ox-2 / cd200 antibodies and use of these |
EP2363711A1 (en) | 2006-01-27 | 2011-09-07 | Tripath Imaging, Inc. | Methods for identifying patients with an increased likelihood of having ovarian cancer and compositions therefor |
US8669345B2 (en) | 2006-01-27 | 2014-03-11 | Biogen Idec Ma Inc. | Nogo receptor antagonists |
EP1987361A4 (en) * | 2006-01-30 | 2009-03-04 | Invitrogen Corp | Compositions and methods for detecting and quantifying toxic substances in disease states |
US20070175313A1 (en) * | 2006-01-31 | 2007-08-02 | Kevin Vandervliet | MP3 player holder assembly |
CN101400703B (en) | 2006-02-01 | 2013-05-08 | 赛法隆澳大利亚控股有限公司 | Domain antibody construct |
EP1991247B1 (en) | 2006-02-14 | 2015-10-14 | President and Fellows of Harvard College | Bifunctional histone deacetylase inhibitors |
ZA200807714B (en) | 2006-02-17 | 2010-01-27 | Genentech Inc | Gene disruptions, compositions and methods relating thereto |
CA2950465A1 (en) | 2006-02-20 | 2007-08-30 | Phylogica Limited | Method of constructing and screening libraries of peptide structures |
TWI417301B (en) | 2006-02-21 | 2013-12-01 | Wyeth Corp | Antibodies against human il-22 and uses therefor |
WO2007100640A2 (en) * | 2006-02-21 | 2007-09-07 | The Regents Of The University Of Michigan | Growth hormone receptor antagonist cancer treatment |
US20080019961A1 (en) * | 2006-02-21 | 2008-01-24 | Regents Of The University Of Michigan | Hedgehog signaling pathway antagonist cancer treatment |
TW200744634A (en) | 2006-02-21 | 2007-12-16 | Wyeth Corp | Methods of using antibodies against human IL-22 |
EP2010677A4 (en) * | 2006-02-24 | 2010-04-14 | Investigen Inc | Methods and compositions for detecting polynucleotides |
EP2650306A1 (en) | 2006-03-06 | 2013-10-16 | Aeres Biomedical Limited | Humanized Anti-CD22 antibodies and their use in treatment of oncology, transplantation and autoimmune disease |
AR059851A1 (en) | 2006-03-16 | 2008-04-30 | Genentech Inc | ANTIBODIES OF EGFL7 AND METHODS OF USE |
EP2010569A4 (en) | 2006-03-20 | 2009-09-09 | Xoma Technology Ltd | Human antibodies specific for gastrin materials and methods |
EP2614839A3 (en) | 2006-04-05 | 2015-01-28 | Genentech, Inc. | Method for using BOC/CDO to modulate hedgehog signaling |
KR20090005315A (en) | 2006-04-05 | 2009-01-13 | 애보트 바이오테크놀로지 리미티드 | Antibody purification |
WO2007121147A2 (en) | 2006-04-10 | 2007-10-25 | Genentech, Inc. | Disheveled pdz modulators |
CA2564435A1 (en) | 2006-04-10 | 2007-10-10 | Abbott Biotechnology Ltd. | Methods for monitoring and treating intestinal disorders |
EP2708242A3 (en) | 2006-04-10 | 2014-03-26 | Abbott Biotechnology Ltd | Uses and compositions for treatment of ankylosing spondylitis |
WO2008063213A2 (en) | 2006-04-10 | 2008-05-29 | Abbott Biotechnology Ltd. | Uses and compositions for treatment of psoriatic arthritis |
US20090288176A1 (en) | 2006-04-19 | 2009-11-19 | Genentech, Inc. | Novel Gene Disruptions, Compositions and Methods Relating Thereto |
US10522240B2 (en) | 2006-05-03 | 2019-12-31 | Population Bio, Inc. | Evaluating genetic disorders |
US7702468B2 (en) | 2006-05-03 | 2010-04-20 | Population Diagnostics, Inc. | Evaluating genetic disorders |
EP2019674B1 (en) | 2006-05-03 | 2016-11-23 | The President and Fellows of Harvard College | Histone deacetylase and tubulin deacetylase inhibitors |
JP2009536527A (en) * | 2006-05-09 | 2009-10-15 | ジェネンテック・インコーポレーテッド | Binding polypeptide with optimized scaffold |
WO2007134132A2 (en) * | 2006-05-12 | 2007-11-22 | Genentech, Inc. | Compositions and methods for the diagnosis and treatment of bladder and urinary tract tumors |
ES2567402T3 (en) | 2006-05-30 | 2016-04-22 | Genentech, Inc. | Anti CD22 antibodies, their immunoconjugates and their uses |
US7862812B2 (en) | 2006-05-31 | 2011-01-04 | Lpath, Inc. | Methods for decreasing immune response and treating immune conditions |
GB0611116D0 (en) | 2006-06-06 | 2006-07-19 | Oxford Genome Sciences Uk Ltd | Proteins |
AU2007319672B2 (en) | 2006-06-06 | 2011-06-30 | Genentech, Inc. | Anti-DLL4 antibodies and methods using same |
CA2652703C (en) | 2006-06-07 | 2018-08-28 | Bioalliance C.V. | Antibodies recognizing a carbohydrate containing epitope on cd-43 and cea expressed on cancer cells and methods using same |
JP2009544761A (en) | 2006-06-14 | 2009-12-17 | マクロジェニクス,インコーポレーテッド | Method of treating autoimmune disease using low toxicity immunosuppressive monoclonal antibody |
HUE030269T2 (en) | 2006-06-26 | 2017-04-28 | Macrogenics Inc | Fc riib-specific antibodies and methods of use thereof |
DE102006030028A1 (en) * | 2006-06-29 | 2008-02-14 | Forschungszentrum Jülich GmbH | A method for finding bait-binding specific molecules and bait-binding molecules and their use |
CN101484199B (en) | 2006-06-30 | 2014-06-25 | 艾伯维生物技术有限公司 | Automatic injection device |
US7572618B2 (en) | 2006-06-30 | 2009-08-11 | Bristol-Myers Squibb Company | Polynucleotides encoding novel PCSK9 variants |
AT503889B1 (en) | 2006-07-05 | 2011-12-15 | Star Biotechnologische Forschungs Und Entwicklungsges M B H F | MULTIVALENT IMMUNE LOBULINE |
KR20150033726A (en) | 2006-07-05 | 2015-04-01 | 카탈리스트 바이오사이언시즈, 인코포레이티드 | Protease screening methods and proteases identified thereby |
JP5622390B2 (en) * | 2006-07-18 | 2014-11-12 | サノフイ | Anti-EPHA2 antagonist antibody for cancer treatment |
JP5638804B2 (en) | 2006-08-04 | 2014-12-10 | アストラゼネカ アクチボラグ | Antibodies against ErbB2 |
AU2007284651B2 (en) | 2006-08-09 | 2014-03-20 | Institute For Systems Biology | Organ-specific proteins and methods of their use |
SG170032A1 (en) | 2006-08-28 | 2011-04-29 | Kyowa Hakko Kirin Co Ltd | Antagonistic human light-specific human monoclonal antibodies |
JP5994121B2 (en) | 2006-09-07 | 2016-09-21 | オタゴ イノベーション リミテッド | Biomarker |
KR101544108B1 (en) | 2006-09-08 | 2015-08-13 | 애브비 바하마스 리미티드 | -13 Interleukin-13 binding proteins |
US9133495B2 (en) * | 2006-09-08 | 2015-09-15 | Ambrx, Inc. | Hybrid suppressor tRNA for vertebrate cells |
JP5451390B2 (en) * | 2006-09-08 | 2014-03-26 | アンブルックス,インコーポレイテッド | Transcription of suppressor TRNA in vertebrate cells |
MX2009002523A (en) | 2006-09-08 | 2009-03-20 | Ambrx Inc | Modified human plasma polypeptide or fc scaffolds and their uses. |
ES2372217T3 (en) | 2006-09-12 | 2012-01-17 | Genentech, Inc. | PROCEDURES AND COMPOSITIONS FOR THE DIAGNOSIS AND TREATMENT OF LUNG CANCER USING THE GENE OF PDGFRA, KIT OR KDR AS A GENETIC MARKER. |
EP2074138A4 (en) * | 2006-09-19 | 2009-12-30 | Phylogica Ltd | Neuroprotective peptide inhibitors of ap-1 signaling and uses therefor |
AU2007302448C1 (en) | 2006-09-26 | 2019-02-14 | Genmab A/S | Combination treatment of CD38-expressing tumors |
EP3357932A1 (en) | 2006-09-29 | 2018-08-08 | OncoMed Pharmaceuticals, Inc. | Compositions and methods for diagnosing and treating cancer |
EP2076287A2 (en) | 2006-10-12 | 2009-07-08 | Wyeth | Methods and compositions with reduced opalescence |
WO2008063776A2 (en) * | 2006-10-12 | 2008-05-29 | Genentech, Inc. | Antibodies to lymphotoxin-alpha |
JP2010506842A (en) | 2006-10-16 | 2010-03-04 | メディミューン,エルエルシー | Molecules with reduced half-life, compositions thereof and uses |
GB0620729D0 (en) | 2006-10-18 | 2006-11-29 | Ucb Sa | Biological products |
EP1914242A1 (en) | 2006-10-19 | 2008-04-23 | Sanofi-Aventis | Novel anti-CD38 antibodies for the treatment of cancer |
US20080096193A1 (en) * | 2006-10-24 | 2008-04-24 | Charles Robert Bupp | Methods and compositions for detecting polynucleotides |
US8436147B2 (en) | 2006-10-27 | 2013-05-07 | Genentech, Inc. | Antibodies and immunoconjugates and uses therefor |
CA2667697A1 (en) | 2006-10-30 | 2008-05-08 | Promega Corporation | Mutant hydrolase proteins with enhanced kinetics and functional expression |
WO2008055260A2 (en) | 2006-11-03 | 2008-05-08 | Wyeth | Glycolysis-inhibiting substances in cell culture |
CA2669095A1 (en) | 2006-11-15 | 2008-05-29 | Functional Genetics, Inc. | Anti-tsg101 antibodies and their uses for treatment of viral infections |
US7488807B2 (en) | 2006-11-22 | 2009-02-10 | 3M Innovative Properties Company | Antibody with protein A selectivity |
WO2008064306A2 (en) * | 2006-11-22 | 2008-05-29 | Curedm, Inc. | Methods and compositions relating to islet cell neogenesis |
US8455626B2 (en) | 2006-11-30 | 2013-06-04 | Abbott Laboratories | Aβ conformer selective anti-aβ globulomer monoclonal antibodies |
DK2102239T3 (en) | 2006-11-30 | 2012-05-29 | Res Dev Foundation | IMPROVED IMMUNOGLOBULIN LIBRARIES |
ES2523915T5 (en) | 2006-12-01 | 2022-05-26 | Seagen Inc | Variant Target Binding Agents and Uses Thereof |
EP2121979B1 (en) | 2006-12-05 | 2017-07-12 | Decode Genetics EHF | Genetic markers for risk management of cardiac arrhythmia |
EP2687232A1 (en) | 2006-12-06 | 2014-01-22 | MedImmune, LLC | Methods of treating systemic lupus erythematosus |
KR20150097813A (en) | 2006-12-19 | 2015-08-26 | 제넨테크, 인크. | Vegf-specific antagonists for adjuvant and neoadjuvant therapy and the treatment of early stage tumors |
US7695718B2 (en) | 2006-12-20 | 2010-04-13 | Xoma Technology Ltd. | Methods for the treatment of IL-1β related diseases |
AR064464A1 (en) * | 2006-12-22 | 2009-04-01 | Genentech Inc | ANTIBODIES ANTI - INSULINAL GROWTH FACTOR RECEIVER |
EP2097448A4 (en) | 2006-12-22 | 2010-07-21 | Univ Utah Res Found | Method of detecting ocular diseases and pathologic conditions and treatment of same |
NZ619576A (en) | 2006-12-27 | 2014-07-25 | Harvard College | Compositions and methods for the treatment of infections and tumors |
US8324350B2 (en) | 2006-12-29 | 2012-12-04 | Abbott Laboratories | Dual-specific IL-1α/IL-1β antibodies |
US8128926B2 (en) | 2007-01-09 | 2012-03-06 | Biogen Idec Ma Inc. | Sp35 antibodies and uses thereof |
EP2740744B1 (en) | 2007-01-09 | 2018-03-28 | Biogen MA Inc. | Sp35 antibodies and uses thereof |
RU2475265C2 (en) | 2007-01-16 | 2013-02-20 | Эбботт Лэборетриз | Methods of treating psoriasis |
WO2008092041A2 (en) | 2007-01-24 | 2008-07-31 | Carnegie Mellon University | Optical biosensors |
EP2106439B1 (en) | 2007-01-24 | 2014-11-12 | The Regents of the University of Michigan | Compositions and methods for treating and diagnosing pancreatic cancer |
IN2009DN05722A (en) | 2007-02-07 | 2015-07-24 | Decode Genetics Ehf | |
US7834154B2 (en) | 2007-02-09 | 2010-11-16 | Genentech, Inc. | Anti-ROBO4 antibodies and uses therefor |
US8685666B2 (en) * | 2007-02-16 | 2014-04-01 | The Board Of Trustees Of Southern Illinois University | ARL-1 specific antibodies and uses thereof |
WO2008101184A2 (en) * | 2007-02-16 | 2008-08-21 | The Board Of Trustees Of Southern Illinois University | Arl-1 specific antibodies |
AU2008218542B2 (en) | 2007-02-21 | 2014-06-26 | Decode Genetics Ehf. | Genetic susceptibility variants associated with cardiovascular disease |
CN101663407B (en) | 2007-02-22 | 2017-08-08 | 健泰科生物技术公司 | method for detecting inflammatory bowel disease |
US7771947B2 (en) * | 2007-02-23 | 2010-08-10 | Investigen, Inc. | Methods and compositions for rapid light-activated isolation and detection of analytes |
WO2008104803A2 (en) | 2007-02-26 | 2008-09-04 | Oxford Genome Sciences (Uk) Limited | Proteins |
EP2447719B1 (en) | 2007-02-26 | 2016-08-24 | Oxford BioTherapeutics Ltd | Proteins |
EP2124952A2 (en) | 2007-02-27 | 2009-12-02 | Abbott GmbH & Co. KG | Method for the treatment of amyloidoses |
PL2121919T3 (en) | 2007-03-22 | 2012-07-31 | Heptares Therapeutics Ltd | Mutant g-protein coupled receptors and methods for selecting them |
JP5721951B2 (en) | 2007-03-22 | 2015-05-20 | バイオジェン アイデック マサチューセッツ インコーポレイテッド | Binding proteins that specifically bind to CD154, including antibodies, antibody derivatives, and antibody fragments, and uses thereof |
US7960139B2 (en) | 2007-03-23 | 2011-06-14 | Academia Sinica | Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells |
RU2476442C2 (en) | 2007-03-29 | 2013-02-27 | Эббот Лэборетриз | Crystalline human il-12 antibodies |
CA2682292A1 (en) | 2007-03-30 | 2008-10-09 | Medimmune, Llc | Aqueous formulation comprising an anti-human interferon alpha antibody |
WO2008121563A2 (en) * | 2007-03-30 | 2008-10-09 | Ambrx, Inc. | Modified fgf-21 polypeptides and their uses |
UY30994A1 (en) | 2007-04-02 | 2008-11-28 | Amgen Fremont Inc | ANTI-IGE ANTIBODIES |
US20100291562A1 (en) * | 2007-04-04 | 2010-11-18 | Michael Adler | Method for the detection of an analyte in biological matrix |
US7807168B2 (en) * | 2007-04-10 | 2010-10-05 | Vaccinex, Inc. | Selection of human TNFα specific antibodies |
NZ593611A (en) | 2007-04-13 | 2012-06-29 | Catalyst Biosciences Inc | Modified factor vii polypeptides and uses thereof |
US8114630B2 (en) | 2007-05-02 | 2012-02-14 | Ambrx, Inc. | Modified interferon beta polypeptides and their uses |
AU2008246442B2 (en) | 2007-05-04 | 2014-07-03 | Technophage, Investigacao E Desenvolvimento Em Biotecnologia, Sa | Engineered rabbit antibody variable domains and uses thereof |
WO2008137915A2 (en) | 2007-05-07 | 2008-11-13 | Medimmune, Llc | Anti-icos antibodies and their use in treatment of oncology, transplantation and autoimmune disease |
HUE036885T2 (en) | 2007-05-14 | 2018-08-28 | Astrazeneca Ab | Methods of reducing basophil levels |
US8318163B2 (en) | 2007-05-17 | 2012-11-27 | Genentech, Inc. | Anti-pan neuropilin antibody and binding fragments thereof |
MX2009012722A (en) | 2007-05-25 | 2009-12-11 | Decode Genetics Ehf | Genetic variants on chr 5pl2 and 10q26 as markers for use in breast cancer risk assessment, diagnosis, prognosis and treatment. |
US7906149B2 (en) * | 2007-05-25 | 2011-03-15 | Boval Company, L.P. | Method for treating allergic dermatitis |
US20090232801A1 (en) * | 2007-05-30 | 2009-09-17 | Abbot Laboratories | Humanized Antibodies Which Bind To AB (1-42) Globulomer And Uses Thereof |
PE20090329A1 (en) * | 2007-05-30 | 2009-03-27 | Abbott Lab | HUMANIZED ANTIBODIES AGAINST GLOBULOMER AB (20-42) AND ITS USES |
US8999337B2 (en) | 2007-06-11 | 2015-04-07 | Abbvie Biotechnology Ltd. | Methods for treating juvenile idiopathic arthritis by inhibition of TNFα |
CA2958672A1 (en) | 2007-06-15 | 2008-12-18 | Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts | Treatment of tumors using specific anti-l1 antibody |
EP2170932A4 (en) * | 2007-06-20 | 2012-10-10 | Phylogica Ltd | Compositions and uses thereof for the treatment of acute respiratory distress syndrome (ards) and clinical disorders associated with therewith |
ES2702087T3 (en) | 2007-06-21 | 2019-02-27 | Macrogenics Inc | Covalent diabodies and their uses |
JP5602625B2 (en) | 2007-06-26 | 2014-10-08 | エフ−スター ビオテヒノロギッシェ フォルシュングス− ウント エントヴィッケルングスゲゼルシャフト ミット ベシュレンクテル ハフツング | Binding substance display |
PL3009148T3 (en) | 2007-07-02 | 2019-03-29 | Oncomed Pharmaceuticals, Inc. | Compositions and methods for treating and diagnosing cancer |
JP5469600B2 (en) | 2007-07-16 | 2014-04-16 | ジェネンテック, インコーポレイテッド | Anti-CD79b antibody and immunoconjugate and method of use thereof |
AR067543A1 (en) | 2007-07-16 | 2009-10-14 | Genentech Inc | ANTI-CD79B ANTIBODIES AND HUMANIZED IMMUNOCATE PLAYERS AND METHODS OF USE |
ES2614735T3 (en) | 2007-07-23 | 2017-06-01 | Janssen Biotech, Inc. | Methods and compositions for treating fibrosis-related disorders using IL-17 antagonists |
BRPI0814645A2 (en) * | 2007-07-25 | 2015-01-27 | Alexion Pharma Inc | METHODS AND COMPOSITIONS FOR TREATING AUTOIMMUNE DISEASE. |
WO2009020477A1 (en) * | 2007-08-06 | 2009-02-12 | Yale University | Modified miniature proteins |
EP2190987B1 (en) | 2007-08-21 | 2012-11-14 | MorphoSys AG | Methods for the formation of disulphide bonds |
DK2193142T3 (en) * | 2007-08-30 | 2015-04-20 | Curedm Group Holdings Llc | Compositions and Methods for Using Project Cell Peptides and Analogs Thereto |
GB0717337D0 (en) | 2007-09-06 | 2007-10-17 | Ucb Pharma Sa | Method of treatment |
BRPI0816785A2 (en) * | 2007-09-14 | 2017-05-02 | Adimab Inc | rationally designed synthetic antibody libraries, and uses thereof |
US8877688B2 (en) * | 2007-09-14 | 2014-11-04 | Adimab, Llc | Rationally designed, synthetic antibody libraries and uses therefor |
EP2535351A3 (en) | 2007-09-26 | 2013-04-03 | UCB Pharma S.A. | Dual specificity antibody fusions |
EP3789400A1 (en) | 2007-09-26 | 2021-03-10 | Chugai Seiyaku Kabushiki Kaisha | Modified antibody constant region |
JP5566293B2 (en) * | 2007-10-04 | 2014-08-06 | バイオノミックス リミテッド | Endothelial cell markers and uses thereof |
SI2219452T1 (en) | 2007-11-05 | 2016-03-31 | Medimmune, Llc | Methods of treating scleroderma |
ES2662845T3 (en) | 2007-11-07 | 2018-04-10 | Genentech, Inc. | IL-22 for use in the treatment of microbial disorders |
EP2728017B1 (en) | 2007-11-19 | 2016-08-24 | Celera Corporation | Lung cancer markers and uses thereof |
US8946148B2 (en) | 2007-11-20 | 2015-02-03 | Ambrx, Inc. | Modified insulin polypeptides and their uses |
WO2009070642A1 (en) * | 2007-11-28 | 2009-06-04 | Medimmune, Llc | Protein formulation |
TWI580694B (en) | 2007-11-30 | 2017-05-01 | 建南德克公司 | Anti-vegf antibodies |
US8697360B2 (en) | 2007-11-30 | 2014-04-15 | Decode Genetics Ehf. | Genetic variants on CHR 11Q and 6Q as markers for prostate and colorectal cancer predisposition |
WO2009079212A2 (en) | 2007-12-03 | 2009-06-25 | Carnegie Mellon University | Linked peptide fluorogenic biosensors |
GB0724051D0 (en) | 2007-12-08 | 2008-01-16 | Medical Res Council | Mutant proteins and methods for producing them |
CN102216329A (en) | 2007-12-17 | 2011-10-12 | 辉瑞有限公司 | Treatment of interstitial cystitis |
NZ585959A (en) | 2007-12-18 | 2012-09-28 | Bioalliance Cv | Antibodies recognizing a carbohydrate containing epitope on cd-43 and cea expressed on cancer cells and methods using same |
GB0724860D0 (en) | 2007-12-20 | 2008-01-30 | Heptares Therapeutics Ltd | Screening |
MX2010006823A (en) | 2007-12-20 | 2010-09-30 | Xoma Technology Ltd | Methods for the treatment of gout. |
NZ586544A (en) | 2007-12-26 | 2012-07-27 | Vaccinex Inc | Anti-c35 antibody in combination with an ani-her2 antibody in cancer therapies and methods |
HUE025560T2 (en) | 2007-12-28 | 2016-03-29 | Prothena Biosciences Ltd | Treatment and prophylaxis of amyloidosis |
GB0800277D0 (en) | 2008-01-08 | 2008-02-13 | Imagination Tech Ltd | Video motion compensation |
CN101918555B (en) | 2008-01-11 | 2013-11-06 | 株式会社遗传科技 | Humanized anti-alpha9 integrin antibodies and the uses thereof |
AU2009205995B2 (en) | 2008-01-18 | 2014-04-03 | Medimmune, Llc | Cysteine engineered antibodies for site-specific conjugation |
PE20091318A1 (en) | 2008-01-31 | 2009-09-16 | Genentech Inc | ANTI-CD79B ANTIBODIES AND IMMUNOCONJUGATES AND METHODS OF USE OF THE SAME |
NZ586947A (en) | 2008-02-08 | 2012-11-30 | Ambrx Inc | Modified leptin polypeptides and their uses |
MX2010008578A (en) | 2008-02-08 | 2010-11-10 | Medimmune Llc | Anti-ifnar1 antibodies with reduced fc ligand affinity. |
GB0802474D0 (en) | 2008-02-11 | 2008-03-19 | Heptares Therapeutics Ltd | Mutant proteins and methods for selecting them |
CA2714521A1 (en) | 2008-02-14 | 2009-08-20 | Decode Genetics Ehf | Susceptibility variants for lung cancer |
EP2271366A4 (en) | 2008-02-28 | 2012-06-20 | 3M Innovative Properties Co | Antibodies to clostridium difficile spores and uses thereof |
US8962803B2 (en) | 2008-02-29 | 2015-02-24 | AbbVie Deutschland GmbH & Co. KG | Antibodies against the RGM A protein and uses thereof |
WO2009109572A2 (en) * | 2008-03-03 | 2009-09-11 | Ablynx Nv | Monovalent phage display of single variable domains |
AU2009220900B2 (en) | 2008-03-04 | 2014-09-11 | Teva Pharmaceuticals International Gmbh | Methods of treating chronic pain |
PL2265288T3 (en) | 2008-03-04 | 2017-01-31 | Labrys Biologics Inc | Methods of treating inflammatory pain |
US20110092452A1 (en) * | 2008-03-05 | 2011-04-21 | The Regents Of The University Of Michigan | Compositions and methods for diagnosing and treating pancreatic cancer |
CA2715921A1 (en) | 2008-03-12 | 2009-09-17 | Otago Innovation Limited | Biomarkers |
CA2715914C (en) | 2008-03-12 | 2019-01-22 | Otago Innovation Limited | Insulin signal peptide fragment biomarkers |
US9873957B2 (en) | 2008-03-13 | 2018-01-23 | Dyax Corp. | Libraries of genetic packages comprising novel HC CDR3 designs |
MX2010010265A (en) | 2008-03-18 | 2010-09-30 | Abbott Lab | Methods for treating psoriasis. |
WO2009118300A1 (en) | 2008-03-25 | 2009-10-01 | Novartis Forschungsstiftung Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Treating cancer by down-regulating frizzled-4 and/or frizzled-1 |
WO2009124090A1 (en) * | 2008-03-31 | 2009-10-08 | Genentech, Inc. | Compositions and methods for treating and diagnosing asthma |
EP2274450A2 (en) | 2008-04-01 | 2011-01-19 | Decode Genetics EHF | Susceptibility variants for peripheral arterial disease and abdominal aortic aneurysm |
CN102046195A (en) | 2008-04-02 | 2011-05-04 | 宏观基因有限公司 | HER2/neu-specific antibodies and methods of using same |
US8669349B2 (en) | 2008-04-02 | 2014-03-11 | Macrogenics, Inc. | BCR-complex-specific antibodies and methods of using same |
DK2276509T3 (en) | 2008-04-11 | 2016-09-19 | Seattle Genetics Inc | DETECTION AND TREATMENT OF CANCER IN PANCREAS, ovarian and other cancers |
GB0807413D0 (en) | 2008-04-23 | 2008-05-28 | Ucb Pharma Sa | Biological products |
CA2721716C (en) | 2008-04-24 | 2019-09-24 | Gene Techno Science Co., Ltd. | Humanized antibodies specific for amino acid sequence rgd of an extracellular matrix protein and the uses thereof |
CA3049612C (en) | 2008-04-24 | 2023-01-10 | Dyax Corp. | Libraries of genetic packages comprising novel hc cdr1, cdr2, and cdr3 and novel lc cdr1, cdr2, and cdr3 designs |
US20090269786A1 (en) * | 2008-04-25 | 2009-10-29 | The Board Of Trustees Of The University Of Illinois | RHO1-Gamma Amino Butyric Acid C Receptor-Specific Antibodies |
EP3348573B1 (en) | 2008-04-25 | 2020-04-22 | Dyax Corp. | Method of producing antibodies against fcrn and use thereof |
BRPI0910482A2 (en) | 2008-04-29 | 2019-09-24 | Abbott Lab | double variable domain immunoglobins and their uses |
EP2113255A1 (en) | 2008-05-02 | 2009-11-04 | f-star Biotechnologische Forschungs- und Entwicklungsges.m.b.H. | Cytotoxic immunoglobulin |
CN102076865B (en) | 2008-05-02 | 2016-03-16 | 西雅图基因公司 | The antibody reduced for the manufacture of core fucosylation and the method and composition of antibody derivatives |
CL2009001083A1 (en) | 2008-05-06 | 2011-01-07 | Genentech Inc | Recipient variants of the c3 complement protein (crig), chimeric molecule and pharmaceutical composition comprising them; use of variants to prepare a medicament to prevent or treat a disease associated with complement, such as an inflammatory or autoimmune disease. |
EP2116556B1 (en) | 2008-05-09 | 2016-03-23 | AbbVie Deutschland GmbH & Co KG | Antibodies to receptor of advanced glycation end products (RAGE) and uses thereof |
SI2279004T1 (en) * | 2008-05-16 | 2015-05-29 | F. Hoffmann-La Roche Ag | Use of biomarkers for assessing treatment of gastrointestinal inflammatory disorders with beta7integrin antagonists |
US8093018B2 (en) | 2008-05-20 | 2012-01-10 | Otsuka Pharmaceutical Co., Ltd. | Antibody identifying an antigen-bound antibody and an antigen-unbound antibody, and method for preparing the same |
EP2599793A1 (en) | 2008-05-29 | 2013-06-05 | Nuclea Biotechnologies, Inc. | Anti-phospho-akt antibodies |
SG191625A1 (en) | 2008-06-03 | 2013-07-31 | Abbott Lab | Dual variable domain immunoglobulins and uses thereof |
EP2297209A4 (en) | 2008-06-03 | 2012-08-01 | Abbott Lab | Dual variable domain immunoglobulins and uses thereof |
CA2726845C (en) | 2008-06-04 | 2017-09-26 | Macrogenics, Inc. | Antibodies with altered binding to fcrn and methods of using same |
WO2009150623A1 (en) | 2008-06-13 | 2009-12-17 | Pfizer Inc | Treatment of chronic prostatitis |
CN102144036B (en) | 2008-07-07 | 2014-07-16 | 解码遗传学私营有限责任公司 | Genetic variants for breast cancer risk assessment |
AU2009269095B2 (en) | 2008-07-08 | 2016-05-19 | Oncomed Pharmaceuticals, Inc. | Notch-binding agents and antagonists and methods of use thereof |
US8822645B2 (en) | 2008-07-08 | 2014-09-02 | Abbvie Inc. | Prostaglandin E2 dual variable domain immunoglobulins and uses thereof |
CN104829718A (en) | 2008-07-08 | 2015-08-12 | 艾伯维公司 | Prostaglandin E2 binding proteins and uses thereof |
DK2982695T3 (en) * | 2008-07-09 | 2019-05-13 | Biogen Ma Inc | COMPOSITIONS CONCERNING ANTIBODIES AGAINST LINGO OR FRAGMENTS THEREOF |
ES2442024T3 (en) | 2008-07-15 | 2014-02-07 | Academia Sinica | Glucan matrices on glass slides coated with PTFE type aluminum and related methods |
EP2321264B1 (en) | 2008-07-23 | 2016-05-04 | President and Fellows of Harvard College | Deacetylase inhibitors and uses thereof |
US10138283B2 (en) * | 2008-07-23 | 2018-11-27 | Ambrx, Inc. | Modified bovine G-CSF polypeptides and their uses |
US9182406B2 (en) * | 2008-08-04 | 2015-11-10 | Biodesy, Inc. | Nonlinear optical detection of molecules comprising an unnatural amino acid possessing a hyperpolarizability |
KR20110039348A (en) | 2008-08-06 | 2011-04-15 | 노보 노르디스크 헬스 케어 악티엔게젤샤프트 | Conjugated proteins with prolonged in vivo efficacy |
WO2010019702A2 (en) | 2008-08-12 | 2010-02-18 | Oncomed Pharmaceuticals, Inc. | Ddr1-binding agents and methods of use thereof |
CA2733642A1 (en) | 2008-08-14 | 2010-02-18 | Cephalon Australia Pty Ltd | Anti-il-12/il-23 antibodies |
CA3059768A1 (en) | 2008-09-05 | 2010-03-11 | President And Fellows Of Harvard College | Continuous directed evolution of proteins and nucleic acids |
TWI445716B (en) | 2008-09-12 | 2014-07-21 | Rinat Neuroscience Corp | Pcsk9 antagonists |
AU2009293640A1 (en) * | 2008-09-22 | 2010-03-25 | Calmune Corporation | Methods and vectors for display of 2G12 -derived domain exchanged antibodies |
WO2010033237A2 (en) * | 2008-09-22 | 2010-03-25 | Calmune Corporation | Methods for creating diversity in libraries and libraries, display vectors and methods, and displayed molecules |
EP2352841A2 (en) | 2008-09-23 | 2011-08-10 | President and Fellows of Harvard College | Sirt4 and uses thereof |
CA2739357A1 (en) | 2008-09-23 | 2010-04-08 | Wyeth Llc | Methods for predicting production of activating signals by cross-linked binding proteins |
CN102224238B (en) | 2008-09-26 | 2015-06-10 | Ambrx公司 | Non-natural amino acid replication-dependent microorganisms and vaccines |
US8313942B2 (en) | 2008-09-26 | 2012-11-20 | Wyeth Llc | Compatible display vector systems |
CA2998281C (en) | 2008-09-26 | 2022-08-16 | Dana-Farber Cancer Institute, Inc. | Human anti-pd-1 antobodies and uses therefor |
US8551789B2 (en) | 2010-04-01 | 2013-10-08 | OncoMed Pharmaceuticals | Frizzled-binding agents and their use in screening for WNT inhibitors |
CN102232085A (en) | 2008-09-26 | 2011-11-02 | Ambrx公司 | Modified animal erythropoietin polypeptides and their uses |
CA2738485A1 (en) | 2008-09-26 | 2010-04-01 | Oncomed Pharmaceuticals, Inc. | Frizzled-binding agents and uses thereof |
PT3335728T (en) | 2008-10-10 | 2020-02-19 | Childrens Medical Center | Biochemically stabilized hiv-1 env trimer vaccine |
AU2009303453B2 (en) | 2008-10-14 | 2015-02-26 | Dyax Corp. | Use of IGF-II/IGF-IIE binding for the treatment and prevention of systemic sclerosis associated pulmonary fibrosis |
US10131693B2 (en) | 2008-10-20 | 2018-11-20 | Gwangju Institute Of Science And Technology | Bipodal-peptide binder |
ES2535734T3 (en) | 2008-10-20 | 2015-05-14 | Abbvie Inc. | Isolation and purification of antibodies by affinity chromatography with protein A |
WO2010048615A2 (en) | 2008-10-24 | 2010-04-29 | The Government Of The United States Of America As Represented By The Secretary, Department Of Health & Human Services, Center For Disease Control And Prevention | Human ebola virus species and compositions and methods thereof |
AU2009333791B2 (en) | 2008-10-29 | 2013-04-04 | Ablynx N.V. | Formulations of single domain antigen binding molecules |
CN102272154A (en) | 2008-10-29 | 2011-12-07 | 惠氏有限责任公司 | Methods for purification of single domain antigen binding molecules |
US8821880B2 (en) | 2008-10-29 | 2014-09-02 | China Synthetic Rubber Corporation | Methods and agents for the diagnosis and treatment of hepatocellular carcinoma |
CA3149920A1 (en) | 2008-10-31 | 2010-05-06 | Janssen Biotech, Inc. | Fibronectin type iii domain based scaffold compositions, methods and uses |
AU2009313551B2 (en) | 2008-11-07 | 2015-12-17 | Fabrus Llc | Anti-DLL4 antibodies and uses thereof |
EP2894165B1 (en) | 2008-11-10 | 2023-01-04 | Alexion Pharmaceuticals, Inc. | Methods and compositions for treating complement-associated disorders |
KR101588547B1 (en) | 2008-11-11 | 2016-01-28 | 더 리젠츠 오브 더 유니버시티 오브 미시간 | Anti-cxcr1 compositions and methods |
ES2719496T3 (en) | 2008-11-12 | 2019-07-10 | Medimmune Llc | Antibody formulation |
DK2189539T4 (en) | 2008-11-21 | 2018-09-17 | Chimera Biotec Gmbh | Conjugate complexes for analyte detection |
SI2361085T2 (en) | 2008-11-22 | 2018-11-30 | F. Hoffmann-La Roche Ag | Use of anti-vegf antibody in combination with chemotherapy for treating breast cancer |
CA2744555A1 (en) | 2008-11-26 | 2010-06-03 | Five Prime Therapeutics, Inc. | Compositions and methods for regulating collagen and smooth muscle actin expression by serpine2 |
US9598491B2 (en) | 2008-11-28 | 2017-03-21 | Emory University | Methods for the treatment of infections and tumors |
SG171812A1 (en) * | 2008-12-04 | 2011-07-28 | Abbott Lab | Dual variable domain immunoglobulins and uses thereof |
DK2376535T3 (en) | 2008-12-09 | 2017-06-12 | Hoffmann La Roche | ANTI-PD-L1 ANTIBODIES AND THEIR USE TO PROMOTE T CELL FUNCTION |
PL2786762T3 (en) | 2008-12-19 | 2019-09-30 | Macrogenics, Inc. | Covalent diabodies and uses thereof |
CA2746120A1 (en) | 2008-12-23 | 2010-07-01 | Genentech, Inc. | Methods and compositions for diagnostic use in cancer patients |
WO2010078526A1 (en) | 2008-12-31 | 2010-07-08 | Biogen Idec Ma Inc. | Anti-lymphotoxin antibodies |
US9181315B2 (en) | 2009-01-08 | 2015-11-10 | Dana-Farber Cancer Institute, Inc. | Compositions and methods for induced brown fat differentiation |
GB0900425D0 (en) | 2009-01-12 | 2009-02-11 | Ucb Pharma Sa | Biological products |
WO2010082134A1 (en) | 2009-01-14 | 2010-07-22 | Iq Therapeutics Bv | Combination antibodies for the treatment and prevention of disease caused by bacillus anthracis and related bacteria and their toxins |
WO2010085510A1 (en) | 2009-01-20 | 2010-07-29 | Zadeh Homayoun H | Antibody mediated osseous regeneration |
WO2010084408A2 (en) | 2009-01-21 | 2010-07-29 | Oxford Biotherapeutics Ltd. | Pta089 protein |
CA2747825A1 (en) | 2009-01-22 | 2010-07-29 | Novo Nordisk Health Care Ag | Stable growth hormone compounds |
US20100310591A1 (en) * | 2009-01-28 | 2010-12-09 | Robert Humphreys | Ii-KEY HYBRID PEPTIDES THAT MODULATE THE IMMUNE RESPONSE TO INFLUENZA |
US8383778B2 (en) | 2009-01-29 | 2013-02-26 | Abbvie Inc. | IL-1 binding proteins |
US20110165063A1 (en) * | 2009-01-29 | 2011-07-07 | Abbott Laboratories | Il-1 binding proteins |
WO2010086828A2 (en) | 2009-02-02 | 2010-08-05 | Rinat Neuroscience Corporation | Agonist anti-trkb monoclonal antibodies |
US8852608B2 (en) | 2009-02-02 | 2014-10-07 | Medimmune, Llc | Antibodies against and methods for producing vaccines for respiratory syncytial virus |
AU2010213892B2 (en) | 2009-02-12 | 2014-10-23 | Janssen Biotech, Inc. | Fibronectin type III domain based scaffold compositions, methods and uses |
WO2010093993A2 (en) | 2009-02-12 | 2010-08-19 | Human Genome Sciences, Inc. | Use of b lymphocyte stimulator protein antagonists to promote transplantation tolerance |
HUE042114T2 (en) | 2009-02-17 | 2019-06-28 | Ucb Biopharma Sprl | Antibody molecules having specificity for human ox40 |
WO2010096388A2 (en) | 2009-02-18 | 2010-08-26 | Carnegie Mellon University | Quenched dendrimeric dyes for bright detection |
US8030026B2 (en) | 2009-02-24 | 2011-10-04 | Abbott Laboratories | Antibodies to troponin I and methods of use thereof |
JP5836807B2 (en) | 2009-03-05 | 2015-12-24 | アッヴィ・インコーポレイテッド | IL-17 binding protein |
WO2010102167A1 (en) | 2009-03-05 | 2010-09-10 | Becton, Dickinson And Company | Matrix metalloproteinase-7 (mmp-7) monoclonal antibodies and methods for their use in the detection of ovarian cancer |
EP2405920A1 (en) | 2009-03-06 | 2012-01-18 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Novel therapy for anxiety |
EP2403875A1 (en) | 2009-03-06 | 2012-01-11 | Tripath Imaging, Inc. | Glycodelin monoclonal antibodies and methods for their use in the detection of ovarian cancer |
CN102333791B (en) | 2009-03-10 | 2014-06-25 | 株式会社遗传科技 | Generation, expression and characterization of the humanized k33n monoclonal antibody |
GB0904214D0 (en) | 2009-03-11 | 2009-04-22 | Ucb Pharma Sa | Biological products |
BRPI1006270B1 (en) | 2009-03-25 | 2022-08-16 | Genentech, Inc | ANTI-A5SS1 ANTIBODY, IMMUNOCONJUGATE, PHARMACEUTICAL COMPOSITION, IN VITRO OR EX VIVO METHOD TO DETECT A5SS1 PROTEIN, USE OF AN ANTIBODY AND KIT TO DETECT A5SS1 PROTEIN |
WO2010111367A1 (en) | 2009-03-25 | 2010-09-30 | Genentech, Inc. | Anti-fgfr3 antibodies and methods using same |
US8466260B2 (en) | 2009-04-01 | 2013-06-18 | Genentech, Inc. | Anti-FcRH5 antibodies and immunoconjugates and methods of use |
US8795963B2 (en) | 2009-04-03 | 2014-08-05 | Decode Genetics Ehf. | Genetic markers for risk management of atrial fibrillation and stroke |
EP2241323A1 (en) | 2009-04-14 | 2010-10-20 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Tenascin-W and brain cancers |
MA33276B1 (en) | 2009-04-20 | 2012-05-02 | Oxford Biotherapeutics Ltd | Cadherine-17 antibodies |
ES2708124T3 (en) | 2009-04-27 | 2019-04-08 | Oncomed Pharm Inc | Procedure for preparing heteromultimeric molecules |
US8765916B2 (en) | 2009-04-29 | 2014-07-01 | The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. | ERG monoclonal antibodies |
MX2011011541A (en) | 2009-04-29 | 2012-02-28 | Abbott Biotech Ltd | Automatic injection device. |
KR101224468B1 (en) | 2009-05-20 | 2013-01-23 | 주식회사 파멥신 | Bispecific antibody having a novel form and use thereof |
BRPI1011195B1 (en) * | 2009-05-20 | 2020-10-13 | Novimmune S.A | methods to produce a collection of nucleic acids |
US8680055B2 (en) | 2009-06-03 | 2014-03-25 | University Of Southern California | Methods for decreasing steroidogenesis in prostate cancer cells |
EP3329932A1 (en) | 2009-06-10 | 2018-06-06 | New York University | Immunological targeting of pathological tau proteins |
EP2261242A1 (en) | 2009-06-10 | 2010-12-15 | Universite Catholique De Louvain | Aspartate-N-acetyltransferase enzyme, diagnostic method and therapeutic method |
WO2010146511A1 (en) | 2009-06-17 | 2010-12-23 | Pfizer Limited | Treatment of overactive bladder |
BRPI1015234A2 (en) | 2009-06-22 | 2018-02-20 | Medimmune Llc | fc regions designed for site specific conjugation. |
GB0910725D0 (en) | 2009-06-22 | 2009-08-05 | Heptares Therapeutics Ltd | Mutant proteins and methods for producing them |
EP2272979A1 (en) | 2009-06-30 | 2011-01-12 | Centre National de la Recherche Scientifique (CNRS) | Method for testing a subject thought to be predisposed to having cancer |
US20120226119A1 (en) | 2009-07-09 | 2012-09-06 | Hoffmann-La Roche Inc. | Vivo tumor vasculature imaging |
US8796182B2 (en) | 2009-07-10 | 2014-08-05 | Decode Genetics Ehf. | Genetic markers associated with risk of diabetes mellitus |
TW201106972A (en) | 2009-07-27 | 2011-03-01 | Genentech Inc | Combination treatments |
SG178177A1 (en) | 2009-07-31 | 2012-03-29 | Genentech Inc | Inhibition of tumor metastasis using bv8- or g-csf-antagonists |
US9259476B2 (en) | 2009-07-31 | 2016-02-16 | Wayne State University | Monophosphorylated lipid A derivatives |
WO2011014771A1 (en) | 2009-07-31 | 2011-02-03 | Wayne State University | Monophosphorylated lipid a derivatives |
CN102612376A (en) | 2009-08-06 | 2012-07-25 | 诺沃-诺迪斯克保健股份有限公司 | Growth hormones with prolonged in-vivo efficacy |
PT2464725T (en) | 2009-08-11 | 2020-05-21 | Hoffmann La Roche | Production of proteins in glutamine-free cell culture media |
WO2011019393A2 (en) | 2009-08-11 | 2011-02-17 | President And Fellows Of Harvard College | Class- and isoform-specific hdac inhibitors and uses thereof |
JP5762408B2 (en) * | 2009-08-13 | 2015-08-12 | クルセル ホランド ベー ヴェー | Antibodies against human respiratory syncytial virus (RSV) and methods of use |
WO2011022264A1 (en) | 2009-08-15 | 2011-02-24 | Genentech, Inc. | Anti-angiogenesis therapy for the treatment of previously treated breast cancer |
EP2292266A1 (en) | 2009-08-27 | 2011-03-09 | Novartis Forschungsstiftung, Zweigniederlassung | Treating cancer by modulating copine III |
IN2012DN02521A (en) | 2009-08-28 | 2015-08-28 | Rinat Neuroscience Corp | |
CA2952742A1 (en) | 2009-08-29 | 2011-03-03 | Abbvie Inc. | Therapeutic dll4 binding proteins |
KR20120060877A (en) | 2009-09-01 | 2012-06-12 | 아보트 러보러터리즈 | Dual variable domain immunoglobulins and uses thereof |
US20110059111A1 (en) | 2009-09-01 | 2011-03-10 | Los Angeles Biomedical Research Institute At Harbor-Ucla Medical Center | Mammalian receptors as targets for antibody and active vaccination therapy against mold infections |
CA2772715C (en) | 2009-09-02 | 2019-03-26 | Genentech, Inc. | Mutant smoothened and methods of using the same |
US20120283415A1 (en) | 2009-09-10 | 2012-11-08 | Ucb Pharma S.A. | Multivalent Antibodies |
KR20140048229A (en) | 2009-09-14 | 2014-04-23 | 애브비 인코포레이티드 | Methods for treating psoriasis |
US20110082054A1 (en) * | 2009-09-14 | 2011-04-07 | Dyax Corp. | Libraries of genetic packages comprising novel hc cdr3 designs |
MX2012002909A (en) | 2009-09-17 | 2012-04-19 | Hoffmann La Roche | Methods and compositions for diagnostics use in cancer patients. |
US20110189183A1 (en) | 2009-09-18 | 2011-08-04 | Robert Anthony Williamson | Antibodies against candida, collections thereof and methods of use |
US20120244170A1 (en) | 2009-09-22 | 2012-09-27 | Rafal Ciosk | Treating cancer by modulating mex-3 |
WO2011038290A2 (en) | 2009-09-25 | 2011-03-31 | The U. S. A., As Represented By The Secretary, Department Of Health And Human Services | Neutralizing antibodies to hiv-1 and their use |
GB201005063D0 (en) | 2010-03-25 | 2010-05-12 | Ucb Pharma Sa | Biological products |
CN102597775A (en) | 2009-09-25 | 2012-07-18 | 佐马技术有限公司 | Screening methods |
WO2011036555A1 (en) | 2009-09-25 | 2011-03-31 | University Of Oslo | Multivalent phage display systems and methods |
US8926976B2 (en) | 2009-09-25 | 2015-01-06 | Xoma Technology Ltd. | Modulators |
AU2010300531A1 (en) | 2009-09-30 | 2012-05-24 | President And Fellows Of Harvard College | Methods for modulation of autophagy through the modulation of autophagy-inhibiting gene products |
TW201116297A (en) | 2009-10-02 | 2011-05-16 | Sanofi Aventis | Antibodies that specifically bind to the EphA2 receptor |
EP2470569A1 (en) | 2009-10-13 | 2012-07-04 | Oxford Biotherapeutics Ltd. | Antibodies against epha10 |
JP2013507928A (en) | 2009-10-15 | 2013-03-07 | アボット・ラボラトリーズ | Dual variable domain immunoglobulins and uses thereof |
WO2011045352A2 (en) | 2009-10-15 | 2011-04-21 | Novartis Forschungsstiftung | Spleen tyrosine kinase and brain cancers |
PT2488204E (en) | 2009-10-16 | 2016-06-09 | Oncomed Pharm Inc | Therapeutic combination and use of dll4 antagonist antibodies and anti-hypertensive agents |
KR101830596B1 (en) | 2009-10-20 | 2018-02-22 | 애브비 인코포레이티드 | Isolation and purification of anti-il-13 antibodies using protein a affinity chromatography |
WO2011050194A1 (en) | 2009-10-22 | 2011-04-28 | Genentech, Inc. | Methods and compositions for modulating hepsin activation of macrophage-stimulating protein |
WO2011050188A1 (en) | 2009-10-22 | 2011-04-28 | Genentech, Inc. | Anti-hepsin antibodies and methods using same |
WO2011056494A1 (en) | 2009-10-26 | 2011-05-12 | Genentech, Inc. | Activin receptor-like kinase-1 antagonist and vegfr3 antagonist combinations |
WO2011056502A1 (en) | 2009-10-26 | 2011-05-12 | Genentech, Inc. | Bone morphogenetic protein receptor type ii compositions and methods of use |
WO2011056497A1 (en) | 2009-10-26 | 2011-05-12 | Genentech, Inc. | Activin receptor type iib compositions and methods of use |
US9234037B2 (en) | 2009-10-27 | 2016-01-12 | Ucb Biopharma Sprl | Method to generate antibodies to ion channels |
US20110098862A1 (en) | 2009-10-27 | 2011-04-28 | ExxonMobil Research Engineering Company Law Department | Multi-stage processes and control thereof |
GB0922434D0 (en) | 2009-12-22 | 2010-02-03 | Ucb Pharma Sa | antibodies and fragments thereof |
GB0922435D0 (en) | 2009-12-22 | 2010-02-03 | Ucb Pharma Sa | Method |
CA2778673A1 (en) * | 2009-10-27 | 2011-05-05 | Karen Margrete Miller | Function modifying nav 1.7 antibodies |
UY32979A (en) | 2009-10-28 | 2011-02-28 | Abbott Lab | IMMUNOGLOBULINS WITH DUAL VARIABLE DOMAIN AND USES OF THE SAME |
US20120213801A1 (en) | 2009-10-30 | 2012-08-23 | Ekaterina Gresko | Phosphorylated Twist1 and cancer |
WO2011053707A1 (en) | 2009-10-31 | 2011-05-05 | Abbott Laboratories | Antibodies to receptor for advanced glycation end products (rage) and uses thereof |
US20120282177A1 (en) | 2009-11-02 | 2012-11-08 | Christian Rohlff | ROR1 as Therapeutic and Diagnostic Target |
JP2013509874A (en) | 2009-11-04 | 2013-03-21 | エラスムス ユニバーシティ メディカル センター ロッテルダム | Novel compounds for modulating angiogenesis and methods of treatment using these compounds |
NZ598901A (en) | 2009-11-05 | 2014-08-29 | Genentech Inc | Methods and composition for secretion of heterologous polypeptides |
CA2780024A1 (en) | 2009-11-11 | 2011-05-19 | Gentian As | Immunoassay for assessing related analytes of different origin |
EP2499159B1 (en) | 2009-11-13 | 2017-01-04 | Dana-Farber Cancer Institute, Inc. | Compositions, kits, and methods for the diagnosis, prognosis, monitoring, treatment and modulation of post-transplant lymphoproliferative disorders and hypoxia associated angiogenesis disorders using galectin-1 |
GB0920127D0 (en) | 2009-11-17 | 2009-12-30 | Ucb Pharma Sa | Antibodies |
GB0920324D0 (en) | 2009-11-19 | 2010-01-06 | Ucb Pharma Sa | Antibodies |
TWI507524B (en) | 2009-11-30 | 2015-11-11 | Genentech Inc | Compositions and methods for the diagnosis and treatment of tumor |
US10087236B2 (en) | 2009-12-02 | 2018-10-02 | Academia Sinica | Methods for modifying human antibodies by glycan engineering |
US11377485B2 (en) | 2009-12-02 | 2022-07-05 | Academia Sinica | Methods for modifying human antibodies by glycan engineering |
ES2562832T3 (en) | 2009-12-08 | 2016-03-08 | Abbvie Deutschland Gmbh & Co Kg | Monoclonal antibodies against the RGM protein for use in the treatment of degeneration of the retinal nerve fiber layer |
CN102741290B (en) | 2009-12-09 | 2015-04-22 | 国家健康和医学研究院 | Monoclonal antibodies that bind B7H6 and uses thereof |
TWI505836B (en) | 2009-12-11 | 2015-11-01 | Genentech Inc | Anti-vegf-c antibodies and methods using same |
US8937159B2 (en) * | 2009-12-16 | 2015-01-20 | Abbvie Biotherapeutics Inc. | Anti-HER2 antibodies and their uses |
CN107095846A (en) | 2009-12-21 | 2017-08-29 | 霍夫曼-拉罗奇有限公司 | Antibody formulations |
NZ600361A (en) | 2009-12-21 | 2014-06-27 | Ambrx Inc | Modified bovine somatotropin polypeptides and their uses |
CN107056929A (en) | 2009-12-21 | 2017-08-18 | Ambrx 公司 | Porcine somatotropin polypeptide and its purposes by modification |
US20110165161A1 (en) * | 2009-12-23 | 2011-07-07 | Shih-Yao Lin | Anti-epcam antibodies that induce apoptosis of cancer cells and methods using same |
HUE027713T2 (en) | 2009-12-23 | 2016-10-28 | Hoffmann La Roche | Anti-bv8 antibodies and uses thereof |
HUE057244T2 (en) | 2010-01-06 | 2022-04-28 | Takeda Pharmaceuticals Co | Plasma kallikrein binding proteins |
RU2012134369A (en) | 2010-01-11 | 2014-02-20 | Алексион Фармасьютикалз, Инк | BIOMARKERS OF IMMUNOMODULATING EFFECTS IN PEOPLE EXPOSED TO ANTIMETAL TREATMENT AGAINST CD200 |
GB201000467D0 (en) | 2010-01-12 | 2010-02-24 | Ucb Pharma Sa | Antibodies |
TWI535445B (en) | 2010-01-12 | 2016-06-01 | 安可美德藥物股份有限公司 | Wnt antagonists and methods of treatment and screening |
WO2011088215A2 (en) | 2010-01-13 | 2011-07-21 | Oncomed Pharmaceuticals, Inc. | Notch1 binding agents and methods of use thereof |
US9745589B2 (en) | 2010-01-14 | 2017-08-29 | Cornell University | Methods for modulating skeletal remodeling and patterning by modulating SHN2 activity, SHN3 activity, or SHN2 and SHN3 activity in combination |
JP5980689B2 (en) | 2010-01-22 | 2016-08-31 | ノヴォ・ノルディスク・ヘルス・ケア・アーゲー | Stable growth hormone compound |
US10429384B2 (en) | 2010-01-22 | 2019-10-01 | Dana-Farber Cancer Institute, Inc. | Compositions, kits, and methods for identification, assessment, prevention, and therapy of metabolic disorders |
RU2605627C2 (en) | 2010-01-22 | 2016-12-27 | Ново Нордиск Хелс Кеа Аг | Growth hormones with prolonged efficacy in vivo |
US20120014956A1 (en) | 2010-02-02 | 2012-01-19 | Hartmut Kupper | Methods and compositions for predicting responsiveness to treatment with tnf-alpha inhibitor |
TWI518325B (en) | 2010-02-04 | 2016-01-21 | 自治醫科大學 | Identification, assessment, and therapy of cancers with innate or acquired resistance to alk inhibitors |
CA2789629A1 (en) | 2010-02-10 | 2011-08-18 | Immunogen, Inc. | Cd20 antibodies and uses thereof |
WO2011103330A2 (en) | 2010-02-17 | 2011-08-25 | The Johns Hopkins University | Novel phosphorylation of cardiac troponin i as a monitor for cardiac injury |
WO2011101328A2 (en) | 2010-02-18 | 2011-08-25 | Roche Glycart Ag | Treatment with a humanized igg class anti egfr antibody and an antibody against insulin like growth factor 1 receptor |
ES2519348T3 (en) | 2010-02-18 | 2014-11-06 | Genentech, Inc. | Neurregulin antagonists and their use in cancer treatment |
SG183335A1 (en) | 2010-02-23 | 2012-09-27 | Genentech Inc | Compositions and methods for the diagnosis and treatment of tumor |
RU2012140447A (en) | 2010-02-23 | 2014-03-27 | Дженентек, Инк. | ANTIANGIOGENIC THERAPY FOR TREATMENT OF OVARIAN CANCER |
SA114360064B1 (en) | 2010-02-24 | 2016-01-05 | رينات نيوروساينس كوربوريشن | Antagonist anti-il-7 receptor antibodies and methods |
KR101637138B1 (en) | 2010-02-24 | 2016-07-06 | 이뮤노젠 아이엔씨 | Folate receptor 1 antibodies and immunoconjugates and uses thereof |
WO2011109298A2 (en) | 2010-03-02 | 2011-09-09 | Abbott Laboratories | Therapeutic dll4 binding proteins |
US20130004519A1 (en) | 2010-03-05 | 2013-01-03 | Ruth Chiquet-Ehrismann | Smoci, tenascin-c and brain cancers |
CN105218674A (en) | 2010-03-11 | 2016-01-06 | 瑞纳神经科学公司 | The antibody combined in pH dependence antigen |
MY173839A (en) | 2010-03-12 | 2020-02-24 | Debiopharm Int Sa | Cd37-binding molecules and immunoconjugates thereof |
WO2011116026A2 (en) | 2010-03-15 | 2011-09-22 | The Board Of Trustees Of The University Of Illinois | Inhibitors of beta integrin-g protein alpha subunit binding interactions |
US20130045871A1 (en) * | 2010-03-18 | 2013-02-21 | Cornell University | Engineering correctly folded antibodies using inner membrane display of twin-arginine translocation intermediates |
WO2011119484A1 (en) | 2010-03-23 | 2011-09-29 | Iogenetics, Llc | Bioinformatic processes for determination of peptide binding |
MX2012010853A (en) | 2010-03-24 | 2013-01-29 | Genentech Inc | Anti-lrp6 antibodies. |
AU2011230619C1 (en) | 2010-03-25 | 2016-06-23 | Oregon Health & Science University | CMV glycoproteins and recombinant vectors |
GB201005064D0 (en) | 2010-03-25 | 2010-05-12 | Ucb Pharma Sa | Biological products |
TR201903279T4 (en) | 2010-03-25 | 2019-03-21 | Ucb Biopharma Sprl | Disulfide stabilized DVD-IG molecules. |
WO2011130332A1 (en) | 2010-04-12 | 2011-10-20 | Academia Sinica | Glycan arrays for high throughput screening of viruses |
CN102933601B (en) | 2010-04-15 | 2016-06-08 | Abbvie公司 | Amyloid beta is in conjunction with albumen |
US20130034543A1 (en) | 2010-04-19 | 2013-02-07 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Resear | Modulating xrn1 |
EP2380909A1 (en) | 2010-04-26 | 2011-10-26 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | PTK-7 protein involved in breast cancer |
CA2797856C (en) | 2010-04-30 | 2018-09-18 | Alexion Pharmaceuticals, Inc. | Anti-c5a antibodies and methods for using the antibodies |
WO2011140114A2 (en) | 2010-05-03 | 2011-11-10 | University Of Rochester | Anti-glucosaminidase passive immunization for staphylococcus aureus infections |
SG185027A1 (en) | 2010-05-03 | 2012-11-29 | Genentech Inc | Compositions and methods for the diagnosis and treatment of tumor |
WO2011141823A2 (en) | 2010-05-14 | 2011-11-17 | Orega Biotech | Methods of treating and/or preventing cell proliferation disorders with il-17 antagonists |
TWI615405B (en) | 2010-05-14 | 2018-02-21 | 艾伯維有限公司 | Il-1 binding proteins |
US9995679B2 (en) | 2010-05-25 | 2018-06-12 | Carnegie Mellon University | Targeted probes of cellular physiology |
WO2011153243A2 (en) | 2010-06-02 | 2011-12-08 | Genentech, Inc. | Anti-angiogenesis therapy for treating gastric cancer |
NZ701208A (en) | 2010-06-03 | 2016-05-27 | Genentech Inc | Immuno-pet imaging of antibodies and immunoconjugates and uses thereof |
JP5944382B2 (en) | 2010-06-03 | 2016-07-05 | アッヴィ バイオテクノロジー リミテッド | Uses and compositions for the treatment of sweat gland abscess (HS) |
WO2012047324A2 (en) | 2010-06-10 | 2012-04-12 | President And Fellows Of Harvard College | Systems and methods for amplification and phage display |
EP2580239A1 (en) | 2010-06-10 | 2013-04-17 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Treating cancer by modulating mammalian sterile 20-like kinase 3 |
TW201207383A (en) | 2010-06-16 | 2012-02-16 | Abbott Lab | Comparison of protein samples |
RU2577986C2 (en) | 2010-06-18 | 2016-03-20 | Дженентек, Инк. | Antibodies against axl and their application |
US20120009196A1 (en) | 2010-07-08 | 2012-01-12 | Abbott Laboratories | Monoclonal antibodies against hepatitis c virus core protein |
SG186983A1 (en) | 2010-07-09 | 2013-02-28 | Genentech Inc | Anti-neuropilin antibodies and methods of use |
UY33492A (en) | 2010-07-09 | 2012-01-31 | Abbott Lab | IMMUNOGLOBULINS WITH DUAL VARIABLE DOMAIN AND USES OF THE SAME |
WO2012006635A1 (en) | 2010-07-09 | 2012-01-12 | Biogen Idec Hemophilia Inc. | Processable single chain molecules and polypeptides made using same |
EA027835B1 (en) | 2010-07-09 | 2017-09-29 | Круселл Холланд Б.В. | Anti-human respiratory syncytial virus (rsv) antibodies and methods of use thereof |
US20120100166A1 (en) | 2010-07-15 | 2012-04-26 | Zyngenia, Inc. | Ang-2 Binding Complexes and Uses Thereof |
WO2012007880A2 (en) | 2010-07-16 | 2012-01-19 | Ablynx Nv | Modified single domain antigen binding molecules and uses thereof |
EP4219805A1 (en) | 2010-07-16 | 2023-08-02 | Adimab, LLC | Antibody libraries |
ES2629850T3 (en) | 2010-07-19 | 2017-08-16 | Otago Innovation Limited | Signal biomarkers |
WO2012010582A1 (en) | 2010-07-21 | 2012-01-26 | Roche Glycart Ag | Anti-cxcr5 antibodies and methods of use |
JP2013533264A (en) | 2010-07-22 | 2013-08-22 | ノヴォ・ノルディスク・ヘルス・ケア・アーゲー | Growth hormone conjugate |
AU2011280969A1 (en) | 2010-07-23 | 2013-02-07 | Trustees Of Boston University | Anti-Despr inhibitors as therapeutics for inhibition of pathological angiogenesis and tumor cell invasiveness and for molecular imaging and targeted delivery |
WO2012015758A2 (en) | 2010-07-30 | 2012-02-02 | Saint Louis University | Methods of treating pain |
JP5964300B2 (en) | 2010-08-02 | 2016-08-03 | マクロジェニクス,インコーポレーテッド | Covalently bonded diabody and its use |
WO2012018387A2 (en) | 2010-08-02 | 2012-02-09 | Population Diagnotics, Inc. | Compositions and methods for discovery of causative mutations in genetic disorders |
JP2013541501A (en) | 2010-08-03 | 2013-11-14 | エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト | Biomarkers for chronic lymphocytic leukemia (CLL) |
CA2807014A1 (en) | 2010-08-03 | 2012-02-09 | Abbvie Inc. | Dual variable domain immunoglobulins and uses thereof |
WO2012019024A2 (en) | 2010-08-04 | 2012-02-09 | Immunogen, Inc. | Her3-binding molecules and immunoconjugates thereof |
BR112013002532A2 (en) | 2010-08-05 | 2016-05-31 | Hoffmann La Roche | anti-mhc antibody anti-viral cytokine fusion protein |
WO2012019061A2 (en) | 2010-08-05 | 2012-02-09 | Stem Centrx, Inc. | Novel effectors and methods of use |
EP2420250A1 (en) | 2010-08-13 | 2012-02-22 | Universitätsklinikum Münster | Anti-Syndecan-4 antibodies |
PL2603530T3 (en) | 2010-08-13 | 2018-03-30 | Roche Glycart Ag | Anti-fap antibodies and methods of use |
KR101653030B1 (en) | 2010-08-13 | 2016-08-31 | 로슈 글리카트 아게 | Anti-tenascin-c a2 antibodies and methods of use |
US9062101B2 (en) | 2010-08-14 | 2015-06-23 | AbbVie Deutschland GmbH & Co. KG | Amyloid-beta binding proteins |
US9567386B2 (en) | 2010-08-17 | 2017-02-14 | Ambrx, Inc. | Therapeutic uses of modified relaxin polypeptides |
SG187736A1 (en) | 2010-08-17 | 2013-03-28 | Ambrx Inc | Modified relaxin polypeptides and their uses |
CA2802278A1 (en) | 2010-08-19 | 2012-02-23 | Veit Peter Grunert | An assay for measurement of antibodies binding to a therapeutic monoclonal antibody |
HUE058226T2 (en) | 2010-08-19 | 2022-07-28 | Zoetis Belgium S A | Anti-ngf antibodies and their use |
GB201014033D0 (en) | 2010-08-20 | 2010-10-06 | Ucb Pharma Sa | Biological products |
US9046513B2 (en) | 2010-08-26 | 2015-06-02 | Abbvie Inc. | Dual variable domain immunoglobulins and uses thereof |
WO2012027723A1 (en) | 2010-08-27 | 2012-03-01 | Stem Centrx, Inc | Notum protein modulators and methods of use |
EP2612151B1 (en) | 2010-08-31 | 2017-08-09 | Genentech, Inc. | Biomarkers and methods of treatment |
EP2611464B1 (en) | 2010-09-03 | 2018-04-25 | AbbVie Stemcentrx LLC | Novel modulators and methods of use |
US8551479B2 (en) | 2010-09-10 | 2013-10-08 | Oncomed Pharmaceuticals, Inc. | Methods for treating melanoma |
EP2614080A1 (en) | 2010-09-10 | 2013-07-17 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Phosphorylated twist1 and metastasis |
CN105753933A (en) | 2010-09-20 | 2016-07-13 | Abbvie 公司 | Purification Of Antibodies By Using Simulated Moving Bed Chromatography |
TWI480288B (en) | 2010-09-23 | 2015-04-11 | Lilly Co Eli | Formulations for bovine granulocyte colony stimulating factor and variants thereof |
EP2446898A1 (en) | 2010-09-30 | 2012-05-02 | Laboratorios Del. Dr. Esteve, S.A. | Use of growth hormone to enhance the immune response in immunosuppressed patients |
US9005907B2 (en) | 2010-10-01 | 2015-04-14 | St. Jude Children's Research Hospital | Methods and compositions for typing molecular subgroups of medulloblastoma |
JP5974012B2 (en) | 2010-10-05 | 2016-08-23 | ジェネンテック, インコーポレイテッド | Mutant smoothened and method of using the same |
WO2012052391A1 (en) | 2010-10-19 | 2012-04-26 | Glaxo Group Limited | Polypeptide with jmjd3 catalytic activity |
CN103298489A (en) | 2010-10-29 | 2013-09-11 | 伊缪诺金公司 | Novel EGFR-binding molecules and immunoconjugates thereof |
EP2632947A4 (en) | 2010-10-29 | 2015-03-18 | Immunogen Inc | Non-antagonistic egfr-binding molecules and immunoconjugates thereof |
WO2012064836A1 (en) | 2010-11-10 | 2012-05-18 | Genentech, Inc. | Methods and compositions for neural disease immunotherapy |
EP2640738A1 (en) | 2010-11-15 | 2013-09-25 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Anti-fungal agents |
EP2640831A1 (en) | 2010-11-17 | 2013-09-25 | Sea Lane Biotechnologies,llc. | Influenza virus neutralizing agents that mimic the binding site of an influenza neutralizing antibody |
US9072766B2 (en) | 2010-11-18 | 2015-07-07 | Beth Israel Deaconess Medical Center, Inc. | Methods of treating obesity by inhibiting nicotinamide N-methyl transferase (NNMT) |
WO2012071436A1 (en) | 2010-11-24 | 2012-05-31 | Genentech, Inc. | Method of treating autoimmune inflammatory disorders using il-23r loss-of-function mutants |
PE20140673A1 (en) | 2010-12-08 | 2014-06-14 | Stem Centrx Inc | NEW MODULATORS AND METHODS FOR THEIR USE |
BR122020012255B1 (en) | 2010-12-16 | 2022-08-09 | Genentech, Inc | USE OF AN ANTI-IL-13 ANTIBODY, USES OF A TH2 PATHWAY INHIBITOR AND ANTI-PERIOSTIN ANTIBODIES |
US9029502B2 (en) | 2010-12-20 | 2015-05-12 | The Regents Of The University Of Michigan | Inhibitors of the epidermal growth factor receptor-heat shock protein 90 binding interaction |
NZ610976A (en) | 2010-12-20 | 2015-07-31 | Genentech Inc | Anti-mesothelin antibodies and immunoconjugates |
TW201249865A (en) | 2010-12-21 | 2012-12-16 | Abbott Lab | Dual variable domain immunoglobulins and uses thereof |
WO2012088094A2 (en) | 2010-12-21 | 2012-06-28 | Abbott Laboratories | Il-1 binding proteins |
JP2014511106A (en) | 2010-12-22 | 2014-05-08 | ジェネンテック, インコーポレイテッド | Anti-PCSK9 antibody and method of use |
MX2013007392A (en) | 2010-12-22 | 2013-11-01 | Cephalon Australia Pty Ltd | Modified antibody with improved half-life. |
US9394537B2 (en) | 2010-12-22 | 2016-07-19 | President And Fellows Of Harvard College | Continuous directed evolution |
JP2014504503A (en) | 2010-12-28 | 2014-02-24 | ゾーマ テクノロジー リミテッド | Cell surface display using PDZ domains |
WO2012089814A1 (en) | 2010-12-30 | 2012-07-05 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Antigen binding formats for use in therapeutic treatments or diagnostic assays |
WO2012092539A2 (en) | 2010-12-31 | 2012-07-05 | Takeda Pharmaceutical Company Limited | Antibodies to dll4 and uses thereof |
MX2013007559A (en) | 2011-01-03 | 2013-07-29 | Hoffmann La Roche | A pharmaceutical composition of a complex of an anti-dig antibody and digoxigenin that is conjugated to a peptide. |
US10208349B2 (en) | 2011-01-07 | 2019-02-19 | Ucb Biopharma Sprl | Lipocalin 2 as a biomarker for IL-17 inhibitor therapy efficacy |
GB201100282D0 (en) | 2011-01-07 | 2011-02-23 | Ucb Pharma Sa | Biological methods |
SG10201510762YA (en) | 2011-01-14 | 2016-01-28 | Ucb Pharma Sa | Antibody molecules which bind il-17a and il-17f |
ES2637979T3 (en) | 2011-01-24 | 2017-10-18 | Abbvie Biotechnology Ltd | Automatic injection devices with overmolded grip surfaces |
WO2012103165A2 (en) | 2011-01-26 | 2012-08-02 | Kolltan Pharmaceuticals, Inc. | Anti-kit antibodies and uses thereof |
US9447187B2 (en) | 2011-02-03 | 2016-09-20 | Alexion Pharmaceuticals, Inc. | Use of an anti-CD200 antibody for prolonging the survival of allografts |
WO2012109238A2 (en) | 2011-02-07 | 2012-08-16 | President And Fellows Of Harvard College | Methods for increasing immune responses using agents that directly bind to and activate ire-1 |
SA112330278B1 (en) | 2011-02-18 | 2015-10-09 | ستيم سينتركس، انك. | Novel modulators and methods of use |
PE20140627A1 (en) | 2011-03-02 | 2014-05-30 | Berg Llc | CELL-BASED INTERROGATORY TESTS AND THE USE OF THEM |
AU2012225574A1 (en) | 2011-03-07 | 2013-09-26 | University Of Louisville Research Foundation, Inc. | Predictive marker of DNMT1 inhibitor therapeutic efficacy and methods of using the marker |
WO2012125735A1 (en) | 2011-03-15 | 2012-09-20 | Abott Laboratories | An integrated approach to the isolation and purification of antibodies |
CA2831136A1 (en) | 2011-03-21 | 2012-09-27 | Biodesy, Llc | Classification of kinase inhibitors using nonlinear optical techniques |
WO2012128810A1 (en) | 2011-03-23 | 2012-09-27 | Abbott Laboratories | Methods and systems for the analysis of protein samples |
MX336740B (en) | 2011-03-29 | 2016-01-29 | Roche Glycart Ag | Antibody fc variants. |
WO2012131053A1 (en) | 2011-03-30 | 2012-10-04 | Ablynx Nv | Methods of treating immune disorders with single domain antibodies against tnf-alpha |
US9777332B2 (en) | 2011-03-31 | 2017-10-03 | St. Jude Children's Research Hospital | Methods and compositions for identifying minimal residual disease in acute lymphoblastic leukemia |
CN103596983B (en) | 2011-04-07 | 2016-10-26 | 霍夫曼-拉罗奇有限公司 | Anti-FGFR4 antibody and using method |
NZ616308A (en) | 2011-04-08 | 2016-03-31 | Biogen Ma Inc | Biomarkers predictive of therapeutic responsiveness to ifnβ and uses thereof |
WO2012142164A1 (en) | 2011-04-12 | 2012-10-18 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Human monoclonal antibodies that bind insulin-like growth factor (igf) i and ii |
WO2012142526A1 (en) | 2011-04-14 | 2012-10-18 | Modiano Jaime | Use of tumor fas expression to determine response to anti-cancer therapy |
EP3403672A1 (en) | 2011-04-20 | 2018-11-21 | Medlmmune, LLC | Antibodies and other molecules that bind b7-h1 and pd-1 |
EP2699597B1 (en) | 2011-04-21 | 2016-06-01 | Garvan Institute of Medical Research | Modified variable domain molecules and methods for producing and using them b |
EP2707389B1 (en) | 2011-05-12 | 2019-10-30 | The Johns Hopkins University | Assay reagents for a neurogranin diagnostic kit |
ES2567276T3 (en) | 2011-05-12 | 2016-04-21 | Genentech, Inc. | LC-MS / MS method of monitoring multiple reactions to detect therapeutic antibodies in animal samples using frame-changing peptides |
US9085626B2 (en) | 2011-05-16 | 2015-07-21 | Genentech, Inc. | FGFR1 agonists and methods of use |
EA201391753A1 (en) | 2011-05-21 | 2014-08-29 | Макродженикс, Инк. | DOMAINS CONNECTING WITH DEIMMUNIZED SERUM AND THEIR APPLICATION TO INCREASE THE TIME OF HALF-DURATION |
EP2714738B1 (en) | 2011-05-24 | 2018-10-10 | Zyngenia, Inc. | Multivalent and monovalent multispecific complexes and their uses |
US9359438B2 (en) | 2011-06-02 | 2016-06-07 | Dyax Corporation | Human neonatal Fc receptor antibodies and methods of use thereof |
LT2714735T (en) | 2011-06-03 | 2021-12-10 | Xoma Technology Ltd. | Antibodies specific for tgf-beta |
EP2717911A1 (en) | 2011-06-06 | 2014-04-16 | Novartis Forschungsstiftung, Zweigniederlassung | Protein tyrosine phosphatase, non-receptor type 11 (ptpn11) and triple-negative breast cancer |
WO2012170740A2 (en) | 2011-06-07 | 2012-12-13 | University Of Hawaii | Biomarker of asbestos exposure and mesothelioma |
WO2012170742A2 (en) | 2011-06-07 | 2012-12-13 | University Of Hawaii | Treatment and prevention of cancer with hmgb1 antagonists |
LT2691530T (en) | 2011-06-10 | 2018-08-10 | Oregon Health & Science University | Cmv glycoproteins and recombinant vectors |
LT2718320T (en) | 2011-06-10 | 2018-04-10 | Medimmune Limited | Anti-pseudomonas psl binding molecules and uses thereof |
KR101629073B1 (en) | 2011-06-15 | 2016-06-09 | 에프. 호프만-라 로슈 아게 | Anti-human epo receptor antibodies and methods of use |
HUE033713T2 (en) | 2011-06-28 | 2017-12-28 | Oxford Biotherapeutics Ltd | Therapeutic and diagnostic target |
EP2726508B1 (en) | 2011-06-28 | 2017-08-09 | Oxford BioTherapeutics Ltd | Antibodies to adp-ribosyl cyclase 2 |
MX2013014687A (en) | 2011-06-30 | 2014-02-17 | Genentech Inc | Anti-c-met antibody formulations. |
US20140341913A1 (en) | 2011-07-13 | 2014-11-20 | Abbvie Inc. | Methods and compositions for treating asthma using anti-il-13 antibodies |
GB201112056D0 (en) | 2011-07-14 | 2011-08-31 | Univ Leuven Kath | Antibodies |
US20140161821A1 (en) | 2011-07-14 | 2014-06-12 | Pfizer Inc. | Treatment with anti-pcsk9 antibodies |
US9738707B2 (en) | 2011-07-15 | 2017-08-22 | Biogen Ma Inc. | Heterodimeric Fc regions, binding molecules comprising same, and methods relating thereto |
KR20140048276A (en) | 2011-07-15 | 2014-04-23 | 온코메드 파마슈티칼스, 인크. | Rspo binding agents and uses thereof |
US20130022551A1 (en) | 2011-07-22 | 2013-01-24 | Trustees Of Boston University | DEspR ANTAGONISTS AND AGONISTS AS THERAPEUTICS |
KR20190133790A (en) | 2011-08-01 | 2019-12-03 | 제넨테크, 인크. | Methods of treating cancer using pd-1 axis binding antagonists and mek inhibitors |
EP2756094B1 (en) | 2011-08-15 | 2017-12-27 | Medlmmune, LLC | Anti-b7-h4 antibodies and their uses |
MX2014001766A (en) | 2011-08-17 | 2014-05-01 | Genentech Inc | Neuregulin antibodies and uses thereof. |
MX2014002053A (en) | 2011-08-23 | 2014-04-25 | Roche Glycart Ag | Anti-mcsp antibodies. |
EP3564261A1 (en) | 2011-08-23 | 2019-11-06 | Foundation Medicine, Inc. | Kif5b-ret fusion molecules and uses thereof |
CN103889452B (en) | 2011-08-23 | 2017-11-03 | 罗切格利卡特公司 | To T cell activation antigen and the bispecific antibody and application method of specific for tumour antigen |
WO2013026835A1 (en) | 2011-08-23 | 2013-02-28 | Roche Glycart Ag | Fc-free antibodies comprising two fab fragments and methods of use |
US20130058947A1 (en) | 2011-09-02 | 2013-03-07 | Stem Centrx, Inc | Novel Modulators and Methods of Use |
EP2753697A1 (en) | 2011-09-05 | 2014-07-16 | ETH Zürich | Biosynthetic gene cluster for the production of peptide/protein analogues |
US9447192B2 (en) | 2011-09-09 | 2016-09-20 | Medimmune Limited | Anti-Siglec-15 antibodies and uses thereof |
AU2012216792A1 (en) | 2011-09-12 | 2013-03-28 | International Aids Vaccine Initiative | Immunoselection of recombinant vesicular stomatitis virus expressing HIV-1 proteins by broadly neutralizing antibodies |
WO2013039996A1 (en) | 2011-09-13 | 2013-03-21 | Dana-Farber Cancer Institute, Inc. | Compositions and methods for brown fat induction and activity using fndc5 |
JP2014533927A (en) | 2011-09-15 | 2014-12-18 | ジェネンテック, インコーポレイテッド | How to promote differentiation |
EP2771349B1 (en) | 2011-09-16 | 2020-02-26 | Iogenetics, LLC. | Bioinformatic processes for determination of peptide binding |
WO2013043715A1 (en) | 2011-09-19 | 2013-03-28 | Genentech, Inc. | Combination treatments comprising c-met antagonists and b-raf antagonists |
US8858941B2 (en) | 2011-09-23 | 2014-10-14 | Oncomed Pharmaceuticals, Inc. | VEGF/DLL4 binding agents and uses thereof |
US9663573B2 (en) | 2011-10-05 | 2017-05-30 | Genentech, Inc. | Methods of treating liver conditions using Notch2 antagonists |
CA2851261A1 (en) | 2011-10-06 | 2013-04-11 | The Board Of Trustees Of The University Of Illinois | Myosin binding protein-c for use in methods relating to diastolic heart failure |
EP2766483B1 (en) | 2011-10-10 | 2022-03-23 | The Hospital For Sick Children | Methods and compositions for screening and treating developmental disorders |
WO2013054320A1 (en) | 2011-10-11 | 2013-04-18 | Tel Hashomer Medical Research Infrastructure And Services Ltd. | Antibodies to carcinoembryonic antigen-related cell adhesion molecule (ceacam) |
AU2012322618A1 (en) | 2011-10-14 | 2014-05-29 | Genentech, Inc. | Anti-HtrA1 antibodies and methods of use |
KR20140075000A (en) | 2011-10-14 | 2014-06-18 | 제넨테크, 인크. | Zymogen activators |
EP2766028B1 (en) | 2011-10-14 | 2017-08-16 | F. Hoffmann-La Roche AG | Peptide inhibitors of bace1 |
KR20140084164A (en) | 2011-10-15 | 2014-07-04 | 제넨테크, 인크. | Scd1 antagonists for treating cancer |
WO2013059531A1 (en) | 2011-10-20 | 2013-04-25 | Genentech, Inc. | Anti-gcgr antibodies and uses thereof |
US8999331B2 (en) | 2011-10-24 | 2015-04-07 | Abbvie Inc. | Immunobinders directed against sclerostin |
JP2014534218A (en) | 2011-10-24 | 2014-12-18 | アッヴィ・インコーポレイテッド | Immunobinding agents targeting TNF |
EP2586461A1 (en) | 2011-10-27 | 2013-05-01 | Christopher L. Parks | Viral particles derived from an enveloped virus |
CA2850034A1 (en) | 2011-10-28 | 2013-05-02 | Genentech, Inc. | Therapeutic combinations and methods of treating melanoma |
CN104053671A (en) | 2011-11-01 | 2014-09-17 | 生态学有限公司 | Antibodies and methods of treating cancer |
WO2013067057A1 (en) | 2011-11-01 | 2013-05-10 | Bionomics, Inc. | Anti-gpr49 antibodies |
US9221907B2 (en) | 2011-11-01 | 2015-12-29 | Bionomics Inc. | Anti-GPR49 monoclonal antibodies |
EP2773373B1 (en) | 2011-11-01 | 2018-08-22 | Bionomics, Inc. | Methods of blocking cancer stem cell growth |
AU2012332777C1 (en) | 2011-11-02 | 2015-12-24 | University Of Rochester | Anti-glucosaminidase passive immunization for Staphylococcus aureus infections |
US10527526B2 (en) | 2011-11-03 | 2020-01-07 | Tripath Imaging, Inc. | Methods and compositions for preparing samples for immunostaining |
US11180807B2 (en) | 2011-11-04 | 2021-11-23 | Population Bio, Inc. | Methods for detecting a genetic variation in attractin-like 1 (ATRNL1) gene in subject with Parkinson's disease |
BR112014011028B1 (en) | 2011-11-07 | 2021-03-02 | Medimmune, Llc | bispecific antibody, composition, and, use of composition |
EP2776022A1 (en) | 2011-11-08 | 2014-09-17 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | New treatment for neurodegenerative diseases |
EP2776838A1 (en) | 2011-11-08 | 2014-09-17 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Early diagnostic of neurodegenerative diseases |
BR112014011115A2 (en) | 2011-11-08 | 2017-06-13 | Pfizer | Methods for treating inflammatory disorders using anti-csf antibodies |
WO2013071233A1 (en) | 2011-11-10 | 2013-05-16 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Methods for detecting infectious agents and a novel virus detected thereby |
US8871908B2 (en) | 2011-11-11 | 2014-10-28 | Rinat Neuroscience Corp. | Antibodies specific for Trop-2 and their uses |
KR102048382B1 (en) | 2011-11-11 | 2019-11-25 | 유씨비 바이오파마 에스피알엘 | Albumin binding antibodies and binding fragments thereof |
AR088920A1 (en) | 2011-11-21 | 2014-07-16 | Genentech Inc | ANTI-C-MET ANTIBODY PURIFICATION |
CA2856411A1 (en) | 2011-11-21 | 2013-05-30 | Immunogen, Inc. | Method of treatment of tumors that are resistant to egfr therapies by egfr antibody cytotoxic agent conjugate |
AU2012340623A1 (en) | 2011-11-23 | 2014-07-17 | Igenica Biotherapeutics, Inc. | Anti-CD98 antibodies and methods of use thereof |
KR102080535B1 (en) | 2011-11-23 | 2020-02-24 | 메디뮨 엘엘씨 | Binding molecules specific for her3 and uses thereof |
WO2013080050A2 (en) | 2011-11-30 | 2013-06-06 | Universitaetsklinikum Erlangen | Methods and compositions for determining responsiveness to treatment with a tnf-alpha inhibitor |
US20140335084A1 (en) | 2011-12-06 | 2014-11-13 | Hoffmann-La Roche Inc. | Antibody formulation |
EP2791175A2 (en) | 2011-12-14 | 2014-10-22 | Abbvie Deutschland GmbH & Co. KG | Composition and method for the diagnosis and treatment of iron-related disorders |
EP3800200A1 (en) | 2011-12-14 | 2021-04-07 | AbbVie Deutschland GmbH & Co. KG | Composition and method for the diagnosis and treatment of iron-related disorders |
CN104144946A (en) | 2011-12-19 | 2014-11-12 | 爱克索马美国有限责任公司 | Methods for treating acne |
KR102048556B1 (en) | 2011-12-22 | 2019-11-26 | 에프. 호프만-라 로슈 아게 | Expression vector element combinations, novel production cell generation methods and their use for the recombinant production of polypeptides |
SG11201403223PA (en) | 2011-12-22 | 2014-07-30 | Hoffmann La Roche | Expression vector organization, novel production cell generation methods and their use for the recombinant production of polypeptides |
CA2854246A1 (en) | 2011-12-22 | 2013-06-27 | F. Hoffmann-La Roche Ag | Full length antibody display system for eukaryotic cells and its use |
WO2013093809A1 (en) | 2011-12-23 | 2013-06-27 | Pfizer Inc. | Engineered antibody constant regions for site-specific conjugation and methods and uses therefor |
AR089434A1 (en) | 2011-12-23 | 2014-08-20 | Genentech Inc | PROCEDURE TO PREPARE FORMULATIONS WITH HIGH CONCENTRATION OF PROTEINS |
MX2014008101A (en) | 2011-12-30 | 2014-09-25 | Abbvie Inc | Dual variable domain immunoglobulins against il-13 and/or il-17. |
EP2800583A1 (en) | 2012-01-02 | 2014-11-12 | Novartis AG | Cdcp1 and breast cancer |
JP2015509091A (en) | 2012-01-09 | 2015-03-26 | ザ スクリプス リサーチ インスティテュート | Humanized antibody |
WO2013106485A2 (en) | 2012-01-09 | 2013-07-18 | The Scripps Research Institute | Ultralong complementarity determining regions and uses thereof |
PL2802606T3 (en) | 2012-01-10 | 2018-09-28 | Biogen Ma Inc. | Enhancement of transport of therapeutic molecules across the blood brain barrier |
SG11201404198TA (en) | 2012-01-18 | 2014-08-28 | Genentech Inc | Anti-lrp5 antibodies and methods of use |
RU2014133547A (en) | 2012-01-18 | 2016-03-10 | Дженентек, Инк. | WAYS OF APPLICATION OF FGF19 MODULATORS |
GB201201332D0 (en) | 2012-01-26 | 2012-03-14 | Imp Innovations Ltd | Method |
WO2013112922A1 (en) | 2012-01-27 | 2013-08-01 | AbbVie Deutschland GmbH & Co. KG | Composition and method for diagnosis and treatment of diseases associated with neurite degeneration |
US10407724B2 (en) | 2012-02-09 | 2019-09-10 | The Hospital For Sick Children | Methods and compositions for screening and treating developmental disorders |
CN104254778A (en) | 2012-02-10 | 2014-12-31 | 西雅图遗传学公司 | Detection and treatment of cd30+ cancers |
AU2013216753B2 (en) | 2012-02-11 | 2017-09-21 | Genentech, Inc. | R-spondin translocations and methods using the same |
US9550830B2 (en) | 2012-02-15 | 2017-01-24 | Novo Nordisk A/S | Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1) |
LT2814844T (en) | 2012-02-15 | 2017-10-25 | Novo Nordisk A/S | Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (trem-1) |
WO2013120929A1 (en) | 2012-02-15 | 2013-08-22 | F. Hoffmann-La Roche Ag | Fc-receptor based affinity chromatography |
CN108530535B (en) | 2012-02-15 | 2021-02-26 | 诺和诺德股份有限公司 | Antibody binding to peptidoglycan-recognizing protein 1 |
GB201203051D0 (en) | 2012-02-22 | 2012-04-04 | Ucb Pharma Sa | Biological products |
GB201203071D0 (en) | 2012-02-22 | 2012-04-04 | Ucb Pharma Sa | Biological products |
CN104520324A (en) | 2012-02-24 | 2015-04-15 | 施特姆森特Rx股份有限公司 | DLL3 modulators and methods of use |
CN104334577A (en) | 2012-03-13 | 2015-02-04 | 霍夫曼-拉罗奇有限公司 | Combination therapy for the treatment of ovarian cancer |
US9139863B2 (en) | 2012-03-16 | 2015-09-22 | Genentech, Inc. | Engineered conformationally-stabilized proteins |
MX2014010943A (en) | 2012-03-16 | 2014-11-26 | Genentech Inc | Engineered conformationally-stabilized proteins. |
BR112014020173A8 (en) | 2012-03-16 | 2017-07-11 | Hoffmann La Roche | METHODS FOR THE TREATMENT OF A MELANOMA, USES OF AN INHIBITOR, COMPOSITIONS, KIT, METHOD OF INHIBITION, METHOD OF IDENTIFICATION, METHOD OF ADJUSTING THE TREATMENT AND INVENTION |
EP2828282B1 (en) | 2012-03-20 | 2017-12-27 | Otago Innovation Limited | Biomarkers |
WO2013139956A1 (en) | 2012-03-22 | 2013-09-26 | Thrombogenics Nv | Antibodies abrogating cell binding to lactadherin |
WO2013142808A1 (en) | 2012-03-23 | 2013-09-26 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Pathogenic phlebovirus isolates and compositions and methods of use |
AU2013240261A1 (en) | 2012-03-27 | 2014-09-18 | Genentech, Inc. | Diagnosis and treatments relating to HER3 inhibitors |
CN104334583A (en) | 2012-03-28 | 2015-02-04 | 弗·哈夫曼-拉罗切有限公司 | Anti-HCMV idiotypic antibodies and uses thereof |
US20150266961A1 (en) | 2012-03-29 | 2015-09-24 | Novartis Forschungsstiftung, Zweigniederlassung, Fridrich Miescher Institute | Inhibition of interleukin-8 and/or its receptor cxcr1 in the treatment of her2/her3-overexpressing breast cancer |
AR090549A1 (en) | 2012-03-30 | 2014-11-19 | Genentech Inc | ANTI-LGR5 AND IMMUNOCATE PLAYERS |
US10061887B2 (en) | 2012-04-02 | 2018-08-28 | Berg Llc | Interrogatory cell-based assays and uses thereof |
WO2013151649A1 (en) | 2012-04-04 | 2013-10-10 | Sialix Inc | Glycan-interacting compounds |
US10130714B2 (en) | 2012-04-14 | 2018-11-20 | Academia Sinica | Enhanced anti-influenza agents conjugated with anti-inflammatory activity |
US9181572B2 (en) | 2012-04-20 | 2015-11-10 | Abbvie, Inc. | Methods to modulate lysine variant distribution |
WO2013158279A1 (en) | 2012-04-20 | 2013-10-24 | Abbvie Inc. | Protein purification methods to reduce acidic species |
PL2838918T3 (en) | 2012-04-20 | 2019-11-29 | Merus Nv | Methods and means for the production of heterodimeric ig-like molecules |
WO2013158275A1 (en) | 2012-04-20 | 2013-10-24 | Abbvie Inc. | Cell culture methods to reduce acidic species |
KR20150005631A (en) | 2012-04-24 | 2015-01-14 | 쓰롬보제닉스 엔.브이. | Anti-pdgf-c antibodies |
CN104583776B (en) | 2012-04-25 | 2016-09-07 | 比奥德赛公司 | For the method detecting the allosteric modulators of protein |
TW201402609A (en) | 2012-05-01 | 2014-01-16 | Genentech Inc | Anti-PMEL17 antibodies and immunoconjugates |
WO2013170191A1 (en) | 2012-05-11 | 2013-11-14 | Genentech, Inc. | Methods of using antagonists of nad biosynthesis from nicotinamide |
JP2015518829A (en) | 2012-05-14 | 2015-07-06 | バイオジェン・エムエイ・インコーポレイテッドBiogen MA Inc. | LINGO-2 antagonist for treatment of conditions involving motor neurons |
US9512223B2 (en) | 2012-05-15 | 2016-12-06 | Morphotek, Inc. | Methods for treatment of gastric cancer |
TW201348247A (en) | 2012-05-21 | 2013-12-01 | Abbvie Inc | Novel purification of non-human antibodies using protein a affinity chromatography |
WO2013177470A1 (en) | 2012-05-23 | 2013-11-28 | Genentech, Inc. | Selection method for therapeutic agents |
WO2013176754A1 (en) | 2012-05-24 | 2013-11-28 | Abbvie Inc. | Novel purification of antibodies using hydrophobic interaction chromatography |
PL2855528T3 (en) | 2012-05-31 | 2019-10-31 | Hoffmann La Roche | Methods of treating cancer using pd-l1 axis binding antagonists and vegf antagonists |
JP2015520192A (en) | 2012-06-06 | 2015-07-16 | オンコメッド ファーマシューティカルズ インコーポレイテッド | Binding agents that modulate the Hippo pathway and uses thereof |
US9617334B2 (en) | 2012-06-06 | 2017-04-11 | Zoetis Services Llc | Caninized anti-NGF antibodies and methods thereof |
MX2014014830A (en) | 2012-06-15 | 2015-05-11 | Genentech Inc | Anti-pcsk9 antibodies, formulations, dosing, and methods of use. |
ES2631608T3 (en) | 2012-06-27 | 2017-09-01 | International Aids Vaccine Initiative | Env-glycoprotein variant of HIV-1 |
EP2867674B1 (en) | 2012-06-28 | 2018-10-10 | UCB Biopharma SPRL | A method for identifying compounds of therapeutic interest |
EP2866831A1 (en) | 2012-06-29 | 2015-05-06 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Treating diseases by modulating a specific isoform of mkl1 |
EP3138578B1 (en) | 2012-07-04 | 2022-01-12 | F. Hoffmann-La Roche AG | Anti-theophylline antibodies and methods of use |
RU2684595C2 (en) | 2012-07-04 | 2019-04-09 | Ф.Хоффманн-Ля Рош Аг | Kovalent-related conjuates of antigen-antibody |
EP3339328A1 (en) | 2012-07-04 | 2018-06-27 | F. Hoffmann-La Roche AG | Anti-biotin antibodies and methods of use |
WO2014006114A1 (en) | 2012-07-05 | 2014-01-09 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | New treatment for neurodegenerative diseases |
MX356162B (en) | 2012-07-05 | 2018-05-16 | Genentech Inc | Expression and secretion system. |
US20150224190A1 (en) | 2012-07-06 | 2015-08-13 | Mohamed Bentires-Alj | Combination of a phosphoinositide 3-kinase inhibitor and an inhibitor of the IL-8/CXCR interaction |
AU2013288932A1 (en) | 2012-07-09 | 2014-12-11 | Genentech, Inc. | Immunoconjugates comprising anti - CD79b antibodies |
MX2015000315A (en) | 2012-07-09 | 2015-07-06 | Genentech Inc | Immunoconjugates comprising anti-cd22 antibodies. |
JP2015527318A (en) | 2012-07-09 | 2015-09-17 | ジェネンテック, インコーポレイテッド | Immune complex comprising anti-CD22 |
MX2015000359A (en) | 2012-07-09 | 2015-04-14 | Genentech Inc | Immunoconjugates comprising anti-cd79b antibodies. |
UY34905A (en) | 2012-07-12 | 2014-01-31 | Abbvie Inc | IL-1 UNION PROTEINS |
HRP20211641T1 (en) | 2012-07-13 | 2022-02-04 | Roche Glycart Ag | Bispecific anti-vegf/anti-ang-2 antibodies and their use in the treatment of ocular vascular diseases |
GB201213652D0 (en) | 2012-08-01 | 2012-09-12 | Oxford Biotherapeutics Ltd | Therapeutic and diagnostic target |
US9297806B2 (en) | 2012-08-01 | 2016-03-29 | The Johns Hopkins University | 5-hydroxymethylcytosine in human cancer |
ES2771324T3 (en) | 2012-08-03 | 2020-07-06 | Dana Farber Cancer Inst Inc | Medical uses of agents that modulate immune cell activation and associated detection methods |
WO2014025813A1 (en) | 2012-08-07 | 2014-02-13 | Genentech, Inc. | Combination therapy for the treatment of glioblastoma |
US9914956B2 (en) | 2012-08-18 | 2018-03-13 | Academia Sinica | Cell-permeable probes for identification and imaging of sialidases |
SG11201500938XA (en) | 2012-08-31 | 2015-04-29 | Immunogen Inc | Diagnostic assays and kits for detection of folate receptor 1 |
WO2014039860A2 (en) | 2012-09-07 | 2014-03-13 | University Of Louisville Research Foundation, Inc. | Compositions and methods for modulating dnmt1 inhibitor activity |
DK2895621T3 (en) | 2012-09-14 | 2020-11-30 | Population Bio Inc | METHODS AND COMPOSITION FOR DIAGNOSIS, FORECAST AND TREATMENT OF NEUROLOGICAL CONDITIONS |
US10233495B2 (en) | 2012-09-27 | 2019-03-19 | The Hospital For Sick Children | Methods and compositions for screening and treating developmental disorders |
WO2014055442A2 (en) | 2012-10-01 | 2014-04-10 | The Trustees Of The University Of Pennsylvania | Compositions and methods for targeting stromal cells for the treatment of cancer |
RU2015117393A (en) | 2012-10-08 | 2016-12-10 | Роше Гликарт Аг | Deprived fc antibodies containing two Fab fragments, and methods for their use |
EP2906598A1 (en) | 2012-10-09 | 2015-08-19 | Igenica Biotherapeutics, Inc. | Anti-c16orf54 antibodies and methods of use thereof |
CN109364250A (en) | 2012-10-09 | 2019-02-22 | 比奥根Ma公司 | Combination therapy and purposes for treating demyelinating disorder |
CA3201072A1 (en) | 2012-10-12 | 2014-04-17 | The Brigham And Women's Hospital, Inc. | Enhancement of the immune response |
US9266959B2 (en) | 2012-10-23 | 2016-02-23 | Oncomed Pharmaceuticals, Inc. | Methods of treating neuroendocrine tumors using frizzled-binding agents |
JP6371294B2 (en) | 2012-10-31 | 2018-08-08 | オンコメッド ファーマシューティカルズ インコーポレイテッド | Methods and monitoring of treatment with DLL4 antagonists |
RU2636043C2 (en) | 2012-11-01 | 2017-11-17 | Эббви Инк. | Anti-vegf/dll4-immunoglobulins with double variable domains and their application |
CA2890207A1 (en) | 2012-11-05 | 2014-05-08 | Foundation Medicine, Inc. | Novel ntrk1 fusion molecules and uses thereof |
US20150284472A1 (en) | 2012-11-05 | 2015-10-08 | Genzyme Corporation | Compositions and methods for treating proteinopathies |
WO2014074942A1 (en) | 2012-11-08 | 2014-05-15 | Illumina, Inc. | Risk variants of alzheimer's disease |
JP6480338B2 (en) | 2012-11-08 | 2019-03-06 | セセン バイオ, インコーポレイテッド | IL-6 antagonists and uses thereof |
WO2014072306A1 (en) | 2012-11-08 | 2014-05-15 | F. Hoffmann-La Roche Ag | Her3 antigen binding proteins binding to the beta-hairpin of her3 |
TWI657095B (en) | 2012-11-13 | 2019-04-21 | 美商建南德克公司 | Anti-hemagglutinin antibodies and methods of use |
WO2014085821A2 (en) | 2012-11-30 | 2014-06-05 | The Regents Of The University Of California | Fully human antibodies and fragments recognizing human c-met |
US9902775B2 (en) | 2012-12-10 | 2018-02-27 | Biogen Ma Inc. | Anti-blood dendritic cell antigen 2 antibodies and uses thereof |
WO2014100762A1 (en) | 2012-12-21 | 2014-06-26 | Biolliance C.V. | Hydrophilic self-immolative linkers and conjugates thereof |
US9550986B2 (en) | 2012-12-21 | 2017-01-24 | Abbvie Inc. | High-throughput antibody humanization |
GB201223276D0 (en) | 2012-12-21 | 2013-02-06 | Ucb Pharma Sa | Antibodies and methods of producing same |
EP2935332B1 (en) | 2012-12-21 | 2021-11-10 | MedImmune, LLC | Anti-h7cr antibodies |
AU2013202668B2 (en) | 2012-12-24 | 2014-12-18 | Adelaide Research & Innovation Pty Ltd | Inhibition of cancer growth and metastasis |
WO2014107739A1 (en) | 2013-01-07 | 2014-07-10 | Eleven Biotherapeutics, Inc. | Antibodies against pcsk9 |
US10717965B2 (en) | 2013-01-10 | 2020-07-21 | Gloriana Therapeutics, Inc. | Mammalian cell culture-produced neublastin antibodies |
CA2898326C (en) | 2013-01-18 | 2022-05-17 | Foundation Medicine, Inc. | Methods of treating cholangiocarcinoma |
WO2014116749A1 (en) | 2013-01-23 | 2014-07-31 | Genentech, Inc. | Anti-hcv antibodies and methods of using thereof |
EP2948178A4 (en) | 2013-01-25 | 2016-07-20 | Thymon Llc | Compositions for selective reduction of circulating bioactive soluble tnf and methods for treating tnf-mediated disease |
US9359444B2 (en) | 2013-02-04 | 2016-06-07 | Oncomed Pharmaceuticals Inc. | Methods and monitoring of treatment with a Wnt pathway inhibitor |
GB201302447D0 (en) | 2013-02-12 | 2013-03-27 | Oxford Biotherapeutics Ltd | Therapeutic and diagnostic target |
WO2014129895A1 (en) | 2013-02-19 | 2014-08-28 | Stichting Vu-Vumc | Means and method for increasing the sensitivity of cancers for radiotherapy |
AU2014218730B2 (en) | 2013-02-22 | 2018-12-13 | Abbvie Stemcentrx Llc | Novel antibody conjugates and uses thereof |
JP2016509045A (en) | 2013-02-22 | 2016-03-24 | エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト | How to treat cancer and prevent drug resistance |
RU2015140573A (en) | 2013-02-25 | 2017-03-30 | Дженентек, Инк. | METHODS AND COMPOSITIONS FOR DETECTION AND TREATMENT OF DRUG-RESISTANT MUTANT RESISTANT TO MEDICINES |
KR20150123811A (en) | 2013-02-26 | 2015-11-04 | 로슈 글리카트 아게 | Anti-mcsp antibodies |
US9925240B2 (en) | 2013-03-06 | 2018-03-27 | Genentech, Inc. | Methods of treating and preventing cancer drug resistance |
CN105229035A (en) * | 2013-03-11 | 2016-01-06 | 诺和诺德保健股份有限公司 | Growth hormone compound |
WO2014139994A1 (en) * | 2013-03-11 | 2014-09-18 | Novo Nordisk Health Care Ag | Growth hormone compounds |
HUE039948T2 (en) | 2013-03-13 | 2019-02-28 | Hoffmann La Roche | Formulations with reduced oxidation |
MY189047A (en) | 2013-03-13 | 2022-01-21 | Genentech Inc | Antibody formulations |
WO2014160497A1 (en) | 2013-03-13 | 2014-10-02 | Genentech, Inc. | Formulations with reduced oxidation |
US10653779B2 (en) | 2013-03-13 | 2020-05-19 | Genentech, Inc. | Formulations with reduced oxidation |
AR095398A1 (en) | 2013-03-13 | 2015-10-14 | Genentech Inc | FORMULATIONS WITH REDUCED OXIDATION |
US9168300B2 (en) | 2013-03-14 | 2015-10-27 | Oncomed Pharmaceuticals, Inc. | MET-binding agents and uses thereof |
AU2014240431A1 (en) | 2013-03-14 | 2015-08-27 | Abbvie Inc. | Low acidic species compositions and methods for producing the same using displacement chromatography |
MX2015012825A (en) | 2013-03-14 | 2016-06-10 | Abbott Lab | Hcv core lipid binding domain monoclonal antibodies. |
CN105246508A (en) | 2013-03-14 | 2016-01-13 | 基因泰克公司 | Combinations of a mek inhibitor compound with an her3/egfr inhibitor compound and methods of use |
CA2906421C (en) | 2013-03-14 | 2022-08-16 | George J. Dawson | Hcv antigen-antibody combination assay and methods and compositions for use therein |
KR20150127216A (en) | 2013-03-14 | 2015-11-16 | 제넨테크, 인크. | Methods of treating cancer and preventing cancer drug resistance |
US10150813B2 (en) | 2013-03-14 | 2018-12-11 | Genentech, Inc. | Anti-B7-H4 antibodies and immunoconjugates |
CA2926384A1 (en) | 2013-03-14 | 2014-10-02 | Abbvie Inc. | Low acidic species compositions and methods for producing and using the same |
WO2014142882A1 (en) | 2013-03-14 | 2014-09-18 | Abbvie Inc. | Protein purification using displacement chromatography |
JP2016520527A (en) | 2013-03-14 | 2016-07-14 | パーカシュ ギル, | Treatment of cancer using antibodies that bind to cell surface GRP78 |
US9790478B2 (en) | 2013-03-14 | 2017-10-17 | Abbott Laboratories | HCV NS3 recombinant antigens and mutants thereof for improved antibody detection |
US9562099B2 (en) | 2013-03-14 | 2017-02-07 | Genentech, Inc. | Anti-B7-H4 antibodies and immunoconjugates |
PE20151750A1 (en) | 2013-03-15 | 2015-12-07 | Genentech Inc | COMPOSITIONS AND METHODS FOR THE DIAGNOSIS AND TREATMENT OF HEPATIC CANCER |
CN105163763B (en) | 2013-03-15 | 2019-07-02 | 艾伯维德国有限责任两合公司 | Anti-egfr antibodies drug conjugates preparation |
WO2014144600A2 (en) | 2013-03-15 | 2014-09-18 | Viktor Roschke | Multivalent and monovalent multispecific complexes and their uses |
SG11201507432XA (en) | 2013-03-15 | 2015-10-29 | Abbvie Inc | Antibody drug conjugate (adc) purification |
EP4079760A3 (en) | 2013-03-15 | 2023-01-25 | Sanofi Pasteur Inc. | Antibodies against clostridium difficile toxins and methods of using the same |
EP2970475A1 (en) | 2013-03-15 | 2016-01-20 | Biogen MA Inc. | Treatment and prevention of acute kidney injury using anti-alpha v beta 5 antibodies |
PT2970422T (en) | 2013-03-15 | 2018-07-06 | Hoffmann La Roche | Il-22 polypeptides and il-22 fc fusion proteins and methods of use |
BR112015023797A2 (en) | 2013-03-15 | 2017-10-24 | Abbvie Inc | dual specificity binding proteins directed against il-1b and / or il-17 |
NZ712314A (en) | 2013-03-15 | 2021-07-30 | Genentech Inc | Biomarkers and methods of treating pd-1 and pd-l1 related conditions |
CN105143258B (en) | 2013-03-15 | 2020-06-23 | Ac免疫有限公司 | anti-Tau antibodies and methods of use |
CN105143265A (en) | 2013-03-15 | 2015-12-09 | 豪夫迈·罗氏有限公司 | Anti-crth2 antibodies and their use |
US20160143910A1 (en) | 2013-03-15 | 2016-05-26 | Constellation Pharmaceuticals, Inc. | Methods of treating cancer and preventing cancer drug resistance |
SG10201912621TA (en) | 2013-03-15 | 2020-02-27 | Genentech Inc | Cell culture compositions with antioxidants and methods for polypeptide production |
CA2899089C (en) | 2013-03-15 | 2021-10-26 | Biogen Ma Inc. | Factor ix polypeptide formulations |
RU2015144020A (en) | 2013-03-15 | 2017-04-21 | Дженентек, Инк. | ENVIRONMENTS FOR CULTIVATION OF CELLS AND METHODS FOR PRODUCING ANTIBODIES |
US9469686B2 (en) | 2013-03-15 | 2016-10-18 | Abbott Laboratories | Anti-GP73 monoclonal antibodies and methods of obtaining the same |
CN105120887A (en) | 2013-04-05 | 2015-12-02 | 诺和诺德保健股份有限公司 | Growth hormone compound formulation |
WO2014168933A1 (en) | 2013-04-08 | 2014-10-16 | Cytodyn Inc. | Felinized antibodies and methods of treating retroviral infections in felines |
UA118674C2 (en) | 2013-04-29 | 2019-02-25 | Ф. Хоффманн-Ля Рош Аг | Fcrn-binding abolished anti-igf-1r antibodies and their use in the treatment of vascular eye diseases |
PE20151807A1 (en) | 2013-04-29 | 2015-12-02 | Hoffmann La Roche | MODIFIED ANTIBODIES OF BINDING TO HUMAN FCRN AND METHOD OF USE |
JP6618893B2 (en) | 2013-04-29 | 2019-12-11 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | Asymmetric antibodies with altered FC receptor binding and methods of use |
EP2992331A4 (en) | 2013-04-30 | 2017-03-29 | Université de Montréal | Novel biomarkers for acute myeloid leukemia |
HUE052232T2 (en) | 2013-05-06 | 2021-04-28 | Scholar Rock Inc | Compositions and methods for growth factor modulation |
KR102293064B1 (en) | 2013-05-20 | 2021-08-23 | 제넨테크, 인크. | Anti-transferrin receptor antibodies and methods of use |
BR112015029395A2 (en) | 2013-05-24 | 2017-09-19 | Medimmune Llc | ANTI-B7-H5 ANTIBODIES AND THEIR USES |
NZ714765A (en) | 2013-06-06 | 2021-12-24 | Pf Medicament | Anti-c10orf54 antibodies and uses thereof |
CA2913490A1 (en) | 2013-06-06 | 2014-12-11 | Dana-Farber Cancer Institute, Inc. | Compositions and methods for identification, assessment, prevention, and treatment of cancer using pd-l1 isoforms |
CA2914566A1 (en) | 2013-06-07 | 2014-12-11 | Duke University | Inhibitors of complement factor h |
EP3008091A1 (en) | 2013-06-13 | 2016-04-20 | Fast Forward Pharmaceuticals B.V. | Cd40 signalling inhibitor and a further compound, wherein the further compound is a bile acid, a bile acid derivative, an tgr5-receptor agonist, an fxr agonist or a combination thereof, for the treatment of chronic inflammation, and the prevention of gastrointestinal cancer or fibrosis. |
WO2014210397A1 (en) | 2013-06-26 | 2014-12-31 | Academia Sinica | Rm2 antigens and use thereof |
US9981030B2 (en) | 2013-06-27 | 2018-05-29 | Academia Sinica | Glycan conjugates and use thereof |
WO2015009856A2 (en) | 2013-07-16 | 2015-01-22 | Genentech, Inc. | Methods of treating cancer using pd-1 axis binding antagonists and tigit inhibitors |
US20160168231A1 (en) | 2013-07-18 | 2016-06-16 | Fabrus, Inc. | Antibodies with ultralong complementarity determining regions |
WO2015010100A2 (en) | 2013-07-18 | 2015-01-22 | Fabrus, Inc. | Humanized antibodies with ultralong complementarity determining regions |
US20160175401A1 (en) | 2013-07-31 | 2016-06-23 | Dana-Farber Cancer Institute Inc. | Compoitions and methods for modulating thermogenesis using pth-related and egf-related compounds |
CA2919790C (en) | 2013-08-02 | 2018-06-19 | Pfizer Inc. | Anti-cxcr4 antibodies and antibody-drug conjugates |
EP3030902B1 (en) | 2013-08-07 | 2019-09-25 | Friedrich Miescher Institute for Biomedical Research | New screening method for the treatment friedreich's ataxia |
WO2015026846A1 (en) | 2013-08-19 | 2015-02-26 | Biogen Idec Ma Inc. | Control of protein glycosylation by culture medium supplementation and cell culture process parameters |
EP3906945A3 (en) | 2013-08-26 | 2022-03-16 | BioNTech Research and Development, Inc. | Nucleic acids encoding human antibodies to sialyl-lewis a |
MX2016002574A (en) | 2013-08-28 | 2016-06-14 | Stemcentrx Inc | Novel sez6 modulators and methods of use. |
RU2016111131A (en) | 2013-08-28 | 2017-10-03 | ЭББВИ СТЕМСЕНТРКС ЭлЭлСи | METHODS FOR CONJUGATION OF SITE-SPECIFIC ANTIBODIES AND COMPOSITIONS |
AU2014312086B2 (en) | 2013-08-30 | 2020-03-12 | Immunogen, Inc. | Antibodies and assays for detection of folate receptor 1 |
US10456470B2 (en) | 2013-08-30 | 2019-10-29 | Genentech, Inc. | Diagnostic methods and compositions for treatment of glioblastoma |
US10617755B2 (en) | 2013-08-30 | 2020-04-14 | Genentech, Inc. | Combination therapy for the treatment of glioblastoma |
US20150065381A1 (en) | 2013-09-05 | 2015-03-05 | International Aids Vaccine Initiative | Methods of identifying novel hiv-1 immunogens |
AU2014317889B2 (en) | 2013-09-06 | 2020-03-05 | Academia Sinica | Human iNKT cell activation using glycolipids with altered glycosyl groups |
KR20160055252A (en) | 2013-09-17 | 2016-05-17 | 제넨테크, 인크. | Methods of using anti-lgr5 antibodies |
WO2015048331A1 (en) | 2013-09-25 | 2015-04-02 | Cornell University | Compounds for inducing anti-tumor immunity and methods thereof |
WO2015048330A2 (en) | 2013-09-25 | 2015-04-02 | Biogen Idec Ma Inc. | On-column viral inactivation methods |
EP3757130A1 (en) | 2013-09-26 | 2020-12-30 | Costim Pharmaceuticals Inc. | Methods for treating hematologic cancers |
EP3049437A1 (en) | 2013-09-27 | 2016-08-03 | F. Hoffmann-La Roche AG | Thermus thermophilus slyd fkbp domain specific antibodies |
LT3049441T (en) | 2013-09-27 | 2020-02-10 | F. Hoffmann-La Roche Ag | Anti-pdl1 antibody formulations |
EP2873423B1 (en) | 2013-10-07 | 2017-05-31 | International Aids Vaccine Initiative | Soluble hiv-1 envelope glycoprotein trimers |
PT3055332T (en) | 2013-10-08 | 2019-12-09 | Immunogen Inc | Anti-folr1 immunoconjugate dosing regimens |
WO2015054670A1 (en) | 2013-10-11 | 2015-04-16 | Genentech, Inc. | Nsp4 inhibitors and methods of use |
EP3057615B1 (en) | 2013-10-18 | 2021-02-24 | F.Hoffmann-La Roche Ag | Anti-rspo antibodies and methods of use |
WO2015057939A1 (en) | 2013-10-18 | 2015-04-23 | Biogen Idec Ma Inc. | Anti-s1p4 antibodies and uses thereof |
RU2016119425A (en) | 2013-10-23 | 2017-11-28 | Дженентек, Инк. | METHODS FOR DIAGNOSIS AND TREATMENT OF EOSINOPHILIC DISEASES |
US10344319B2 (en) | 2013-10-28 | 2019-07-09 | Dots Technology Corp. | Allergen detection |
EP4331590A2 (en) | 2013-10-29 | 2024-03-06 | President and Fellows of Harvard College | Nuclear factor erythroid 2-like 2 (nrf2) for use in treatment of age-related macular degeneration |
US20160272674A1 (en) | 2013-11-07 | 2016-09-22 | Abbvie Inc. | Isolation and purification of antibodies |
DK3511422T3 (en) | 2013-11-12 | 2023-02-06 | Population Bio Inc | METHODS AND COMPOSITIONS FOR DIAGNOSING, PROGNOSIS AND TREATMENT OF ENDOMETRIOSIS |
WO2015071759A1 (en) | 2013-11-15 | 2015-05-21 | Institut Pasteur | A molecular marker of plasmodium falciparum artemisinin resistance |
PT3071597T (en) | 2013-11-21 | 2020-10-08 | Hoffmann La Roche | Anti-alpha-synuclein antibodies and methods of use |
PL3074035T3 (en) | 2013-11-25 | 2021-03-08 | Famewave Ltd. | Compositions comprising anti-ceacam1 and anti-pd antibodies for cancer therapy |
US20160297875A1 (en) | 2013-12-07 | 2016-10-13 | Case Western Reserve University | Compositions and methods of treating thrombosis |
BR112016013514B1 (en) | 2013-12-13 | 2022-04-19 | Stora Enso Oyj (Fi) | MULTI-LAYER CARDBOARD |
US20160333063A1 (en) | 2013-12-13 | 2016-11-17 | The General Hospital Corporation | Soluble high molecular weight (hmw) tau species and applications thereof |
PE20160712A1 (en) | 2013-12-13 | 2016-07-26 | Genentech Inc | ANTI-CD33 ANTIBODIES AND IMMUNOCONJUGATES |
US9067998B1 (en) | 2014-07-15 | 2015-06-30 | Kymab Limited | Targeting PD-1 variants for treatment of cancer |
AU2014364587B9 (en) | 2013-12-17 | 2023-02-16 | Genentech, Inc. | Methods of treating cancers using PD-1 axis binding antagonists and taxanes |
US9914769B2 (en) | 2014-07-15 | 2018-03-13 | Kymab Limited | Precision medicine for cholesterol treatment |
US8992927B1 (en) | 2014-07-15 | 2015-03-31 | Kymab Limited | Targeting human NAV1.7 variants for treatment of pain |
EP3083687A2 (en) | 2013-12-17 | 2016-10-26 | F. Hoffmann-La Roche AG | Combination therapy comprising ox40 binding agonists and pd-1 axis binding antagonists |
AU2014364593A1 (en) | 2013-12-17 | 2016-07-07 | Genentech, Inc. | Methods of treating cancer using PD-1 axis binding antagonists and an anti-CD20 antibody |
US8986694B1 (en) | 2014-07-15 | 2015-03-24 | Kymab Limited | Targeting human nav1.7 variants for treatment of pain |
RS60443B1 (en) | 2013-12-17 | 2020-07-31 | Genentech Inc | Anti-cd3 antibodies and methods of use |
US9045545B1 (en) | 2014-07-15 | 2015-06-02 | Kymab Limited | Precision medicine by targeting PD-L1 variants for treatment of cancer |
CN116478927A (en) | 2013-12-19 | 2023-07-25 | 诺华股份有限公司 | Human mesothelin chimeric antigen receptor and application thereof |
US20160289633A1 (en) | 2013-12-20 | 2016-10-06 | Biogen Ma Inc. | Use of Perfusion Seed Cultures to Improve Biopharmaceutical Fed-Batch Production Capacity and Product Quality |
US11708411B2 (en) | 2013-12-20 | 2023-07-25 | Wake Forest University Health Sciences | Methods and compositions for increasing protective antibody levels induced by pneumococcal polysaccharide vaccines |
EP2960252A1 (en) | 2014-06-26 | 2015-12-30 | Institut Pasteur | Phospholipase for treatment of immunosuppression |
TWI670283B (en) | 2013-12-23 | 2019-09-01 | 美商建南德克公司 | Antibodies and methods of use |
KR102534681B1 (en) | 2013-12-24 | 2023-05-18 | 잔센파마슈티카엔.브이. | Anti-vista antibodies and fragments |
RU2682754C2 (en) | 2014-01-03 | 2019-03-21 | Ф. Хоффманн-Ля Рош Аг | Covalent bonded polypeptide toxin and antibody conjugates |
WO2015103549A1 (en) | 2014-01-03 | 2015-07-09 | The United States Of America, As Represented By The Secretary Department Of Health And Human Services | Neutralizing antibodies to hiv-1 env and their use |
CN111228509A (en) | 2014-01-03 | 2020-06-05 | 豪夫迈·罗氏有限公司 | Bispecific anti-hapten/anti-blood brain barrier receptor antibodies, complexes thereof and their use as blood brain barrier shuttles |
CA2930154A1 (en) | 2014-01-03 | 2015-07-09 | F. Hoffmann-La Roche Ag | Covalently linked helicar-anti-helicar antibody conjugates and uses thereof |
CN111057151B (en) | 2014-01-06 | 2022-05-03 | 豪夫迈·罗氏有限公司 | Monovalent blood brain barrier shuttle modules |
CN105899534B (en) | 2014-01-15 | 2020-01-07 | 豪夫迈·罗氏有限公司 | Fc region variants with modified FCRN and maintained protein A binding properties |
US10150818B2 (en) | 2014-01-16 | 2018-12-11 | Academia Sinica | Compositions and methods for treatment and detection of cancers |
WO2015184009A1 (en) | 2014-05-27 | 2015-12-03 | Academia Sinica | Compositions and methods relating to universal glycoforms for enhanced antibody efficacy |
CA2937123A1 (en) | 2014-01-16 | 2015-07-23 | Academia Sinica | Compositions and methods for treatment and detection of cancers |
WO2016114819A1 (en) | 2015-01-16 | 2016-07-21 | Academia Sinica | Compositions and methods for treatment and detection of cancers |
EP3097196B1 (en) | 2014-01-20 | 2019-09-11 | President and Fellows of Harvard College | Negative selection and stringency modulation in continuous evolution systems |
KR20160111469A (en) | 2014-01-24 | 2016-09-26 | 제넨테크, 인크. | Methods of using anti-steap1 antibodies and immunoconjugates |
JOP20200094A1 (en) | 2014-01-24 | 2017-06-16 | Dana Farber Cancer Inst Inc | Antibody molecules to pd-1 and uses thereof |
SI3097122T1 (en) | 2014-01-24 | 2020-07-31 | Ngm Biopharmaceuticals, Inc. | Antibodies binding beta klotho domain 2 and methods of use thereof |
JOP20200096A1 (en) | 2014-01-31 | 2017-06-16 | Children’S Medical Center Corp | Antibody molecules to tim-3 and uses thereof |
WO2015116902A1 (en) | 2014-01-31 | 2015-08-06 | Genentech, Inc. | G-protein coupled receptors in hedgehog signaling |
US11648335B2 (en) | 2014-01-31 | 2023-05-16 | Wake Forest University Health Sciences | Organ/tissue decellularization, framework maintenance and recellularization |
AU2015214264B2 (en) | 2014-02-04 | 2018-12-20 | Curis, Inc. | Mutant Smoothened and methods of using the same |
WO2015120187A1 (en) | 2014-02-05 | 2015-08-13 | The University Of Chicago | Chimeric antigen receptors recognizing cancer-spevific tn glycopeptide variants |
CA2938731A1 (en) | 2014-02-08 | 2015-08-13 | Genentech, Inc. | Methods of treating alzheimer's disease |
MY179105A (en) | 2014-02-08 | 2020-10-28 | Genentech Inc | Methods of treating alzheimer's disease |
JP6764348B2 (en) | 2014-02-11 | 2020-09-30 | ビステラ, インコーポレイテッド | Antibody molecules against dengue virus and their use |
WO2015123325A1 (en) | 2014-02-12 | 2015-08-20 | Genentech, Inc. | Anti-jagged1 antibodies and methods of use |
CN106550593A (en) | 2014-02-21 | 2017-03-29 | 艾伯维施特姆森特克斯有限责任公司 | For melanomatous anti-DLL3 antibody and drug conjugate |
BR112016018980A2 (en) | 2014-02-21 | 2017-10-10 | Genentech Inc | method of treating a disorder, multispecific antibody, isolated nucleic acid, host cell, methods of producing an antibody, producing an antibody half or multispecific antibody, and producing a multispecific, immunoconjugate antibody and pharmaceutical formulation |
GB201403775D0 (en) | 2014-03-04 | 2014-04-16 | Kymab Ltd | Antibodies, uses & methods |
EP3116999B1 (en) | 2014-03-14 | 2021-09-15 | F. Hoffmann-La Roche AG | Methods and compositions for secretion of heterologous polypeptides |
US9738702B2 (en) | 2014-03-14 | 2017-08-22 | Janssen Biotech, Inc. | Antibodies with improved half-life in ferrets |
MY187246A (en) | 2014-03-14 | 2021-09-14 | Novartis Ag | Antibody molecules to lag-3 and uses thereof |
US20170107294A1 (en) | 2014-03-21 | 2017-04-20 | Nordlandssykehuset Hf | Anti-cd14 antibodies and uses thereof |
US10556945B2 (en) | 2014-03-21 | 2020-02-11 | Teva Pharmaceuticals International Gmbh | Antagonist antibodies directed against calcitonin gene-related peptide and methods using same |
FI3119431T3 (en) | 2014-03-21 | 2024-03-20 | Teva Pharmaceuticals Int Gmbh | Antagonist antibodies directed against calcitonin gene-related peptide and methods using same |
US11124760B2 (en) | 2014-03-24 | 2021-09-21 | Biogen Ma Inc. | Methods for overcoming glutamine deprivation during mammalian cell culture |
RU2016141385A (en) | 2014-03-24 | 2018-04-28 | Дженентек, Инк. | CANCER TREATMENT WITH C-MET ANTAGONISTS AND THEIR CORRELATION WITH HGF EXPRESSION |
WO2015148915A1 (en) | 2014-03-27 | 2015-10-01 | Academia Sinica | Reactive labelling compounds and uses thereof |
KR20160145624A (en) | 2014-03-31 | 2016-12-20 | 제넨테크, 인크. | Anti-ox40 antibodies and methods of use |
CA2943834A1 (en) | 2014-03-31 | 2015-10-08 | Genentech, Inc. | Combination therapy comprising anti-angiogenesis agents and ox40 binding agonists |
SG10202107077QA (en) | 2014-04-02 | 2021-07-29 | Hoffmann La Roche | Method for detecting multispecific antibody light chain mispairing |
KR102352573B1 (en) | 2014-04-04 | 2022-01-18 | 바이오노믹스 인코포레이티드 | Humanized antibodies that bind lgr5 |
WO2015157238A2 (en) | 2014-04-08 | 2015-10-15 | Medimmune, Llc | Binding molecules specific for il-21 and uses thereof |
TW201542594A (en) | 2014-04-11 | 2015-11-16 | Medimmune Llc | Bispecific HER2 antibodies |
MA52909A (en) | 2014-04-18 | 2021-04-21 | Acceleron Pharma Inc | METHODS FOR INCREASING RED BLOOD CELLS AND SICKLE CELL TREATMENT |
WO2015164615A1 (en) | 2014-04-24 | 2015-10-29 | University Of Oslo | Anti-gluten antibodies and uses thereof |
US11427647B2 (en) | 2014-04-27 | 2022-08-30 | Famewave Ltd. | Polynucleotides encoding humanized antibodies against CEACAM1 |
CR20160534A (en) | 2014-04-27 | 2017-04-25 | Ccam Biotherapeutics Ltd | HUMANIZED ANTIBODIES AGAINST THE CELLULAR ADHESION MOLECULE RELATED TO CARCINOEMBRIONIC ANTIGEN 1 (CEACAM1) |
US9753036B2 (en) | 2014-04-29 | 2017-09-05 | Edp Biotech Corporation | Methods and compositions for screening and detecting biomarkers |
EP3711780A3 (en) | 2014-04-30 | 2020-12-09 | Pfizer Inc | Anti-ptk7 antibody-drug conjugates |
WO2015171523A1 (en) | 2014-05-05 | 2015-11-12 | Regeneron Pharmaceuticals, Inc. | Humanized c5 and c3 animals |
CA2946606C (en) | 2014-05-13 | 2023-06-06 | Bavarian Nordic A/S | Combination therapy for treating cancer with a poxvirus expressing a tumor antigen and an antagonist of tim-3 |
EP3142700B1 (en) | 2014-05-16 | 2021-03-03 | Medimmune, LLC | Molecules with altered neonate fc receptor binding having enhanced therapeutic and diagnostic properties |
JP2017522861A (en) | 2014-05-22 | 2017-08-17 | ジェネンテック, インコーポレイテッド | Anti-GPC3 antibody and immunoconjugate |
MX2016015163A (en) | 2014-05-23 | 2017-03-03 | Genentech Inc | Mit biomarkers and methods using the same. |
JP2017518989A (en) | 2014-05-27 | 2017-07-13 | アカデミア シニカAcademia Sinica | Anti-CD20 glycoengineered antibody group and use thereof |
US10005847B2 (en) | 2014-05-27 | 2018-06-26 | Academia Sinica | Anti-HER2 glycoantibodies and uses thereof |
US10118969B2 (en) | 2014-05-27 | 2018-11-06 | Academia Sinica | Compositions and methods relating to universal glycoforms for enhanced antibody efficacy |
US11332523B2 (en) | 2014-05-28 | 2022-05-17 | Academia Sinica | Anti-TNF-alpha glycoantibodies and uses thereof |
CA2949998A1 (en) | 2014-05-28 | 2015-12-03 | Agenus Inc. | Anti-gitr antibodies and methods of use thereof |
GB201409558D0 (en) | 2014-05-29 | 2014-07-16 | Ucb Biopharma Sprl | Method |
KR102015451B1 (en) | 2014-06-03 | 2019-08-28 | 엑스바이오테크, 인크. | Compositions and methods for treating and preventing staphylococcus aureus infections |
US9856324B2 (en) | 2014-06-04 | 2018-01-02 | MabVax Therapeutics, Inc. | Human monoclonal antibodies to ganglioside GD2 |
CA2949982A1 (en) | 2014-06-11 | 2015-12-17 | Genentech, Inc. | Anti-lgr5 antibodies and uses thereof |
US20170137824A1 (en) | 2014-06-13 | 2017-05-18 | Indranil BANERJEE | New treatment against influenza virus |
CN107073121A (en) | 2014-06-13 | 2017-08-18 | 基因泰克公司 | Treatment and the method for prevention cancer drug resistance |
MA40008A (en) | 2014-06-13 | 2021-05-05 | Acceleron Pharma Inc | ANTAGONIST ACTRII FOR THE TREATMENT AND PREVENTION OF SKIN ULCER IN A SUBJECT WITH ANEMIA |
CA2952876A1 (en) | 2014-06-20 | 2015-12-23 | Bioalliance C.V. | Anti-folate receptor alpha (fra) antibody-drug conjugates and methods of using thereof |
WO2015198202A1 (en) | 2014-06-23 | 2015-12-30 | Friedrich Miescher Institute For Biomedical Research | Methods for triggering de novo formation of heterochromatin and or epigenetic silencing with small rnas |
GB201411320D0 (en) | 2014-06-25 | 2014-08-06 | Ucb Biopharma Sprl | Antibody construct |
TW201623329A (en) | 2014-06-30 | 2016-07-01 | 亞佛瑞司股份有限公司 | Vaccines and monoclonal antibodies targeting truncated variants of osteopontin and uses thereof |
US20170165261A1 (en) | 2014-07-01 | 2017-06-15 | Brian Arthur Hemmings | Combination of a brafv600e inhibitor and mertk inhibitor to treat melanoma |
EP3166966A1 (en) | 2014-07-10 | 2017-05-17 | Affiris AG | Substances and methods for the use in prevention and/or treatment in huntington's disease |
MX2017000363A (en) | 2014-07-11 | 2017-04-27 | Genentech Inc | Notch pathway inhibition. |
WO2016011052A1 (en) | 2014-07-14 | 2016-01-21 | Genentech, Inc. | Diagnostic methods and compositions for treatment of glioblastoma |
US9139648B1 (en) | 2014-07-15 | 2015-09-22 | Kymab Limited | Precision medicine by targeting human NAV1.9 variants for treatment of pain |
PL3169361T3 (en) | 2014-07-15 | 2019-11-29 | Hoffmann La Roche | Compositions for treating cancer using pd-1 axis binding antagonists and mek inhibitors |
GB201412658D0 (en) | 2014-07-16 | 2014-08-27 | Ucb Biopharma Sprl | Molecules |
GB201412659D0 (en) | 2014-07-16 | 2014-08-27 | Ucb Biopharma Sprl | Molecules |
SG10201913702WA (en) | 2014-07-17 | 2020-03-30 | Novo Nordisk As | Site directed mutagenesis of trem-1 antibodies for decreasing viscosity |
US9777061B2 (en) | 2014-07-21 | 2017-10-03 | Novartis Ag | Treatment of cancer using a CD33 chimeric antigen receptor |
EP3511413B1 (en) | 2014-07-25 | 2022-09-07 | Theravectys | Lentiviral vectors for regulated expression of a chimeric antigen receptor molecule |
JP2017523980A (en) | 2014-08-06 | 2017-08-24 | ライナット ニューロサイエンス コーポレイション | Method for lowering LDL-cholesterol |
WO2016020799A1 (en) | 2014-08-06 | 2016-02-11 | Rinat Neuroscience Corp. | Methods for reducing ldl-cholesterol |
JP6919118B2 (en) | 2014-08-14 | 2021-08-18 | ノバルティス アーゲー | Treatment of cancer with GFRα-4 chimeric antigen receptor |
JP7084138B2 (en) | 2014-08-19 | 2022-06-14 | ノバルティス アーゲー | Anti-CD123 Chimeric Antigen Receptor (CAR) for use in cancer treatment |
CA2996445A1 (en) | 2014-09-05 | 2016-03-10 | Eli Hatchwell | Methods and compositions for inhibiting and treating neurological conditions |
EP3188746A4 (en) | 2014-09-05 | 2018-05-02 | The Johns Hopkins University | Targeting capn9/capns2 activity as a therapeutic strategy for the treatment of myofibroblast differentiation and associated pathologies |
AU2015315294B2 (en) | 2014-09-08 | 2020-09-17 | Academia Sinica | Human iNKT cell activation using glycolipids |
CN113698485A (en) | 2014-09-12 | 2021-11-26 | 基因泰克公司 | anti-B7-H4 antibodies and immunoconjugates |
PE20170670A1 (en) | 2014-09-12 | 2017-06-06 | Genentech Inc | ANTI-CLL-1 ANTIBODIES AND IMMUNOCONJUGATES |
SG10201809668TA (en) | 2014-09-12 | 2018-11-29 | Genentech Inc | Anti-her2 antibodies and immunoconjugates |
EP3191127A1 (en) | 2014-09-13 | 2017-07-19 | Novartis AG | Combination therapies of egfr inhibitors |
KR20170057339A (en) | 2014-09-15 | 2017-05-24 | 제넨테크, 인크. | Antibody formulations |
EP3194444B1 (en) | 2014-09-16 | 2019-07-10 | Symphogen A/S | Anti-met antibodies and compositions |
MX2017003472A (en) | 2014-09-17 | 2017-10-31 | Genentech Inc | Immunoconjugates comprising anti-her2 antibodies and pyrrolobenzodiazepines. |
US10222386B2 (en) | 2014-09-19 | 2019-03-05 | The Johns Hopkins University | Biomarkers of congnitive dysfunction |
ES2796903T3 (en) | 2014-09-23 | 2020-11-30 | Hoffmann La Roche | Procedure for the use of anti-CD79b immunoconjugates |
EP3197492A1 (en) | 2014-09-23 | 2017-08-02 | Pfizer Inc | Treatment with anti-pcsk9 antibodies |
EP3197557A1 (en) | 2014-09-24 | 2017-08-02 | Friedrich Miescher Institute for Biomedical Research | Lats and breast cancer |
SI3200822T1 (en) | 2014-09-30 | 2021-10-29 | Deutsches Krebsforschungszentrum Stiftung Des Oeffentlichen Rechts | Binding molecules, especially antibodies, binding to l1cam (cd171) |
KR20230153495A (en) | 2014-10-01 | 2023-11-06 | 메디뮨 리미티드 | Antibodies to ticagrelor and methods of use |
BR112017006664A2 (en) | 2014-10-03 | 2017-12-26 | Novartis Ag | combination therapies |
JP6877339B2 (en) | 2014-10-14 | 2021-05-26 | ノバルティス アーゲー | Antibody molecule against PD-L1 and its use |
WO2016061389A2 (en) | 2014-10-16 | 2016-04-21 | Genentech, Inc. | Anti-alpha-synuclein antibodies and methods of use |
US11124822B2 (en) | 2014-10-17 | 2021-09-21 | Carnegie Mellon University | Enhanced biomolecule detection assays based on tyramide signal amplification and gammaPNA probes |
MA41685A (en) | 2014-10-17 | 2017-08-22 | Biogen Ma Inc | COPPER SUPPLEMENT FOR THE REGULATION OF GLYCOSYLATION IN A MAMMAL CELL CULTURE PROCESS |
US10920208B2 (en) | 2014-10-22 | 2021-02-16 | President And Fellows Of Harvard College | Evolution of proteases |
EP3209697A4 (en) | 2014-10-23 | 2018-05-30 | La Trobe University | Fn14-binding proteins and uses thereof |
TW202124419A (en) | 2014-10-24 | 2021-07-01 | 美商必治妥美雅史谷比公司 | Modified fgf-21 polypeptides and uses thereof |
MA40864A (en) | 2014-10-31 | 2017-09-05 | Biogen Ma Inc | HYPOTAURINE, GABA, BETA-ALANINE AND CHOLINE FOR THE REGULATION OF THE ACCUMULATION OF RESIDUAL BY-PRODUCTS IN MAMMAL CELL CULTURE PROCESSES |
US10626176B2 (en) | 2014-10-31 | 2020-04-21 | Jounce Therapeutics, Inc. | Methods of treating conditions with antibodies that bind B7-H4 |
MX2017005258A (en) | 2014-10-31 | 2017-07-26 | Oncomed Pharm Inc | Combination therapy for treatment of disease. |
BR112017009151A2 (en) | 2014-11-03 | 2018-03-06 | Genentech, Inc. | Assays for Detecting Immune T-Cell Subgroups and Methods of Using Them |
AU2015343339A1 (en) | 2014-11-03 | 2017-06-15 | Genentech, Inc. | Methods and biomarkers for predicting efficacy and evaluation of an OX40 agonist treatment |
KR102626877B1 (en) | 2014-11-05 | 2024-01-19 | 애넥슨, 인코포레이티드 | Humanized Anti-Complement Factor C1Q Antibodies and Uses Thereof |
CA2966558C (en) | 2014-11-05 | 2024-03-12 | Genentech, Inc. | Methods of producing two chain proteins in bacteria |
MX2017003478A (en) | 2014-11-05 | 2018-02-01 | Genentech Inc | Anti-fgfr2/3 antibodies and methods using same. |
CN113699203A (en) | 2014-11-05 | 2021-11-26 | 基因泰克公司 | Method for producing double-stranded proteins in bacteria |
JP6707090B2 (en) | 2014-11-06 | 2020-06-10 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | Fc region variants with altered FcRn binding and methods of use |
WO2016073157A1 (en) | 2014-11-06 | 2016-05-12 | Genentech, Inc. | Anti-ang2 antibodies and methods of use thereof |
US20160152720A1 (en) | 2014-11-06 | 2016-06-02 | Genentech, Inc. | Combination therapy comprising ox40 binding agonists and tigit inhibitors |
KR20170076697A (en) | 2014-11-06 | 2017-07-04 | 에프. 호프만-라 로슈 아게 | Fc-region variants with modified fcrn- and protein a-binding properties |
WO2016073894A1 (en) | 2014-11-07 | 2016-05-12 | Eleven Biotherapeutics, Inc. | Therapeutic agents with increased ocular retention |
EP3215530B9 (en) | 2014-11-07 | 2020-09-09 | Sesen Bio, Inc. | Improved il-6 antibodies |
CA2960297A1 (en) | 2014-11-10 | 2016-05-19 | Genentech, Inc. | Anti-interleukin-33 antibodies and uses thereof |
WO2016077369A1 (en) | 2014-11-10 | 2016-05-19 | Genentech, Inc. | Animal model for nephropathy and agents for treating the same |
PL3218406T3 (en) | 2014-11-10 | 2021-10-04 | Medimmune Limited | Binding molecules specific for cd73 and uses thereof |
JP6847037B2 (en) | 2014-11-11 | 2021-03-24 | メディミューン リミテッド | Concomitant therapeutic agents containing anti-CD73 antibody and A2A receptor inhibitor and their use |
US9879087B2 (en) | 2014-11-12 | 2018-01-30 | Siamab Therapeutics, Inc. | Glycan-interacting compounds and methods of use |
ES2941897T3 (en) | 2014-11-12 | 2023-05-26 | Seagen Inc | Compounds that interact with glycans and procedures for use |
EP3875481A1 (en) | 2014-11-14 | 2021-09-08 | The U.S.A. as represented by the Secretary, Department of Health and Human Services | Neutralizing antibodies to ebola virus glycoprotein and their use |
CN107429075B (en) | 2014-11-17 | 2022-11-01 | 卡内基梅隆大学 | Activatable two-component photosensitizer |
CA2967368A1 (en) | 2014-11-17 | 2016-05-26 | Genentech, Inc. | Combination therapy comprising ox40 binding agonists and pd-1 axis binding antagonists |
JP6779876B2 (en) | 2014-11-19 | 2020-11-04 | ジェネンテック, インコーポレイテッド | Anti-transferrin receptor antibody and how to use it |
CN107108745B (en) | 2014-11-19 | 2021-01-12 | 基因泰克公司 | Antibodies against BACE1 and their use for immunotherapy of neurological diseases |
JP6993228B2 (en) | 2014-11-19 | 2022-03-03 | ジェネンテック, インコーポレイテッド | Anti-transferrin receptor / anti-BACE1 multispecific antibody and usage |
CN107206072B (en) | 2014-11-20 | 2022-01-21 | 豪夫迈·罗氏有限公司 | Combination therapy of the T cell activating bispecific antigen binding molecule CD3 ABD folate receptor 1(FolR1) and a PD-1 axis binding antagonist |
ES2832802T3 (en) | 2014-11-21 | 2021-06-11 | Univ Maryland | Systems of directed administration of the specific particulate of a structure |
MA41119A (en) | 2014-12-03 | 2017-10-10 | Acceleron Pharma Inc | METHODS OF TREATMENT OF MYELODYSPLASIC SYNDROMES AND SIDEROBLASTIC ANEMIA |
SG11201704449VA (en) | 2014-12-05 | 2017-06-29 | Genentech Inc | ANTI-CD79b ANTIBODIES AND METHODS OF USE |
EP3227337A1 (en) | 2014-12-05 | 2017-10-11 | F. Hoffmann-La Roche AG | Methods and compositions for treating cancer using pd-1 axis antagonists and hpk1 antagonists |
RU2017120039A (en) | 2014-12-10 | 2019-01-10 | Дженентек, Инк. | ANTIBODIES TO HEMATOENCEPHALIC BARRIER RECEPTORS AND METHODS OF APPLICATION |
CN108064167A (en) | 2014-12-11 | 2018-05-22 | 皮埃尔法布雷医药公司 | Anti- C10ORF54 antibody and application thereof |
US10093733B2 (en) | 2014-12-11 | 2018-10-09 | Abbvie Inc. | LRP-8 binding dual variable domain immunoglobulin proteins |
US9765135B2 (en) | 2014-12-19 | 2017-09-19 | Chugai Seiyaku Kabushiki Kaisha | Anti-C5 antibodies |
US20170340733A1 (en) | 2014-12-19 | 2017-11-30 | Novartis Ag | Combination therapies |
CA2972048C (en) | 2014-12-22 | 2023-03-07 | The Rockefeller University | Anti-mertk agonistic antibodies and uses thereof |
WO2016106286A1 (en) | 2014-12-23 | 2016-06-30 | Biodesy, Inc. | Attachment of proteins to interfaces for use in nonlinear optical detection |
WO2016111947A2 (en) | 2015-01-05 | 2016-07-14 | Jounce Therapeutics, Inc. | Antibodies that inhibit tim-3:lilrb2 interactions and uses thereof |
MX2017009038A (en) | 2015-01-08 | 2017-10-25 | Biogen Ma Inc | Lingo-1 antagonists and uses for treatment of demyelinating disorders. |
SG10202111844VA (en) | 2015-01-09 | 2021-12-30 | Adalta Ltd | Cxcr4 binding molecules |
US9975965B2 (en) | 2015-01-16 | 2018-05-22 | Academia Sinica | Compositions and methods for treatment and detection of cancers |
US10495645B2 (en) | 2015-01-16 | 2019-12-03 | Academia Sinica | Cancer markers and methods of use thereof |
EP3247723A1 (en) | 2015-01-22 | 2017-11-29 | Chugai Seiyaku Kabushiki Kaisha | A combination of two or more anti-c5 antibodies and methods of use |
CA2972731A1 (en) | 2015-01-24 | 2016-07-28 | Chi-Huey Wong | Cancer markers and methods of use thereof |
EP3789766A1 (en) | 2015-01-24 | 2021-03-10 | Academia Sinica | Novel glycan conjugates and methods of use thereof |
JP6912386B2 (en) | 2015-01-26 | 2021-08-04 | ザ ユニバーシティー オブ シカゴ | CAR T cells that recognize cancer-specific IL13Rα2 |
EP3250609A4 (en) | 2015-01-26 | 2018-07-11 | The University of Chicago | Il13ra alpha 2 binding agents and use thereof in cancer treatment |
WO2016123591A2 (en) | 2015-01-30 | 2016-08-04 | Academia Sinica | Compositions and methods for treatment and detection of cancers |
CN107849123A (en) | 2015-02-02 | 2018-03-27 | i2制药股份有限公司 | Resist alternative light chain antibody |
EP3253875B1 (en) | 2015-02-04 | 2020-01-08 | H. Hoffnabb-La Roche Ag | Tau antisense oligomers and uses thereof |
US10330683B2 (en) | 2015-02-04 | 2019-06-25 | Genentech, Inc. | Mutant smoothened and methods of using the same |
JP6772156B2 (en) | 2015-02-04 | 2020-10-21 | ブリストル−マイヤーズ スクイブ カンパニーBristol−Myers Squibb Company | How to choose a therapeutic molecule |
KR20170110129A (en) | 2015-02-05 | 2017-10-10 | 추가이 세이야쿠 가부시키가이샤 | Antibodies comprising ionic concentration dependent antigen binding domains, Fc region variants, antibodies that bind to IL-8, and their use |
ES2937020T3 (en) | 2015-03-03 | 2023-03-23 | Kymab Ltd | Antibodies, uses and methods |
EP3265491A1 (en) | 2015-03-03 | 2018-01-10 | Xoma (Us) Llc | Treatment of post-prandial hyperinsulinemia and hypoglycemia after bariatric surgery |
EP3735982A1 (en) | 2015-03-10 | 2020-11-11 | The University of Massachusetts | Targeting gdf6 and bmp signaling for anti-melanoma therapy |
CA2977285A1 (en) | 2015-03-16 | 2016-09-22 | F. Hoffmann-La Roche Ag | Methods of detecting and quantifying il-13 and uses in diagnosing and treating th2-associated diseases |
WO2016146833A1 (en) | 2015-03-19 | 2016-09-22 | F. Hoffmann-La Roche Ag | Biomarkers for nad(+)-diphthamide adp ribosyltransferase resistance |
US10174292B2 (en) | 2015-03-20 | 2019-01-08 | International Aids Vaccine Initiative | Soluble HIV-1 envelope glycoprotein trimers |
US10562960B2 (en) | 2015-03-20 | 2020-02-18 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Neutralizing antibodies to gp120 and their use |
MA53297A (en) | 2015-03-23 | 2022-05-04 | Jounce Therapeutics Inc | ANTI-ICOS ANTIBODIES |
EP3072901A1 (en) | 2015-03-23 | 2016-09-28 | International Aids Vaccine Initiative | Soluble hiv-1 envelope glycoprotein trimers |
CA2981068C (en) | 2015-03-26 | 2021-12-14 | Women & Infants Hospital Of Rhode Island | Therapy for malignant disease comprising the inhibition of human epididymal secretory protein e4 and immune checkpoint inhibitors |
WO2016160618A2 (en) | 2015-03-27 | 2016-10-06 | University Of Southern California | Car t-cell therapy directed to lhr for the treatment of solid tumors |
ES2820768T3 (en) | 2015-04-03 | 2021-04-22 | Xoma Technology Ltd | Cancer treatment using TGF-beta and PD-1 inhibitors |
KR20170132793A (en) | 2015-04-03 | 2017-12-04 | 유레카 쎄라퓨틱스, 인코포레이티드 | Constructs targeting AFP peptide / MHC complexes and uses thereof |
WO2016164503A1 (en) | 2015-04-06 | 2016-10-13 | Acceleron Pharma Inc. | Alk7:actriib heteromultimers and uses thereof |
CN108137648A (en) | 2015-04-06 | 2018-06-08 | 哈佛学院校长同事会 | For the composition and method of Nonmyeloablative pretreatment |
MA41919A (en) | 2015-04-06 | 2018-02-13 | Acceleron Pharma Inc | ALK4 HETEROMULTIMERS: ACTRIIB AND THEIR USES |
CA2981851A1 (en) | 2015-04-07 | 2016-10-13 | Alector Llc | Anti-sortilin antibodies and methods of use thereof |
KR20180002653A (en) | 2015-04-07 | 2018-01-08 | 제넨테크, 인크. | Antigen binding complexes having an agonistic activity activity and methods of use |
MY188432A (en) | 2015-04-13 | 2021-12-08 | Pfizer | Chimeric antigen receptors targeting b-cell maturation antigen |
CA3219684A1 (en) | 2015-04-13 | 2016-10-13 | Pfizer Inc. | Anti-bcma antibodies, anti-cd3 antibodies and bi-specific antibodies binding to bcma and cd3 |
US11299729B2 (en) | 2015-04-17 | 2022-04-12 | President And Fellows Of Harvard College | Vector-based mutagenesis system |
CN107969128A (en) | 2015-04-17 | 2018-04-27 | 高山免疫科学股份有限公司 | Immune modulator with adjustable affinity |
WO2016170022A1 (en) | 2015-04-21 | 2016-10-27 | Institut Gustave Roussy | Therapeutic methods, products and compositions inhibiting znf555 |
GB201506869D0 (en) | 2015-04-22 | 2015-06-03 | Ucb Biopharma Sprl | Method |
GB201506870D0 (en) | 2015-04-22 | 2015-06-03 | Ucb Biopharma Sprl | Method |
PL3286315T3 (en) | 2015-04-24 | 2021-11-02 | F. Hoffmann-La Roche Ag | Methods of identifying bacteria comprising binding polypeptides |
US10814012B2 (en) | 2015-04-29 | 2020-10-27 | University Of South Australia | Compositions and methods for administering antibodies |
EP3778640A1 (en) | 2015-05-01 | 2021-02-17 | Genentech, Inc. | Masked anti-cd3 antibodies and methods of use |
WO2016179194A1 (en) | 2015-05-04 | 2016-11-10 | Jounce Therapeutics, Inc. | Lilra3 and method of using the same |
US10844122B2 (en) | 2015-05-06 | 2020-11-24 | Janssen Biotech, Inc. | Prostate specific membrane antigen (PSMA) bispecific binding agents and uses thereof |
JP6867955B2 (en) | 2015-05-07 | 2021-05-12 | アジェナス インコーポレイテッド | Anti-OX40 antibody and how to use it |
CN116196414A (en) | 2015-05-11 | 2023-06-02 | 豪夫迈·罗氏有限公司 | Compositions and methods for treating lupus nephritis |
EP3294334B1 (en) | 2015-05-11 | 2020-07-08 | The Johns Hopkins University | Autoimmune antibodies for use in inhibiting cancer cell growth |
LT3294770T (en) | 2015-05-12 | 2020-12-28 | F. Hoffmann-La Roche Ag | Therapeutic and diagnostic methods for cancer |
CN114907271A (en) | 2015-05-22 | 2022-08-16 | 转化药物开发有限责任公司 | Compositions of benzamide and active compound and methods of use thereof |
WO2016189045A1 (en) | 2015-05-27 | 2016-12-01 | Ucb Biopharma Sprl | Method for the treatment of neurological disease |
WO2016196381A1 (en) | 2015-05-29 | 2016-12-08 | Genentech, Inc. | Pd-l1 promoter methylation in cancer |
JP7144935B2 (en) | 2015-05-29 | 2022-09-30 | ジェネンテック, インコーポレイテッド | Therapeutic and diagnostic methods for cancer |
LT3303394T (en) | 2015-05-29 | 2020-10-12 | Agenus Inc. | Anti-ctla-4 antibodies and methods of use thereof |
PE20180193A1 (en) | 2015-05-29 | 2018-01-26 | Abbvie Inc | ANTI-CD40 ANTIBODIES AND THEIR USES |
JP2018520658A (en) | 2015-05-29 | 2018-08-02 | ジェネンテック, インコーポレイテッド | Humanized anti-Ebola virus glycoprotein antibodies and uses thereof |
ES2784603T3 (en) | 2015-06-02 | 2020-09-29 | Novo Nordisk As | Insulins with recombinant polar extensions |
JP2018516933A (en) | 2015-06-02 | 2018-06-28 | ジェネンテック, インコーポレイテッド | Compositions and methods for treating neurological disorders using anti-IL-34 antibodies |
WO2016196975A1 (en) | 2015-06-03 | 2016-12-08 | The United States Of America, As Represented By The Secretary Department Of Health & Human Services | Neutralizing antibodies to hiv-1 env and their use |
JP7010473B2 (en) | 2015-06-04 | 2022-02-10 | ユニバーシティ オブ サザン カリフォルニア | LYM-1 and LYM-2 Targeted CAR Cell Immunotherapy |
RU2732122C2 (en) | 2015-06-05 | 2020-09-11 | Дженентек, Инк. | Anti-tau protein antibodies and methods of using said antibodies |
AU2016274585A1 (en) | 2015-06-08 | 2017-12-14 | Genentech, Inc. | Methods of treating cancer using anti-OX40 antibodies |
WO2016200835A1 (en) | 2015-06-08 | 2016-12-15 | Genentech, Inc. | Methods of treating cancer using anti-ox40 antibodies and pd-1 axis binding antagonists |
KR20180033502A (en) | 2015-06-12 | 2018-04-03 | 알렉터 엘엘씨 | Anti-CD33 antibodies and methods of use thereof |
US11174313B2 (en) | 2015-06-12 | 2021-11-16 | Alector Llc | Anti-CD33 antibodies and methods of use thereof |
TW201710286A (en) | 2015-06-15 | 2017-03-16 | 艾伯維有限公司 | Binding proteins against VEGF, PDGF, and/or their receptors |
JP2018524295A (en) | 2015-06-15 | 2018-08-30 | ジェネンテック, インコーポレイテッド | Antibodies and immune complexes |
EP3310811B1 (en) | 2015-06-16 | 2021-06-16 | Genentech, Inc. | Anti-cd3 antibodies and methods of use |
WO2016205200A1 (en) | 2015-06-16 | 2016-12-22 | Genentech, Inc. | Anti-cll-1 antibodies and methods of use |
WO2016205520A1 (en) | 2015-06-16 | 2016-12-22 | Genentech, Inc. | Humanized and affinity matured antibodies to fcrh5 and methods of use |
CN107787331B (en) | 2015-06-17 | 2022-01-11 | 豪夫迈·罗氏有限公司 | anti-HER 2 antibodies and methods of use |
WO2016205320A1 (en) | 2015-06-17 | 2016-12-22 | Genentech, Inc. | Methods of treating locally advanced or metastatic breast cancers using pd-1 axis binding antagonists and taxanes |
GB201510758D0 (en) | 2015-06-18 | 2015-08-05 | Ucb Biopharma Sprl | Novel TNFa structure for use in therapy |
US20180188257A1 (en) | 2015-06-19 | 2018-07-05 | University Of Rochester | Septin proteins as novel biomarkers for detection and treatment of müllerian cancers |
BR112017027870A2 (en) | 2015-06-24 | 2018-08-28 | Janssen Pharmaceutica Nv | antibodies and anti-sight fragments |
KR20180021864A (en) | 2015-06-29 | 2018-03-05 | 제넨테크, 인크. | Type II anti-CD20 antibodies for use in organ transplantation |
CN114671951A (en) | 2015-06-29 | 2022-06-28 | 伊缪诺金公司 | anti-CD 123 antibodies and conjugates and derivatives thereof |
GB201601077D0 (en) | 2016-01-20 | 2016-03-02 | Ucb Biopharma Sprl | Antibody molecule |
GB201601073D0 (en) | 2016-01-20 | 2016-03-02 | Ucb Biopharma Sprl | Antibodies |
GB201601075D0 (en) | 2016-01-20 | 2016-03-02 | Ucb Biopharma Sprl | Antibodies molecules |
US10877045B2 (en) | 2015-07-21 | 2020-12-29 | Saint Louis University | Compositions and methods for diagnosing and treating endometriosis-related infertility |
WO2017015545A1 (en) | 2015-07-22 | 2017-01-26 | President And Fellows Of Harvard College | Evolution of site-specific recombinases |
WO2017015559A2 (en) | 2015-07-23 | 2017-01-26 | President And Fellows Of Harvard College | Evolution of bt toxins |
EP3878465A1 (en) | 2015-07-29 | 2021-09-15 | Novartis AG | Combination therapies comprising antibody molecules to tim-3 |
EP3964528A1 (en) | 2015-07-29 | 2022-03-09 | Novartis AG | Combination therapies comprising antibody molecules to lag-3 |
US10612011B2 (en) | 2015-07-30 | 2020-04-07 | President And Fellows Of Harvard College | Evolution of TALENs |
WO2017024171A1 (en) | 2015-08-04 | 2017-02-09 | Acceleron Pharma Inc. | Methods for treating myeloproliferative disorders |
TW202330904A (en) | 2015-08-04 | 2023-08-01 | 美商再生元醫藥公司 | Taurine supplemented cell culture medium and methods of use |
EP3331562A2 (en) | 2015-08-06 | 2018-06-13 | Xoma (Us) Llc | Antibody fragments against the insulin receptor and uses thereof to treat hypoglycemia |
CN105384825B (en) | 2015-08-11 | 2018-06-01 | 南京传奇生物科技有限公司 | A kind of bispecific chimeric antigen receptor and its application based on single domain antibody |
WO2017040342A1 (en) | 2015-08-28 | 2017-03-09 | Genentech, Inc. | Anti-hypusine antibodies and uses thereof |
CN114605548A (en) | 2015-09-01 | 2022-06-10 | 艾吉纳斯公司 | anti-PD-1 antibodies and methods of use thereof |
EP3344806A4 (en) | 2015-09-04 | 2019-03-20 | OBI Pharma, Inc. | Glycan arrays and method of use |
KR20230155021A (en) | 2015-09-15 | 2023-11-09 | 스칼러 락, 인크. | Anti-pro/latent-myostatin antibodies and uses thereof |
MA42844A (en) | 2015-09-17 | 2018-07-25 | Immunogen Inc | THERAPEUTIC COMBINATIONS INCLUDING ANTI-FOLR1 IMMUNOCONJUGATES |
TW202342532A (en) | 2015-09-18 | 2023-11-01 | 日商中外製藥股份有限公司 | Il-8-binding antibodies and uses thereof |
AU2016326721C1 (en) | 2015-09-23 | 2021-06-03 | Cytoimmune Therapeutics, Inc. | FLT3 directed car cells for immunotherapy |
CN116987187A (en) | 2015-09-23 | 2023-11-03 | 豪夫迈·罗氏有限公司 | Optimized variants of anti-VEGF antibodies |
JP6967003B2 (en) | 2015-09-23 | 2021-11-17 | メレオ バイオファーマ 5 インコーポレイテッド | Methods and compositions for the treatment of cancer |
KR20180083313A (en) | 2015-09-24 | 2018-07-20 | 에이비비트로, 엘엘씨 | HIV antibody compositions and methods of use |
CN108289953B (en) | 2015-09-29 | 2022-03-11 | 细胞基因公司 | PD-1 binding proteins and methods of use thereof |
US20180282415A1 (en) | 2015-09-30 | 2018-10-04 | Merck Patent Gmbh | Combination of a PD-1 Axis Binding Antagonist and an ALK Inhibitor for Treating ALK-Negative Cancer |
MA43348A (en) | 2015-10-01 | 2018-08-08 | Novo Nordisk As | PROTEIN CONJUGATES |
MA43345A (en) | 2015-10-02 | 2018-08-08 | Hoffmann La Roche | PYRROLOBENZODIAZEPINE ANTIBODY-DRUG CONJUGATES AND METHODS OF USE |
US20170247454A1 (en) | 2015-10-02 | 2017-08-31 | Hoffmann-La Roche Inc. | Anti-pd1 antibodies and methods of use |
WO2017064716A1 (en) | 2015-10-13 | 2017-04-20 | Rappaport Family Institute For Research | Heparanase-neutralizing monoclonal antibodies |
MA43354A (en) | 2015-10-16 | 2018-08-22 | Genentech Inc | CONJUGATE DRUG CONJUGATES WITH CLOUDY DISULPHIDE |
US11207393B2 (en) | 2015-10-16 | 2021-12-28 | President And Fellows Of Harvard College | Regulatory T cell PD-1 modulation for regulating T cell effector immune responses |
MA45326A (en) | 2015-10-20 | 2018-08-29 | Genentech Inc | CALICHEAMICIN-ANTIBODY-DRUG CONJUGATES AND METHODS OF USE |
CA2998208A1 (en) | 2015-10-22 | 2017-04-27 | Jounce Therapeutics, Inc. | Gene signatures for determining icos expression |
KR20180063325A (en) | 2015-10-23 | 2018-06-11 | 유레카 쎄라퓨틱스, 인코포레이티드 | Antibody / T-cell receptor chimeric constructs and uses thereof |
EP3368570A1 (en) | 2015-10-27 | 2018-09-05 | UCB Biopharma SPRL | Methods of treatment using anti-il-17a/f antibodies |
WO2017072669A1 (en) | 2015-10-28 | 2017-05-04 | Friedrich Miescher Institute For Biomedical Research | Tenascin-w and biliary tract cancers |
EP3184547A1 (en) | 2015-10-29 | 2017-06-28 | F. Hoffmann-La Roche AG | Anti-tpbg antibodies and methods of use |
AU2016343978A1 (en) | 2015-10-29 | 2018-05-17 | Dana-Farber Cancer Institute, Inc. | Methods for identification, assessment, prevention, and treatment of metabolic disorders using PM20D1 and N-lipidated amino acids |
EP3368074A2 (en) | 2015-10-30 | 2018-09-05 | Hoffmann-La Roche AG | Anti-factor d antibodies and conjugates |
CA3001362C (en) | 2015-10-30 | 2020-10-13 | Genentech, Inc. | Anti-htra1 antibodies and methods of use thereof |
GB201519303D0 (en) | 2015-11-02 | 2015-12-16 | Imp Innovations Ltd | Phagemid vector |
WO2017079442A1 (en) | 2015-11-04 | 2017-05-11 | Icahn School Of Medicine At Mount Sinai | Methods of treating tumors and cancer, and identifying candidate subjects for such treatment |
WO2017079768A1 (en) | 2015-11-08 | 2017-05-11 | Genentech, Inc. | Methods of screening for multispecific antibodies |
SG10202103712VA (en) | 2015-11-10 | 2021-05-28 | Medimmune Llc | Binding molecules specific for asct2 and uses thereof |
EP3374391A2 (en) | 2015-11-10 | 2018-09-19 | Visterra, Inc. | Antibody molecule-drug conjugates and uses thereof |
SG11201803213XA (en) | 2015-11-12 | 2018-05-30 | Siamab Therapeutics Inc | Glycan-interacting compounds and methods of use |
EP3380121B1 (en) | 2015-11-23 | 2023-12-20 | Acceleron Pharma Inc. | Actrii antagonist for use in treating eye disorders |
KR20180091849A (en) | 2015-11-25 | 2018-08-16 | 비스테라, 인크. | Antibody molecules against APRIL and uses thereof |
EP3383908A1 (en) | 2015-12-02 | 2018-10-10 | Stsciences, Inc. | Antibodies specific to glycosylated btla (b- and t- lymphocyte attenuator) |
KR20180086246A (en) | 2015-12-02 | 2018-07-30 | 주식회사 에스티큐브앤컴퍼니 | Antibodies and molecules that immunospecifically bind to BTN1A1 and their therapeutic uses |
GB201521393D0 (en) | 2015-12-03 | 2016-01-20 | Ucb Biopharma Sprl | Antibodies |
GB201521382D0 (en) | 2015-12-03 | 2016-01-20 | Ucb Biopharma Sprl | Antibodies |
GB201521391D0 (en) | 2015-12-03 | 2016-01-20 | Ucb Biopharma Sprl | Antibodies |
GB201521389D0 (en) | 2015-12-03 | 2016-01-20 | Ucb Biopharma Sprl | Method |
GB201521383D0 (en) | 2015-12-03 | 2016-01-20 | Ucb Biopharma Sprl And Ucb Celltech | Method |
EP3178848A1 (en) | 2015-12-09 | 2017-06-14 | F. Hoffmann-La Roche AG | Type ii anti-cd20 antibody for reducing formation of anti-drug antibodies |
CA2997406A1 (en) | 2015-12-09 | 2017-06-15 | F. Hoffmann-La Roche Ag | Type ii anti-cd20 antibody for reducing formation of anti-drug antibodies or cytokine release |
US10829562B2 (en) | 2015-12-10 | 2020-11-10 | Katholieke Universiteit Leuven | Haemorrhagic disorder due to ventricular assist device |
JP2019502695A (en) | 2015-12-17 | 2019-01-31 | ノバルティス アーゲー | Combination of antibody molecule against PD-1 and C-Met inhibitor and use thereof |
MX2018007423A (en) | 2015-12-17 | 2018-11-09 | Novartis Ag | Antibody molecules to pd-1 and uses thereof. |
EP4342529A2 (en) | 2015-12-18 | 2024-03-27 | Chugai Seiyaku Kabushiki Kaisha | Anti-c5 antibodies and methods of use |
EP3405190A4 (en) | 2015-12-23 | 2019-10-30 | Moonshot Pharma LLC | Methods for inducing an immune response |
BR112018013071A2 (en) | 2015-12-30 | 2018-12-11 | Genentech Inc | use of tryptophan derivatives for protein formulations |
JP7008023B2 (en) | 2015-12-30 | 2022-01-25 | ジェネンテック, インコーポレイテッド | Formulation with reduced polysorbate degradation |
US20200270365A1 (en) | 2016-01-05 | 2020-08-27 | Jiangsu Hengrui Medicine Co., Ltd. | Pcsk9 antibody, antigen-binding fragment thereof, and medical uses thereof |
US10472422B2 (en) | 2016-01-08 | 2019-11-12 | Abgenomics International Inc. | Tetravalent anti-PSGL-1 antibodies and uses thereof |
CN115814077A (en) | 2016-01-08 | 2023-03-21 | 供石公司 | Anti-pro/latent myostatin antibodies and methods of use thereof |
BR112018011029A2 (en) | 2016-01-08 | 2018-11-21 | Hoffmann La Roche | methods for treating or delaying cancer progression and improving immune function in a cancer individual, uses of a binding antagonist and a bispecific antibody, compositions and kits |
US20190016791A1 (en) | 2016-01-20 | 2019-01-17 | Genentech, Inc. | High dose treatments for alzheimer's disease |
EA201891641A1 (en) | 2016-01-21 | 2019-01-31 | Пфайзер Инк. | CHEMICAL ANTIGENOUS RECEPTORS AIMED AT OPTION III RECEPTOR EPIDERMAL GROWTH FACTOR |
US10221242B2 (en) | 2016-01-21 | 2019-03-05 | Pfizer Inc. | Antibodies specific for epidermal growth factor receptor variant III and their uses |
CN109071625A (en) | 2016-02-04 | 2018-12-21 | 柯瑞斯公司 | Smooth mutant and its application method |
GB201602413D0 (en) | 2016-02-10 | 2016-03-23 | Nascient Ltd | Method |
BR112018016461A2 (en) | 2016-02-12 | 2019-10-01 | Janssen Pharmaceutica Nv | antibodies and anti-sight fragments, their uses and their identification methods |
KR20180119632A (en) | 2016-02-29 | 2018-11-02 | 제넨테크, 인크. | Treatment and Diagnosis Methods for Cancer |
CN109476731A (en) | 2016-02-29 | 2019-03-15 | 基础医药有限公司 | The method for the treatment of cancer |
US10336784B2 (en) | 2016-03-08 | 2019-07-02 | Academia Sinica | Methods for modular synthesis of N-glycans and arrays thereof |
US11072652B2 (en) | 2016-03-10 | 2021-07-27 | Viela Bio, Inc. | ILT7 binding molecules and methods of using the same |
CN116284392A (en) | 2016-03-10 | 2023-06-23 | 艾科赛扬制药股份有限公司 | Activin type 2 receptor binding proteins and uses thereof |
AU2017230103A1 (en) | 2016-03-11 | 2018-09-06 | Scholar Rock, Inc. | TGFβ1-binding immunoglobulins and use thereof |
WO2017160599A1 (en) | 2016-03-14 | 2017-09-21 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Use of cd300b antagonists to treat sepsis and septic shock |
PL3430054T3 (en) | 2016-03-15 | 2022-05-23 | Chugai Seiyaku Kabushiki Kaisha | Methods of treating cancers using pd-1 axis binding antagonists and anti-gpc3 antibodies |
US11186641B2 (en) | 2016-03-17 | 2021-11-30 | Oslo Universitetssykehus Hf | Fusion proteins targeting tumour associated macrophages for treating cancer |
US11291721B2 (en) | 2016-03-21 | 2022-04-05 | Marengo Therapeutics, Inc. | Multispecific and multifunctional molecules and uses thereof |
CN108697799A (en) | 2016-03-22 | 2018-10-23 | 生态学有限公司 | The application of anti-LGR5 monoclonal antibodies |
JP6943872B2 (en) | 2016-03-25 | 2021-10-06 | ジェネンテック, インコーポレイテッド | Multiple whole antibody and antibody complex drug quantification assay |
AU2017238651A1 (en) | 2016-03-25 | 2018-10-04 | Visterra, Inc. | Formulation of antibody molecules to dengue virus |
US10980894B2 (en) | 2016-03-29 | 2021-04-20 | Obi Pharma, Inc. | Antibodies, pharmaceutical compositions and methods |
CA3019164A1 (en) | 2016-03-29 | 2017-10-05 | Janssen Biotech, Inc. | Method of treating psoriasis with increased interval dosing of anti-il12/23 antibody |
TWI780045B (en) | 2016-03-29 | 2022-10-11 | 台灣浩鼎生技股份有限公司 | Antibodies, pharmaceutical compositions and methods |
WO2017175058A1 (en) | 2016-04-07 | 2017-10-12 | Janssen Pharmaceutica Nv | Anti-vista antibodies and fragments, uses thereof, and methods of identifying same |
WO2017177199A2 (en) | 2016-04-08 | 2017-10-12 | Iti Health, Inc. | Plectin-1 binding antibodies and uses thereof |
US20170319688A1 (en) | 2016-04-14 | 2017-11-09 | Genentech, Inc. | Anti-rspo3 antibodies and methods of use |
US20170298119A1 (en) | 2016-04-15 | 2017-10-19 | Visterra, Inc. | Antibody molecules to zika virus and uses thereof |
JP2019515670A (en) | 2016-04-15 | 2019-06-13 | ジェネンテック, インコーポレイテッド | Methods for monitoring and treating cancer |
CA3019921A1 (en) | 2016-04-15 | 2017-10-19 | Genentech, Inc. | Methods for monitoring and treating cancer |
KR20180128496A (en) | 2016-04-22 | 2018-12-03 | 오비아이 파머 인코퍼레이티드 | Cancer immunotherapy by immune activation or immunomodulation through glycosylated antigen |
WO2017189730A1 (en) | 2016-04-26 | 2017-11-02 | Icahn School Of Medicine At Mount Sinai | Treatment of hippo pathway mutant tumors and methods of identifying subjects as candidates for treatment |
PE20181890A1 (en) | 2016-05-02 | 2018-12-11 | Hoffmann La Roche | CONTORSBODY - A MONOCATENARIO DIANA LEAGUE |
CN109071640B (en) | 2016-05-11 | 2022-10-18 | 豪夫迈·罗氏有限公司 | Modified anti-tenascin antibodies and methods of use |
JP7084878B2 (en) | 2016-05-16 | 2022-06-15 | 武田薬品工業株式会社 | Anti-factor IX Padua antibody |
EP3458101B1 (en) | 2016-05-20 | 2020-12-30 | H. Hoffnabb-La Roche Ag | Protac antibody conjugates and methods of use |
US11649291B2 (en) | 2016-05-24 | 2023-05-16 | Insmed Incorporated | Antibodies and methods of making same |
KR102543118B1 (en) | 2016-05-27 | 2023-06-14 | 아게누스 인코포레이티드 | Anti-tim-3 antibodies and methods of use thereof |
JP7022080B2 (en) | 2016-05-27 | 2022-02-17 | ジェネンテック, インコーポレイテッド | Biochemical analytical methods for the characterization of site-specific antibody-drug conjugates |
BR112019022558A2 (en) | 2016-06-02 | 2020-05-19 | Hoffmann La Roche | antibodies, methods to treat or slow the progression of a proliferative disease and to treat or slow the progression of cancer in an individual, pharmaceutical compositions, kit, uses of a combination of an anti-cd20 antibody and an antibody and invention |
EP3252078A1 (en) | 2016-06-02 | 2017-12-06 | F. Hoffmann-La Roche AG | Type ii anti-cd20 antibody and anti-cd20/cd3 bispecific antibody for treatment of cancer |
SG11201810678WA (en) | 2016-06-02 | 2018-12-28 | Abbvie Inc | Glucocorticoid receptor agonist and immunoconjugates thereof |
JP6921943B2 (en) | 2016-06-06 | 2021-08-18 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | Ophthalmic fusion protein with increased eye retention |
CN109476648B (en) | 2016-06-06 | 2022-09-13 | 豪夫迈·罗氏有限公司 | Sevelamer antibody-drug conjugates and methods of use |
WO2017223405A1 (en) | 2016-06-24 | 2017-12-28 | Genentech, Inc. | Anti-polyubiquitin multispecific antibodies |
EP3475446A1 (en) | 2016-06-27 | 2019-05-01 | Juno Therapeutics, Inc. | Method of identifying peptide epitopes, molecules that bind such epitopes and related uses |
MA45491A (en) | 2016-06-27 | 2019-05-01 | Juno Therapeutics Inc | CMH-E RESTRICTED EPITOPES, BINDING MOLECULES AND RELATED METHODS AND USES |
CN109415435B (en) | 2016-07-04 | 2024-01-16 | 豪夫迈·罗氏有限公司 | Novel antibody forms |
US11292801B2 (en) | 2016-07-05 | 2022-04-05 | Blade Therapeutics, Inc. | Calpain modulators and therapeutic uses thereof |
CA3030099A1 (en) | 2016-07-08 | 2018-01-11 | Staten Biotechnology B.V. | Anti-apoc3 antibodies and methods of use thereof |
CN109862911A (en) | 2016-07-13 | 2019-06-07 | 比奥根Ma公司 | The dosage of LINGO-1 antagonist and purposes for treating demyelinating disorder |
JP7219376B2 (en) | 2016-07-15 | 2023-02-08 | ノバルティス アーゲー | Treatment and prevention of cytokine release syndrome using chimeric antigen receptors in combination with kinase inhibitors |
MX2019000643A (en) | 2016-07-15 | 2019-06-13 | Poseida Therapeutics Inc | Chimeric antigen receptors and methods for use. |
EP3496739B1 (en) | 2016-07-15 | 2021-04-28 | Acceleron Pharma Inc. | Compositions comprising actriia polypeptides for use in treating pulmonary hypertension |
WO2018014039A1 (en) | 2016-07-15 | 2018-01-18 | Poseida Therapeutics, Inc. | Chimeric antigen receptors (cars) specific for muc1 and methods for their use |
WO2018014260A1 (en) | 2016-07-20 | 2018-01-25 | Nanjing Legend Biotech Co., Ltd. | Multispecific antigen binding proteins and methods of use thereof |
EP3487880A1 (en) | 2016-07-25 | 2019-05-29 | Biogen MA Inc. | Anti-hspa5 (grp78) antibodies and uses thereof |
AU2017302038B2 (en) | 2016-07-27 | 2024-03-21 | Obi Pharma, Inc. | Immunogenic/therapeutic glycan compositions and uses thereof |
US20180050085A1 (en) | 2016-07-27 | 2018-02-22 | Acceleron Pharma Inc. | Methods and compositions for treating myelofibrosis |
MX2018015721A (en) | 2016-07-29 | 2019-05-27 | Chugai Pharmaceutical Co Ltd | Bispecific antibody exhibiting increased alternative fviii-cofactor-function activity. |
TWI786054B (en) | 2016-07-29 | 2022-12-11 | 台灣浩鼎生技股份有限公司 | Human antibodies, pharmaceutical compositions and methods |
WO2018026748A1 (en) | 2016-08-01 | 2018-02-08 | Xoma (Us) Llc | Parathyroid hormone receptor 1 (pth1r) antibodies and uses thereof |
CN110072887A (en) | 2016-08-02 | 2019-07-30 | 威特拉公司 | Engineered polypeptide and its application |
EP3494141A4 (en) | 2016-08-03 | 2020-04-08 | Bio-Techne Corporation | Identification of vsig3/vista as a novel immune checkpoint and use thereof for immunotherapy |
EP3494139B1 (en) | 2016-08-05 | 2022-01-12 | F. Hoffmann-La Roche AG | Multivalent and multiepitopic anitibodies having agonistic activity and methods of use |
WO2018025982A1 (en) | 2016-08-05 | 2018-02-08 | 中外製薬株式会社 | Composition for prophylaxis or treatment of il-8 related diseases |
CN109689090B (en) | 2016-08-05 | 2023-12-26 | 免疫医疗有限责任公司 | anti-O2 antibodies and uses thereof |
WO2018029124A1 (en) | 2016-08-08 | 2018-02-15 | F. Hoffmann-La Roche Ag | Therapeutic and diagnostic methods for cancer |
JP7093767B2 (en) | 2016-08-11 | 2022-06-30 | ジェネンテック, インコーポレイテッド | Pyrrolobenzodiazepine prodrug and its antibody conjugate |
KR102588027B1 (en) | 2016-08-22 | 2023-10-12 | 초 파마 인크. | Antibodies, binding fragments and methods of use |
SG10201607778XA (en) | 2016-09-16 | 2018-04-27 | Chugai Pharmaceutical Co Ltd | Anti-Dengue Virus Antibodies, Polypeptides Containing Variant Fc Regions, And Methods Of Use |
CN116731197A (en) | 2016-09-19 | 2023-09-12 | 豪夫迈·罗氏有限公司 | Complement factor based affinity chromatography |
EP3515943A4 (en) | 2016-09-19 | 2020-05-06 | Celgene Corporation | Methods of treating vitiligo using pd-1 binding proteins |
JP2019531284A (en) | 2016-09-19 | 2019-10-31 | セルジーン コーポレイション | Methods of treating immune disorders using PD-1 binding proteins |
PE20191148A1 (en) | 2016-09-23 | 2019-09-02 | Teva Pharmaceuticals Int Gmbh | TREATMENT OF REFRACTORY MIGRANA |
CN109906232B (en) | 2016-09-23 | 2023-11-07 | 马伦戈治疗公司 | Multispecific antibody molecules comprising lambda light chain and kappa light chain |
TWI787203B (en) | 2016-09-23 | 2022-12-21 | 美商建南德克公司 | Uses of il-13 antagonists for treating atopic dermatitis |
EP3515488A1 (en) | 2016-09-23 | 2019-07-31 | Teva Pharmaceuticals International GmbH | Treating cluster headache |
WO2018064119A1 (en) | 2016-09-28 | 2018-04-05 | Blade Therapeutics, Inc. | Calpain modulators and therapeutic uses thereof |
BR112019005944A2 (en) | 2016-09-28 | 2019-06-11 | Musc Foudation For Res Development | antibodies that bind to interleukin 2 and their uses |
GB201616596D0 (en) | 2016-09-29 | 2016-11-16 | Nascient Limited | Epitope and antibodies |
CA3037961A1 (en) | 2016-09-30 | 2018-04-05 | Janssen Biotech, Inc. | Safe and effective method of treating psoriasis with anti-il23 specific antibody |
JP2019530875A (en) | 2016-10-03 | 2019-10-24 | アボット・ラボラトリーズAbbott Laboratories | Improved method for assessing UCH-L1 status in patient samples |
JP7050770B2 (en) | 2016-10-05 | 2022-04-08 | エフ・ホフマン-ラ・ロシュ・アクチェンゲゼルシャフト | Method for preparing antibody drug conjugate |
AU2017340504A1 (en) | 2016-10-05 | 2019-04-11 | Acceleron Pharma, Inc. | Compositions and method for treating kidney disease |
CA3038712A1 (en) | 2016-10-06 | 2018-04-12 | Genentech, Inc. | Therapeutic and diagnostic methods for cancer |
MX2019003886A (en) | 2016-10-07 | 2019-08-05 | Novartis Ag | Chimeric antigen receptors for the treatment of cancer. |
WO2018068201A1 (en) | 2016-10-11 | 2018-04-19 | Nanjing Legend Biotech Co., Ltd. | Single-domain antibodies and variants thereof against ctla-4 |
CA3037380A1 (en) | 2016-10-11 | 2018-04-19 | Agenus Inc. | Anti-lag-3 antibodies and methods of use thereof |
WO2018071576A1 (en) | 2016-10-14 | 2018-04-19 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Treatment of tumors by inhibition of cd300f |
WO2018075408A1 (en) | 2016-10-17 | 2018-04-26 | Alexion Pharmaceuticals, Inc. | Methods of treating acute myeloid leukemia (aml) with combinations of anti-cd200 antibodies, cytarabine, and daunorubicin |
SG11201903063UA (en) | 2016-10-19 | 2019-05-30 | Medimmune Llc | Anti-o1 antibodies and uses thereof |
JP2019535250A (en) | 2016-10-29 | 2019-12-12 | ジェネンテック, インコーポレイテッド | Anti-MIC antibody and method of use |
US11249082B2 (en) | 2016-10-29 | 2022-02-15 | University Of Miami | Zika virus assay systems |
WO2018085359A1 (en) | 2016-11-02 | 2018-05-11 | Immunogen, Inc. | Combination treatment with antibody-drug conjugates and parp inhibitors |
TW202321301A (en) | 2016-11-02 | 2023-06-01 | 美商永斯醫療股份有限公司 | Antibodies to pd-1 and uses thereof |
US11779604B2 (en) | 2016-11-03 | 2023-10-10 | Kymab Limited | Antibodies, combinations comprising antibodies, biomarkers, uses and methods |
AU2017353939A1 (en) | 2016-11-07 | 2019-06-06 | Neuracle Science Co., Ltd. | Anti-family with sequence similarity 19, member A5 antibodies and method of use thereof |
MX2019005661A (en) | 2016-11-16 | 2019-10-07 | Janssen Biotech Inc | Method of treating psoriasis with anti-il-23 specific antibody. |
EP3541847A4 (en) | 2016-11-17 | 2020-07-08 | Seattle Genetics, Inc. | Glycan-interacting compounds and methods of use |
TW201829463A (en) | 2016-11-18 | 2018-08-16 | 瑞士商赫孚孟拉羅股份公司 | Anti-hla-g antibodies and use thereof |
CN110290800A (en) | 2016-11-21 | 2019-09-27 | 台湾浩鼎生技股份有限公司 | Conjugating biomolecules, medical composition and method |
AU2017363309A1 (en) | 2016-11-23 | 2019-07-11 | Bioverativ Therapeutics Inc. | Mono- and bispecific antibodies binding to coagulation factor IX and coagulation factor X |
CN110234319B (en) | 2016-11-23 | 2022-09-27 | 转化药物开发有限责任公司 | Compositions of benzamide and active compound and methods of use thereof |
WO2018102594A1 (en) | 2016-12-01 | 2018-06-07 | Alexion Pharmaceuticals, Inc. | Methods of treating solid tumors with anti-cd200 antibodies |
ES2963226T3 (en) | 2016-12-07 | 2024-03-26 | Agenus Inc | ANTI-CTLA-4 antibodies and methods of their use |
TWI789371B (en) | 2016-12-07 | 2023-01-11 | 美商建南德克公司 | Anti-tau antibodies and methods of use |
PE20190921A1 (en) | 2016-12-07 | 2019-06-26 | Agenus Inc | ANTIBODIES AND METHODS OF THEIR USE |
CN110290801B (en) | 2016-12-07 | 2024-01-26 | 基因泰克公司 | anti-TAU antibodies and methods of use |
GB201621635D0 (en) | 2016-12-19 | 2017-02-01 | Ucb Biopharma Sprl | Crystal structure |
BR112019010294A2 (en) | 2016-12-21 | 2019-09-03 | Hoffmann La Roche | method for the enzymatic production of an antibody, antibody and pharmaceutical formulation |
JP6850351B2 (en) | 2016-12-21 | 2021-03-31 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | In vitro sugar chain engineering of antibodies |
AU2017381656B2 (en) | 2016-12-21 | 2020-07-02 | F. Hoffmann-La Roche Ag | Re-use of enzymes in in vitro glycoengineering of antibodies |
WO2018119402A1 (en) | 2016-12-23 | 2018-06-28 | Visterra, Inc. | Binding polypeptides and methods of making the same |
ES2944357T3 (en) | 2017-01-06 | 2023-06-20 | Scholar Rock Inc | Treatment of metabolic diseases by inhibiting myostatin activation |
CR20190350A (en) | 2017-01-06 | 2019-11-15 | Scholar Rock Inc | ISOFORM-SPECIFIC, CONTEXT-PERMISSIVE TGFß1 INHIBITORS AND USE THEREOF |
WO2018129395A1 (en) | 2017-01-06 | 2018-07-12 | Scholar Rock, Inc. | Methods for treating metabolic diseases by inhibiting myostatin activation |
US11274157B2 (en) | 2017-01-12 | 2022-03-15 | Eureka Therapeutics, Inc. | Constructs targeting histone H3 peptide/MHC complexes and uses thereof |
WO2018130660A1 (en) | 2017-01-13 | 2018-07-19 | Academia Sinica | Reloadable hydrogel system for treating myocardial infarction |
TWI659751B (en) | 2017-01-13 | 2019-05-21 | 中央研究院 | Improved reloadable hydrogel system for treating brain conditions |
CN110431150A (en) | 2017-01-18 | 2019-11-08 | 威特拉公司 | Antibody molecule-drug conjugates and application thereof |
MX2019008348A (en) | 2017-01-18 | 2019-10-21 | Genentech Inc | Idiotypic antibodies against anti-pd-l1 antibodies and uses thereof. |
CN110234351A (en) | 2017-01-30 | 2019-09-13 | 詹森生物科技公司 | For treating the anti-TNF antibodies, composition and method of activity psoriatic arthritis |
US10240205B2 (en) | 2017-02-03 | 2019-03-26 | Population Bio, Inc. | Methods for assessing risk of developing a viral disease using a genetic test |
MX2019009359A (en) | 2017-02-06 | 2020-01-30 | Australian Meat & Live Stock | Immunostimulating compositions and uses therefore. |
MX2019009377A (en) | 2017-02-07 | 2019-12-11 | Janssen Biotech Inc | Anti-tnf antibodies, compositions, and methods for the treatment of active ankylosing spondylitis. |
AU2018219283B2 (en) | 2017-02-08 | 2022-05-19 | Bristol-Myers Squibb Company | Modified relaxin polypeptides comprising a pharmacokinetic enhancer and uses thereof |
US11021535B2 (en) | 2017-02-10 | 2021-06-01 | The United States Of America As Represented By The Secretary, Department Of Health And Human Services | Neutralizing antibodies to plasmodium falciparum circumsporozoite protein and their use |
CN110494453B (en) | 2017-02-10 | 2023-05-26 | 豪夫迈·罗氏有限公司 | Anti-tryptase antibodies, compositions thereof and uses thereof |
US20200291089A1 (en) | 2017-02-16 | 2020-09-17 | Elstar Therapeutics, Inc. | Multifunctional molecules comprising a trimeric ligand and uses thereof |
WO2018152496A1 (en) | 2017-02-17 | 2018-08-23 | The Usa, As Represented By The Secretary, Dept. Of Health And Human Services | Compositions and methods for the diagnosis and treatment of zika virus infection |
MX2019010219A (en) | 2017-02-27 | 2020-02-07 | Regeneron Pharma | Humanized model of kidney and liver disorders. |
MX2019010295A (en) | 2017-03-01 | 2019-11-21 | Genentech Inc | Diagnostic and therapeutic methods for cancer. |
WO2018158719A1 (en) | 2017-03-02 | 2018-09-07 | Novartis Ag | Engineered heterodimeric proteins |
MX2019010397A (en) | 2017-03-02 | 2020-08-20 | Beth Israel Deaconess Medical Ct Inc | Selecting headache patients responsive to antibodies directed against calcitonin gene related peptide. |
MA47812A (en) | 2017-03-03 | 2021-04-14 | Seagen Inc | COMPOUNDS INTERACTING WITH GLYCAN AND METHODS OF USE |
US11744861B2 (en) | 2017-03-13 | 2023-09-05 | Poseida Therapeutics, Inc. | Compositions and methods for selective elimination and replacement of hematopoietic stem cells |
AR111249A1 (en) | 2017-03-22 | 2019-06-19 | Genentech Inc | OPTIMIZED ANTIBODY COMPOSITIONS FOR THE TREATMENT OF OCULAR DISORDERS |
CN110891611B (en) | 2017-03-22 | 2024-03-29 | 阿森迪斯制药公司 | Hydrogel crosslinked hyaluronic acid prodrug compositions and methods |
JP7346300B2 (en) | 2017-03-23 | 2023-09-19 | アボット・ラボラトリーズ | Methods for aiding in the diagnosis and determination of the extent of traumatic brain injury in human subjects using the early biomarker ubiquitin carboxy-terminal hydrolase L1 |
EP3599843A4 (en) | 2017-03-24 | 2021-01-13 | The Regents of the University of California | Proteoglycan irregularities in abnormal fibroblasts and therapies based therefrom |
MA49355A (en) | 2017-03-27 | 2020-02-05 | Hoffmann La Roche | Improved antigen binding receptor formats |
TW201900673A (en) | 2017-03-27 | 2019-01-01 | 瑞士商赫孚孟拉羅股份公司 | Modified antigen binding receptor |
BR112019018767A2 (en) | 2017-04-03 | 2020-05-05 | Hoffmann La Roche | antibodies, bispecific antigen binding molecule, one or more isolated polynucleotides, one or more vectors, host cell, method for producing an antibody, pharmaceutical composition, uses, method for treating a disease in an individual and invention |
CA3055769A1 (en) | 2017-04-03 | 2018-10-11 | Oncologie, Inc. | Methods for treating cancer using ps-targeting antibodies with immuno-oncology agents |
WO2018184965A1 (en) | 2017-04-03 | 2018-10-11 | F. Hoffmann-La Roche Ag | Immunoconjugates of il-2 with an anti-pd-1 and tim-3 bispecific antibody |
RU2761377C2 (en) | 2017-04-03 | 2021-12-07 | Ф. Хоффманн-Ля Рош Аг | Immunoconjugates of antibody to pd-1 with il-2 or il-15 mutant |
EP3606560A2 (en) | 2017-04-05 | 2020-02-12 | Novo Nordisk A/S | Oligomer extended insulin-fc conjugates |
HUE060019T2 (en) | 2017-04-05 | 2023-01-28 | Hoffmann La Roche | Anti-lag3 antibodies |
AU2018247794A1 (en) | 2017-04-05 | 2019-08-22 | F. Hoffmann-La Roche Ag | Bispecific antibodies specifically binding to PD1 and LAG3 |
CA3059542A1 (en) | 2017-04-12 | 2018-10-18 | Pfizer Inc. | Antibodies having conditional affinity and methods of use thereof |
MA50956A (en) | 2017-04-13 | 2020-10-14 | Agenus Inc | ANTI-CD137 ANTIBODIES AND RELATED METHODS OF USE |
MX2019012136A (en) | 2017-04-14 | 2020-07-20 | Gamamabs Pharma | Amhrii-binding compounds for preventing or treating lung cancers. |
KR20200014276A (en) | 2017-04-14 | 2020-02-10 | 가마맵스 파마 | AMHRII-binding compounds for preventing or treating cancer |
US10877048B2 (en) | 2017-04-15 | 2020-12-29 | Abbott Laboratories | Methods for aiding in the hyperacute diagnosis and determination of traumatic brain injury in a human subject using early biomarkers |
WO2018195283A1 (en) | 2017-04-19 | 2018-10-25 | Elstar Therapeutics, Inc. | Multispecific molecules and uses thereof |
SG11201909048TA (en) | 2017-04-21 | 2019-11-28 | Genentech Inc | Use of klk5 antagonists for treatment of a disease |
WO2018193427A1 (en) | 2017-04-21 | 2018-10-25 | Staten Biotechnology B.V. | Anti-apoc3 antibodies and methods of use thereof |
GB201706451D0 (en) | 2017-04-24 | 2017-06-07 | Imp Innovations Ltd | Cancer treatment |
EP3615569A1 (en) | 2017-04-25 | 2020-03-04 | The U.S.A. As Represented By The Secretary, Department Of Health And Human Services | Antibodies and methods for the diagnosis and treatment of epstein barr virus infection |
SG10201913655UA (en) | 2017-04-26 | 2020-03-30 | Eureka Therapeutics Inc | Cells expressing chimeric activating receptors and chimeric stimulating receptors and uses thereof |
CA3059820A1 (en) | 2017-04-26 | 2018-11-01 | Eureka Therapeutics, Inc. | Constructs specifically recognizing glypican 3 and uses thereof |
BR112019022515A2 (en) | 2017-04-27 | 2020-06-16 | Tesaro, Inc. | ANTIBODY AGENTS TARGETED AGAINST Lymphocyte Activation Gene 3 (LAG-3) AND USES OF THE SAME |
WO2018201047A1 (en) | 2017-04-28 | 2018-11-01 | Elstar Therapeutics, Inc. | Multispecific molecules comprising a non-immunoglobulin heterodimerization domain and uses thereof |
BR112019022476A2 (en) | 2017-04-28 | 2020-05-12 | Abbott Laboratories | METHODS FOR HYPERAGUDE DIAGNOSTIC AID AND DETERMINATION OF TRAUMATIC BRAIN INJURY USING INITIAL BIOMARKERS IN AT LEAST TWO SAMPLES FROM THE SAME HUMAN |
ES2952961T3 (en) | 2017-05-01 | 2023-11-07 | Agenus Inc | Anti-TIGIT antibodies and methods of their use |
US10865238B1 (en) | 2017-05-05 | 2020-12-15 | Duke University | Complement factor H antibodies |
JOP20190256A1 (en) | 2017-05-12 | 2019-10-28 | Icahn School Med Mount Sinai | Newcastle disease viruses and uses thereof |
US11168129B2 (en) | 2017-05-15 | 2021-11-09 | University Of Rochester | Broadly neutralizing anti-influenza human monoclonal antibody and uses thereof |
WO2018215535A1 (en) | 2017-05-23 | 2018-11-29 | Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) | Novel cd73 antibody, preparation and uses thereof |
AU2018272054A1 (en) | 2017-05-25 | 2019-09-26 | Abbott Laboratories | Methods for aiding in the determination of whether to perform imaging on a human subject who has sustained or may have sustained an injury to the head using early biomarkers |
AU2018275236A1 (en) | 2017-05-30 | 2019-10-31 | Abbott Laboratories | Methods for aiding in diagnosing and evaluating a mild traumatic brain injury in a human subject using cardiac troponin I |
WO2018222901A1 (en) | 2017-05-31 | 2018-12-06 | Elstar Therapeutics, Inc. | Multispecific molecules that bind to myeloproliferative leukemia (mpl) protein and uses thereof |
KR20200024158A (en) | 2017-05-31 | 2020-03-06 | 주식회사 에스티큐브앤컴퍼니 | How to treat cancer using antibodies and molecules that immunospecifically bind to BTN1A1 |
CA3065301A1 (en) | 2017-05-31 | 2018-12-06 | Stcube & Co., Inc. | Antibodies and molecules that immunospecifically bind to btn1a1 and the therapeutic uses thereof |
AU2018275359C1 (en) | 2017-06-02 | 2022-02-03 | Pfizer Inc. | Antibodies specific for FLT3 and their uses |
CA3062328A1 (en) | 2017-06-02 | 2018-12-06 | Pfizer Inc. | Chimeric antigen receptors targeting flt3 |
JP2020522562A (en) | 2017-06-06 | 2020-07-30 | ストキューブ アンド シーオー., インコーポレイテッド | Methods of treating cancer with antibodies and molecules that bind to BTN1A1 or BTN1A1 ligand |
WO2018226336A1 (en) | 2017-06-09 | 2018-12-13 | Providence Health & Services - Oregon | Utilization of cd39 and cd103 for identification of human tumor reactive cells for treatment of cancer |
GB201709379D0 (en) | 2017-06-13 | 2017-07-26 | Univ Leuven Kath | Humanised ADAMTS13 binding antibodies |
US20190062428A1 (en) | 2017-06-19 | 2019-02-28 | Surface Oncology, Inc. | Combination of anti-cd47 antibodies and cell death-inducing agents, and uses thereof |
WO2018237326A1 (en) | 2017-06-22 | 2018-12-27 | 1AlMOONSHOT PHARMA LLC | Methods for treating cancer with compositions comprising amlexanox and immune modulators |
US20200172628A1 (en) | 2017-06-22 | 2020-06-04 | Novartis Ag | Antibody molecules to cd73 and uses thereof |
EP3645042A4 (en) | 2017-06-26 | 2021-03-17 | Bio-Techne Corporation | Hybridoma clones, monoclonal antibodies to vsig-4, and methods of making and using |
US20200223924A1 (en) | 2017-06-27 | 2020-07-16 | Novartis Ag | Dosage regimens for anti-tim-3 antibodies and uses thereof |
JP7454945B2 (en) | 2017-07-03 | 2024-03-25 | アボット・ラボラトリーズ | Improved method for measuring ubiquitin carboxy-terminal hydrolase L1 levels in blood |
WO2019010164A1 (en) | 2017-07-06 | 2019-01-10 | President And Fellows Of Harvard College | Evolution of trna synthetases |
AU2018301393A1 (en) | 2017-07-11 | 2020-02-06 | Compass Therapeutics Llc | Agonist antibodies that bind human CD137 and uses thereof |
CU20200002A7 (en) | 2017-07-14 | 2020-11-30 | Pfizer | ANTIBODIES AGAINST MADCAM |
WO2019018629A1 (en) | 2017-07-19 | 2019-01-24 | The Usa, As Represented By The Secretary, Dept. Of Health And Human Services | Antibodies and methods for the diagnosis and treatment of hepatitis b virus infection |
EP3431496A1 (en) | 2017-07-19 | 2019-01-23 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Anti- isoasp7 amyloid beta antibodies and uses thereof |
WO2019018647A1 (en) | 2017-07-20 | 2019-01-24 | Pfizer Inc. | Anti-gd3 antibodies and antibody-drug conjugates |
CA3070095A1 (en) | 2017-07-20 | 2019-01-24 | Novartis Ag | Dosage regimens of anti-lag-3 antibodies and uses thereof |
AU2018304458B2 (en) | 2017-07-21 | 2021-12-09 | Foundation Medicine, Inc. | Therapeutic and diagnostic methods for cancer |
EP3658583A1 (en) | 2017-07-28 | 2020-06-03 | Scholar Rock, Inc. | Ltbp complex-specific inhibitors of tgf-beta 1 and uses thereof |
HRP20220404T1 (en) | 2017-08-03 | 2022-05-27 | Amgen Inc. | Interleukin-21 muteins and methods of treatment |
AU2018310985A1 (en) | 2017-08-03 | 2019-11-07 | Alector Llc | Anti-CD33 antibodies and methods of use thereof |
WO2019035938A1 (en) | 2017-08-16 | 2019-02-21 | Elstar Therapeutics, Inc. | Multispecific molecules that bind to bcma and uses thereof |
US11485781B2 (en) | 2017-08-17 | 2022-11-01 | Massachusetts Institute Of Technology | Multiple specificity binders of CXC chemokines |
MX2020002076A (en) | 2017-08-25 | 2020-03-24 | Five Prime Therapeutics Inc | B7-h4 antibodies and methods of use thereof. |
EP3679145A2 (en) | 2017-09-08 | 2020-07-15 | Poseida Therapeutics, Inc. | Compositions and methods for chimeric ligand receptor (clr)-mediated conditional gene expression |
CN116003405A (en) | 2017-09-08 | 2023-04-25 | 美国安进公司 | Inhibitors of KRAS G12C and methods of use thereof |
WO2019056002A1 (en) | 2017-09-18 | 2019-03-21 | President And Fellows Of Harvard College | Continuous evolution for stabilized proteins |
TW201922780A (en) | 2017-09-25 | 2019-06-16 | 美商健生生物科技公司 | Safe and effective method of treating Lupus with anti-IL12/IL23 antibody |
WO2019067499A1 (en) | 2017-09-27 | 2019-04-04 | Alexion Pharmaceuticals, Inc. | Biomarker signature for predicting tumor response to anti-cd200 therapy |
KR20200049764A (en) | 2017-09-29 | 2020-05-08 | 추가이 세이야쿠 가부시키가이샤 | Multispecific antigen-binding molecules having blood coagulation factor viii (fviii) cofactor function-substituting activity and pharmaceutical formulations containing such a molecule as an active ingredient |
KR20200058540A (en) | 2017-10-02 | 2020-05-27 | 비스테라, 인크. | Antibody molecules against CD138 and uses thereof |
ES2759622T3 (en) | 2017-10-02 | 2020-05-11 | Certest Biotec S L | Anti-Dps antibodies and test devices for the detection of bacteria of the genus Campylobacter |
EP3692370A2 (en) | 2017-10-04 | 2020-08-12 | OPKO Pharmaceuticals, LLC | Articles and methods directed to personalized therapy of cancer |
US11707522B2 (en) | 2017-10-13 | 2023-07-25 | Boehringer Ingelheim International Gmbh | Human antibodies to Tn antigen |
EP3697816A1 (en) | 2017-10-19 | 2020-08-26 | Debiopharm International S.A. | Combination product for the treatment of cancer |
EP3697809A1 (en) | 2017-10-20 | 2020-08-26 | Institut Curie | Dap10/12 based cars adapted for rush |
US11718679B2 (en) | 2017-10-31 | 2023-08-08 | Compass Therapeutics Llc | CD137 antibodies and PD-1 antagonists and uses thereof |
WO2019089594A1 (en) | 2017-10-31 | 2019-05-09 | Immunogen, Inc. | Combination treatment with antibody-drug conjugates and cytarabine |
PE20211266A1 (en) | 2017-10-31 | 2021-07-19 | Allogene Therapeutics Inc | METHODS AND COMPOSITIONS FOR THE DOSAGE OF T-CELLS WITH ALOGENIC CHIMERIC ANTIGEN RECEPTOR |
JP7039694B2 (en) | 2017-10-31 | 2022-03-22 | スターテン・バイオテクノロジー・ベー・フェー | Anti-APOC3 antibody and how to use it |
EP3704146B1 (en) | 2017-11-01 | 2021-12-15 | F. Hoffmann-La Roche AG | Trifab-contorsbody |
WO2019086394A1 (en) | 2017-11-01 | 2019-05-09 | F. Hoffmann-La Roche Ag | The compbody - a multivalent target binder |
KR20200075860A (en) | 2017-11-06 | 2020-06-26 | 제넨테크, 인크. | How to diagnose and treat cancer |
AU2018364630A1 (en) | 2017-11-09 | 2020-05-21 | Pinteon Therapeutics Inc. | Methods and compositions for the generation and use of humanized conformation-specific phosphorylated tau antibodies |
CA3081602A1 (en) | 2017-11-16 | 2019-05-23 | Novartis Ag | Combination therapies |
WO2019100052A2 (en) | 2017-11-20 | 2019-05-23 | Compass Therapeutics Llc | Cd137 antibodies and tumor antigen-targeting antibodies and uses thereof |
US11401345B2 (en) | 2017-11-27 | 2022-08-02 | Purdue Pharma L.P. | Humanized antibodies targeting human tissue factor |
EP3717517A1 (en) | 2017-11-30 | 2020-10-07 | H. Hoffnabb-La Roche Ag | Anti-pd-l1 antibodies and methods of using the same for detection of pd-l1 |
JP7348899B2 (en) | 2017-12-08 | 2023-09-21 | マレンゴ・セラピューティクス,インコーポレーテッド | Multispecific molecules and their uses |
CA3067055A1 (en) | 2017-12-09 | 2019-06-13 | Abbott Laboratories | Methods for aiding in diagnosing and evaluating a traumatic brain injury in a human subject using a combination of gfap and uch-l1 |
AU2018378971A1 (en) | 2017-12-09 | 2020-01-02 | Abbott Laboratories | Methods for aiding in the diagnosis and evaluation of a subject who has sustained an orthopedic injury and that has or may have sustained an injury to the head, such as mild traumatic brain injury (TBI), using glial fibrillary acidic protein (GFAP) and/or ubiquitin carboxy-terminal hydrolase l1 (UCH-L1) |
US20210087267A1 (en) | 2017-12-20 | 2021-03-25 | Alexion Pharmaceuticals, Inc. | Liquid formulations of anti-cd200 antibodies |
WO2019126536A1 (en) | 2017-12-20 | 2019-06-27 | Alexion Pharmaceuticals Inc. | Humanized anti-cd200 antibodies and uses thereof |
WO2019122046A1 (en) | 2017-12-21 | 2019-06-27 | F. Hoffmann-La Roche Ag | Universal reporter cell assay for specificity test of novel antigen binding moieties |
AU2018390881A1 (en) | 2017-12-21 | 2020-07-02 | F. Hoffmann-La Roche Ag | Antibodies binding to HLA-A2/WT1 |
JP2021508246A (en) | 2017-12-21 | 2021-03-04 | エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト | CAR-T cell assay for specificity testing of novel antigen binding moiety |
EP3728318A2 (en) | 2017-12-22 | 2020-10-28 | Jounce Therapeutics, Inc. | Antibodies for lilrb2 |
EP3728321A1 (en) | 2017-12-22 | 2020-10-28 | F. Hoffmann-La Roche AG | Use of pilra binding agents for treatment of a disease |
EP3732202A4 (en) | 2017-12-28 | 2022-06-15 | Nanjing Legend Biotech Co., Ltd. | Single-domain antibodies and variants thereof against tigit |
CN111542543B (en) | 2017-12-28 | 2023-12-22 | 南京传奇生物科技有限公司 | Antibodies to PD-L1 and variants thereof |
US11440957B2 (en) | 2017-12-29 | 2022-09-13 | Alector Llc | Anti-TMEM106B antibodies and methods of use thereof |
CR20210319A (en) | 2018-01-12 | 2021-07-27 | Amgen Inc | Anti-pd-1 antibodies and methods of treatment |
AU2019207812A1 (en) | 2018-01-12 | 2020-07-23 | Bristol-Myers Squibb Company | Combination therapy with anti-IL-8 antibodies and anti-PD-1 antibodies for treating cancer |
SG11202004233UA (en) | 2018-01-15 | 2020-06-29 | Nanjing Legend Biotech Co Ltd | Single-domain antibodies and variants thereof against pd-1 |
CA3088649A1 (en) | 2018-01-16 | 2019-07-25 | Lakepharma, Inc. | Bispecific antibody that binds cd3 and another target |
JP7268038B2 (en) | 2018-01-31 | 2023-05-02 | アレクトル エルエルシー | ANTI-MS4A4A ANTIBODY AND METHOD OF USE THEREOF |
WO2019152705A1 (en) | 2018-02-01 | 2019-08-08 | Pfizer Inc. | Antibodies specific for cd70 and their uses |
KR20200128018A (en) | 2018-02-01 | 2020-11-11 | 화이자 인코포레이티드 | Chimeric antigen receptor targeting CD70 |
US11787857B2 (en) | 2018-02-02 | 2023-10-17 | Bio-Techne Corporation | Compounds that modulate the interaction of VISTA and VSIG3 and methods of making and using |
WO2019150309A1 (en) | 2018-02-02 | 2019-08-08 | Hammack Scott | Modulators of gpr68 and uses thereof for treating and preventing diseases |
WO2019157131A1 (en) | 2018-02-07 | 2019-08-15 | Dana-Farber Cancer Institute, Inc. | Cell-permeable stapled peptide modules for cellular delivery |
BR112020016169A2 (en) | 2018-02-08 | 2020-12-15 | Genentech, Inc. | MOLECULES FOR BINDING THE BIESPECIFIC ANTIGEN, INSULATED NUCLEIC ACID, VECTOR, HOSTING CELL, METHODS FOR PRODUCING THE BINDING MOLECULE, SET OF NUCLEIC ACIDS, ISOLATED, VEGETABLE CONTAINER, VEGETABLE CONTAINERS, TO TREAT OR DELAY CANCER PROGRESSION, METHODS TO IMPROVE THE IMMUNE FUNCTION AND KIT |
KR20220098056A (en) | 2018-02-09 | 2022-07-08 | 제넨테크, 인크. | Therapeutic and diagnostic methods for mast cell-mediated inflammatory diseases |
TWI829667B (en) | 2018-02-09 | 2024-01-21 | 瑞士商赫孚孟拉羅股份公司 | Antibodies binding to gprc5d |
EP3752195A4 (en) | 2018-02-14 | 2021-11-17 | Viela Bio, Inc. | Antibodies to feline mcdonough sarcoma (fms)-like tyrosine kinase 3 receptor ligand (flt3l) and uses thereof for treating autoimmune and inflammatory diseases |
GB201802486D0 (en) | 2018-02-15 | 2018-04-04 | Ucb Biopharma Sprl | Methods |
TW202000702A (en) | 2018-02-26 | 2020-01-01 | 美商建南德克公司 | Dosing for treatment with anti-TIGIT and anti-PD-L1 antagonist antibodies |
WO2019166453A1 (en) | 2018-03-01 | 2019-09-06 | F. Hoffmann-La Roche Ag | Specificity assay for novel target antigen binding moieties |
CA3091801A1 (en) | 2018-03-02 | 2019-09-06 | Five Prime Therapeutics, Inc. | B7-h4 antibodies and methods of use thereof |
EP3762015A4 (en) | 2018-03-05 | 2022-04-27 | Janssen Biotech, Inc. | Methods of treating crohn's disease with anti-il23 specific antibody |
RU2020129265A (en) | 2018-03-12 | 2022-04-12 | ЗОИТИС СЕРВИСЕЗ ЭлЭлСи | ANTIBODIES AGAINST NGF AND THEIR RELATED METHODS |
NZ767596A (en) | 2018-03-14 | 2022-01-28 | Surface Oncology Inc | Antibodies that bind cd39 and uses thereof |
US20200040103A1 (en) | 2018-03-14 | 2020-02-06 | Genentech, Inc. | Anti-klk5 antibodies and methods of use |
US20210009711A1 (en) | 2018-03-14 | 2021-01-14 | Elstar Therapeutics, Inc. | Multifunctional molecules and uses thereof |
WO2019178362A1 (en) | 2018-03-14 | 2019-09-19 | Elstar Therapeutics, Inc. | Multifunctional molecules that bind to calreticulin and uses thereof |
MX2020009296A (en) | 2018-03-15 | 2020-11-13 | Chugai Pharmaceutical Co Ltd | Anti-dengue virus antibodies having cross-reactivity to zika virus and methods of use. |
WO2019183499A1 (en) | 2018-03-22 | 2019-09-26 | Surface Oncology, Inc. | Anti-il-27 antibodies and uses thereof |
WO2019180272A1 (en) | 2018-03-23 | 2019-09-26 | Fundación Instituto De Investigación Sanitaria De Santiago De Compostela | Anti-leptin affinity reagents for use in the treatment of obesity and other leptin-resistance associated diseases |
DK3775909T3 (en) | 2018-03-26 | 2023-07-24 | Glycanostics S R O | AGENTS AND METHODS FOR GLYCOPROFILING A PROTEIN |
JP7331000B2 (en) | 2018-03-26 | 2023-08-22 | アレクシオン ファーマシューティカルズ, インコーポレイテッド | High throughput method for measuring protease activity of complement C3 convertase |
JP7104458B2 (en) | 2018-04-02 | 2022-07-21 | 上海博威生物医薬有限公司 | Lymphocyte activation gene-3 (LAG-3) -binding antibody and its use |
CN111936520A (en) | 2018-04-02 | 2020-11-13 | 百时美施贵宝公司 | anti-TREM-1 antibodies and uses thereof |
EP3775883A1 (en) | 2018-04-04 | 2021-02-17 | F. Hoffmann-La Roche AG | Diagnostic assays to detect tumor antigens in cancer patients |
CN111742220A (en) | 2018-04-04 | 2020-10-02 | 豪夫迈·罗氏有限公司 | Diagnostic assay for detecting tumor antigens in cancer patients |
TW202011029A (en) | 2018-04-04 | 2020-03-16 | 美商建南德克公司 | Methods for detecting and quantifying FGF21 |
WO2019200357A1 (en) | 2018-04-12 | 2019-10-17 | Surface Oncology, Inc. | Biomarker for cd47 targeting therapeutics and uses therefor |
US20210147547A1 (en) | 2018-04-13 | 2021-05-20 | Novartis Ag | Dosage Regimens For Anti-Pd-L1 Antibodies And Uses Thereof |
AR115052A1 (en) | 2018-04-18 | 2020-11-25 | Hoffmann La Roche | MULTI-SPECIFIC ANTIBODIES AND THE USE OF THEM |
AR114789A1 (en) | 2018-04-18 | 2020-10-14 | Hoffmann La Roche | ANTI-HLA-G ANTIBODIES AND THE USE OF THEM |
US20210230255A1 (en) | 2018-04-27 | 2021-07-29 | Fondazione Ebri Rita Levi-Montalcini | Antibody directed against a tau-derived neurotoxic peptide and uses thereof |
JP7402541B2 (en) | 2018-05-03 | 2023-12-21 | ユニバーシティ オブ ロチェスター | Anti-influenza neuraminidase monoclonal antibody and its use |
AU2019264712A1 (en) | 2018-05-11 | 2021-01-07 | Wuxi Biologics (Shanghai) Co., Ltd. | Fully human antibodies against OX40, method for preparing same, and use thereof |
EP3790587A4 (en) | 2018-05-11 | 2022-01-26 | Janssen Biotech, Inc. | Methods of treating depression using il-23 antibodies |
WO2019222130A1 (en) | 2018-05-15 | 2019-11-21 | Immunogen, Inc. | Combination treatment with antibody-drug conjugates and flt3 inhibitors |
WO2019226658A1 (en) | 2018-05-21 | 2019-11-28 | Compass Therapeutics Llc | Multispecific antigen-binding compositions and methods of use |
TW202003580A (en) | 2018-05-21 | 2020-01-16 | 美商坎伯斯治療有限責任公司 | Compositions and methods for enhancing the killing of target cells by NK cells |
SG11202010934SA (en) | 2018-05-23 | 2020-12-30 | Pfizer | Antibodies specific for gucy2c and uses thereof |
KR102602329B1 (en) | 2018-05-23 | 2023-11-16 | 화이자 인코포레이티드 | Antibodies specific for CD3 and their uses |
MX2020011828A (en) | 2018-05-25 | 2021-02-09 | Alector Llc | Anti-sirpa antibodies and methods of use thereof. |
UY38247A (en) | 2018-05-30 | 2019-12-31 | Novartis Ag | ANTIBODIES AGAINST ENTPD2, COMBINATION THERAPIES AND METHODS OF USE OF ANTIBODIES AND COMBINATION THERAPIES |
US11830582B2 (en) | 2018-06-14 | 2023-11-28 | University Of Miami | Methods of designing novel antibody mimetics for use in detecting antigens and as therapeutic agents |
US11913044B2 (en) | 2018-06-14 | 2024-02-27 | President And Fellows Of Harvard College | Evolution of cytidine deaminases |
CN112533632A (en) | 2018-06-18 | 2021-03-19 | Ucb生物制药有限责任公司 | GREMLIN-1 antagonists for the prevention and treatment of cancer |
PE20210418A1 (en) | 2018-06-19 | 2021-03-08 | Atarga Llc | COMPLEMENT COMPONENT 5 ANTIBODY MOLECULES AND THEIR USES |
US20210347842A1 (en) | 2018-06-19 | 2021-11-11 | Eli Lilly And Company | Compositions and methods of use of il-10 agents in conjunction with chimeric antigen receptor cell therapy |
US20210277118A1 (en) | 2018-06-21 | 2021-09-09 | Daiichi Sankyo Company, Limited | Compositions including cd3 antigen binding fragments and uses thereof |
SG11202012446UA (en) | 2018-06-23 | 2021-01-28 | Genentech Inc | Methods of treating lung cancer with a pd-1 axis binding antagonist, a platinum agent, and a topoisomerase ii inhibitor |
WO2020006176A1 (en) | 2018-06-27 | 2020-01-02 | Obi Pharma, Inc. | Glycosynthase variants for glycoprotein engineering and methods of use |
CN112384532A (en) | 2018-06-29 | 2021-02-19 | 艾利妥 | anti-SIRP-beta 1 antibodies and methods of use thereof |
JP2021530246A (en) | 2018-07-03 | 2021-11-11 | マレンゴ・セラピューティクス,インコーポレーテッド | Anti-TCR antibody molecule and its use |
WO2020008083A1 (en) | 2018-07-05 | 2020-01-09 | Consejo Superior De Investigaciones Científicas | Therapeutic target in chemokine receptors for the screening of compounds useful for the treatment of pathological processes involving chemokine signaling |
EP3677278B1 (en) | 2018-07-11 | 2021-11-10 | Scholar Rock, Inc. | Isoform selective tgfbeta1 inhibitors and use thereof |
US20210340238A1 (en) | 2018-07-11 | 2021-11-04 | Scholar Rock, Inc. | TGFß1 INHIBITORS AND USE THEREOF |
EP3820508A1 (en) | 2018-07-11 | 2021-05-19 | Scholar Rock, Inc. | High-affinity, isoform-selective tgf?1 inhibitors and use thereof |
KR20230065382A (en) | 2018-07-13 | 2023-05-11 | 알렉터 엘엘씨 | Anti-sortilin antibodies and methods of use thereof |
WO2020016838A2 (en) | 2018-07-18 | 2020-01-23 | Janssen Biotech, Inc. | Sustained response predictors after treatment with anti-il23 specific antibody |
WO2020018789A1 (en) | 2018-07-18 | 2020-01-23 | Genentech, Inc. | Methods of treating lung cancer with a pd-1 axis binding antagonist, an antimetabolite, and a platinum agent |
AU2019306165A1 (en) | 2018-07-20 | 2021-02-25 | Pierre Fabre Medicament | Receptor for vista |
WO2020021465A1 (en) | 2018-07-25 | 2020-01-30 | Advanced Accelerator Applications (Italy) S.R.L. | Method of treatment of neuroendocrine tumors |
SI3625368T1 (en) | 2018-08-08 | 2023-04-28 | Pml Screening, Llc | Methods for assessing the risk of developing progressive multifocal leukoencephalopathy caused by john cunningham virus by genetic testing |
AU2019319822A1 (en) | 2018-08-08 | 2021-03-18 | Genentech, Inc. | Use of tryptophan derivatives and L-methionine for protein formulation |
US20210388089A1 (en) | 2018-08-09 | 2021-12-16 | Compass Therapeutics Llc | Antigen binding agents that bind cd277 and uses thereof |
WO2020033925A2 (en) | 2018-08-09 | 2020-02-13 | Compass Therapeutics Llc | Antibodies that bind cd277 and uses thereof |
US20210309746A1 (en) | 2018-08-09 | 2021-10-07 | Compass Therapeutics Llc | Antibodies that bind cd277 and uses thereof |
JP2021533755A (en) | 2018-08-10 | 2021-12-09 | ユーティレックス カンパニー リミテッド | Chimeric antigen receptor and CAR-T cells that bind to HLA-DR |
AR114550A1 (en) | 2018-08-10 | 2020-09-16 | Chugai Pharmaceutical Co Ltd | ANTI-CD137 ANTIGEN BINDING MOLECULES AND THEIR USES |
WO2020041360A1 (en) | 2018-08-21 | 2020-02-27 | Quidel Corporation | Dbpa antibodies and uses thereof |
PE20211096A1 (en) | 2018-08-31 | 2021-06-14 | Alector Llc | ANTI-CD33 ANTIBODIES AND METHODS OF USING THEM |
GB201814281D0 (en) | 2018-09-03 | 2018-10-17 | Femtogenix Ltd | Cytotoxic agents |
US10899826B1 (en) | 2018-09-13 | 2021-01-26 | Teva Pharmaceuticals International Gmbh | Pharmaceutical compositions for an anti-CGRP antagonist antibody |
KR20210063330A (en) | 2018-09-19 | 2021-06-01 | 제넨테크, 인크. | Methods of treatment and diagnosis for bladder cancer |
JP2022500638A (en) | 2018-09-21 | 2022-01-04 | ジェネンテック, インコーポレイテッド | Diagnostic methods for triple-negative breast cancer |
FI3883606T3 (en) | 2018-09-24 | 2023-09-07 | Janssen Biotech Inc | Safe and effective method of treating ulcerative colitis with anti-il12/il23 antibody |
US20220242957A1 (en) | 2018-09-27 | 2022-08-04 | Marengo Therapeutics, Inc. | Csf1r/ccr2 multispecific antibodies |
CA3114295A1 (en) | 2018-09-28 | 2020-04-02 | Kyowa Kirin Co., Ltd. | Il-36 antibodies and uses thereof |
JP2022504287A (en) | 2018-10-03 | 2022-01-13 | スターテン・バイオテクノロジー・ベー・フェー | Antibodies specific for human and cynomolgus monkey APOC3, and methods of their use |
JP2022512595A (en) | 2018-10-05 | 2022-02-07 | バヴァリアン・ノルディック・アクティーゼルスカブ | Combination therapy to treat cancer by intravenous administration of recombinant MVA and immune checkpoint antagonists or immune checkpoint agonists |
US20210395391A1 (en) | 2018-10-11 | 2021-12-23 | Pfizer Inc. | Dosage Regimen for TFPI Antagonists |
UY38407A (en) | 2018-10-15 | 2020-05-29 | Novartis Ag | TREM2 STABILIZING ANTIBODIES |
KR20210079311A (en) | 2018-10-18 | 2021-06-29 | 제넨테크, 인크. | Diagnosis and treatment methods for sarcoma renal cancer |
BR112021007765A2 (en) | 2018-10-23 | 2021-08-03 | Scholar Rock, Inc. | selective rgmc inhibitors and their use |
GB201817309D0 (en) | 2018-10-24 | 2018-12-05 | Ucb Biopharma Sprl | Antibodies |
GB201817311D0 (en) | 2018-10-24 | 2018-12-05 | Ucb Biopharma Sprl | Antibodies |
EP3870235A1 (en) | 2018-10-24 | 2021-09-01 | F. Hoffmann-La Roche AG | Conjugated chemical inducers of degradation and methods of use |
WO2020086408A1 (en) | 2018-10-26 | 2020-04-30 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | A high-yield perfusion-based transient gene expression bioprocess |
EP3877407A1 (en) | 2018-11-05 | 2021-09-15 | F. Hoffmann-La Roche AG | Methods of producing two chain proteins in prokaryotic host cells |
EA202191058A1 (en) | 2018-11-16 | 2021-10-07 | Мемориал Слоун Кеттеринг Кэнсер Сентр | ANTIBODIES AGAINST MUCIN-16 AND METHODS OF THEIR APPLICATION |
WO2020106358A1 (en) | 2018-11-20 | 2020-05-28 | Takeda Vaccines, Inc. | Novel anti-zika virus antibodies and uses thereof |
BR112021009856A8 (en) | 2018-11-20 | 2021-09-08 | Bavarian Nordic As | Therapy for treating cancer with an intratumoral and/or intravenous administration of a recombinant mva encoding 4-1bbl (cd137l) and/or cd40l |
US11548941B2 (en) | 2018-11-20 | 2023-01-10 | Janssen Biotech, Inc. | Safe and effective method of treating psoriasis with anti-IL-23 specific antibody |
WO2020118011A1 (en) | 2018-12-06 | 2020-06-11 | Alexion Pharmaceuticals, Inc. | Anti-alk2 antibodies and uses thereof |
CA3119798A1 (en) | 2018-12-06 | 2020-06-11 | Genentech, Inc. | Combination therapy of diffuse large b-cell lymphoma comprising an anti-cd79b immunoconjugates, an alkylating agent and an anti-cd20 antibody |
CN113227119A (en) | 2018-12-10 | 2021-08-06 | 基因泰克公司 | Photocrosslinked peptides for site-specific conjugation to Fc-containing proteins |
US20200197517A1 (en) | 2018-12-18 | 2020-06-25 | Janssen Biotech, Inc. | Safe and Effective Method of Treating Lupus with Anti-IL12/IL23 Antibody |
EP3898687A1 (en) | 2018-12-20 | 2021-10-27 | Kyowa Kirin Co., Ltd. | Fn14 antibodies and uses thereof |
JP2022513507A (en) | 2018-12-20 | 2022-02-08 | ポセイダ セラピューティクス,インコーポレイティド | Nanotransposon composition and usage |
US20220089694A1 (en) | 2018-12-20 | 2022-03-24 | The U.S.A., As Represented By The Secretary, Department Of Health And Human Services | Ebola virus glycoprotein-specific monoclonal antibodies and uses thereof |
AR117453A1 (en) | 2018-12-20 | 2021-08-04 | Genentech Inc | CF OF MODIFIED ANTIBODIES AND METHODS TO USE THEM |
AU2019400980A1 (en) | 2018-12-20 | 2021-06-24 | Novartis Ag | Pharmaceutical combinations |
TW202039552A (en) | 2018-12-21 | 2020-11-01 | 瑞士商赫孚孟拉羅股份公司 | Antibody that binds to vegf and il-1beta and methods of use |
KR20210107025A (en) | 2018-12-21 | 2021-08-31 | 제넨테크, 인크. | Methods for producing polypeptides using cell lines resistant to apoptosis |
JP2022516505A (en) | 2018-12-28 | 2022-02-28 | スパークス・セラピューティクス・インコーポレイテッド | Claudin 18.2 specific binding molecule, composition and method thereof for the treatment of cancer and other diseases. |
US20220073630A1 (en) | 2018-12-28 | 2022-03-10 | Hoffmann-La Roche, Inc. | A peptide-mhc-i-antibody fusion protein for therapeutic use in a patient with amplified immune response |
AU2020208828A1 (en) | 2019-01-15 | 2021-08-05 | Janssen Biotech, Inc. | Anti-TNF antibody compositions and methods for the treatment of juvenile idiopathic arthritis |
SG11202107538VA (en) | 2019-01-16 | 2021-08-30 | Compass Therapeutics Llc | Formulations of antibodies that bind human cd137 and uses thereof |
GB201900732D0 (en) | 2019-01-18 | 2019-03-06 | Ucb Biopharma Sprl | Antibodies |
EP3914615A1 (en) | 2019-01-23 | 2021-12-01 | F. Hoffmann-La Roche AG | Methods of producing multimeric proteins in eukaryotic host cells |
CA3127748A1 (en) | 2019-01-23 | 2020-07-30 | Janssen Biotech, Inc. | Anti-tnf antibody compositions for use in methods for the treatment of psoriatic arthritis |
WO2020153467A1 (en) | 2019-01-24 | 2020-07-30 | 中外製薬株式会社 | Novel cancer antigens and antibodies of said antigens |
CN113348178A (en) | 2019-01-28 | 2021-09-03 | 枫叶生物技术有限公司 | PSMP antagonists for the treatment of fibrotic diseases of the lung, kidney or liver |
GB201901197D0 (en) | 2019-01-29 | 2019-03-20 | Femtogenix Ltd | G-A Crosslinking cytotoxic agents |
WO2020160291A2 (en) | 2019-01-30 | 2020-08-06 | Scholar Rock, Inc. | LTBP COMPLEX-SPECIFIC INHIBITORS OF TGFβ AND USES THEREOF |
US11738050B2 (en) | 2019-02-01 | 2023-08-29 | Regents Of The University Of Minnesota | Compounds binding to fibroblast activation protein alpha |
EP3693063A1 (en) | 2019-02-06 | 2020-08-12 | Diaccurate | Methods and compositions for treating cancer |
EP3696191A1 (en) | 2019-02-14 | 2020-08-19 | Fundación Instituto de Investigación contra la Leucemia Josep Carreras (IJC) | Car t-cells for the treatment of cd1a-positive cancer |
US10871640B2 (en) | 2019-02-15 | 2020-12-22 | Perkinelmer Cellular Technologies Germany Gmbh | Methods and systems for automated imaging of three-dimensional objects |
SG11202109061YA (en) | 2019-02-21 | 2021-09-29 | Marengo Therapeutics Inc | Multifunctional molecules that bind to t cell related cancer cells and uses thereof |
CN114127113A (en) | 2019-02-21 | 2022-03-01 | 马伦戈治疗公司 | Multifunctional molecules binding to calreticulin and uses thereof |
AU2020224681A1 (en) | 2019-02-21 | 2021-09-16 | Marengo Therapeutics, Inc. | Antibody molecules that bind to NKp30 and uses thereof |
CA3130628A1 (en) | 2019-02-21 | 2020-08-27 | Marengo Therapeutics, Inc. | Multifunctional molecules that bind to t cells and uses thereof to treat autoimmune disorders |
SG11202109122SA (en) | 2019-02-21 | 2021-09-29 | Marengo Therapeutics Inc | Anti-tcr antibody molecules and uses thereof |
EP3930847B1 (en) | 2019-02-26 | 2024-02-14 | Inspirna, Inc. | High-affinity anti-mertk antibodies and uses thereof |
CN113710706A (en) | 2019-02-27 | 2021-11-26 | 豪夫迈·罗氏有限公司 | Administration for anti-TIGIT antibody and anti-CD 20 antibody or anti-CD 38 antibody treatment |
MX2021010565A (en) | 2019-03-08 | 2021-10-13 | Genentech Inc | Methods for detecting and quantifying membrane-associated proteins on extracellular vesicles. |
WO2020183269A1 (en) | 2019-03-14 | 2020-09-17 | Janssen Biotech, Inc. | Manufacturing methods for producing anti-tnf antibody compositions |
US20220144934A1 (en) | 2019-03-14 | 2022-05-12 | Janssen Biotech, Inc. | Methods for Producing Anti-TNF Antibody Compositions |
CN113825765A (en) | 2019-03-14 | 2021-12-21 | 詹森生物科技公司 | Method for producing anti-IL 12/IL23 antibody composition |
CA3133388A1 (en) | 2019-03-14 | 2020-09-17 | Janssen Biotech, Inc. | Methods for producing anti-tnf antibody compositions |
EA202192459A1 (en) | 2019-03-18 | 2021-11-25 | Янссен Байотек, Инк. | METHOD FOR TREATMENT OF PSORIASIS WITH ANTIBODY TO IL12 / IL23 IN CHILDREN |
JP2022524215A (en) | 2019-03-28 | 2022-04-28 | ダニスコ・ユーエス・インク | Modified antibody |
MA55519A (en) | 2019-03-29 | 2022-02-09 | Atarga Llc | ANTI-FGF23 ANTIBODIES |
EP3948289A1 (en) | 2019-03-29 | 2022-02-09 | F. Hoffmann-La Roche AG | Modulators of cell surface protein interactions and methods and compositions related to same |
SG11202111345PA (en) | 2019-04-19 | 2021-11-29 | Chugai Pharmaceutical Co Ltd | Chimeric receptor that recognizes engineered site in antibody |
CN114364703A (en) | 2019-04-19 | 2022-04-15 | 豪夫迈·罗氏有限公司 | Anti-merk antibodies and methods of use thereof |
WO2020227228A2 (en) | 2019-05-03 | 2020-11-12 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Neutralizing antibodies to plasmodium falciparum circumsporozoite protein and their use |
AU2020275415A1 (en) | 2019-05-14 | 2021-11-25 | Genentech, Inc. | Methods of using anti-CD79B immunoconjugates to treat follicular lymphoma |
US20230085439A1 (en) | 2019-05-21 | 2023-03-16 | University Of Georgia Research Foundation, Inc. | Antibodies that bind human metapneumovirus fusion protein and their use |
CA3138241A1 (en) | 2019-05-23 | 2020-11-26 | Janssen Biotech, Inc. | Method of treating inflammatory bowel disease with a combination therapy of antibodies to il-23 and tnf alpha |
US20220241412A1 (en) | 2019-05-24 | 2022-08-04 | Pfizer Inc. | Combination therapies using cdk inhibitors |
JP2022535534A (en) | 2019-06-03 | 2022-08-09 | ヤンセン バイオテツク,インコーポレーテツド | Anti-TNF Antibodies, Compositions and Methods for Treating Active Ankylosing Spondylitis |
MX2021014885A (en) | 2019-06-03 | 2022-04-06 | Janssen Biotech Inc | Anti-tnf antibody compositions, and methods for the treatment of psoriatic arthritis. |
MX2021015212A (en) | 2019-06-11 | 2022-04-06 | Alector Llc | Anti-sortilin antibodies for use in therapy. |
CA3142021A1 (en) | 2019-06-17 | 2020-12-24 | Visterra, Inc. | Humanized antibody molecules to cd138 and uses thereof |
CN114051500A (en) | 2019-07-02 | 2022-02-15 | 豪夫迈·罗氏有限公司 | Immunoconjugates comprising interleukin-2 mutants and anti-CD 8 antibodies |
EP3998083A4 (en) | 2019-07-12 | 2023-08-23 | Chugai Seiyaku Kabushiki Kaisha | Anti-mutation type fgfr3 antibody and use therefor |
AR119393A1 (en) | 2019-07-15 | 2021-12-15 | Hoffmann La Roche | ANTIBODIES THAT BIND NKG2D |
BR112021005478A2 (en) | 2019-07-24 | 2021-06-15 | H. Lundbeck A/S | anti-mglur5 antibodies and their use |
WO2021021606A1 (en) | 2019-07-26 | 2021-02-04 | Visterra, Inc. | Interleukin-2 agents and uses thereof |
KR20220058540A (en) | 2019-07-31 | 2022-05-09 | 알렉터 엘엘씨 | Anti-MS4A4A antibodies and methods of use thereof |
JP2022543553A (en) | 2019-07-31 | 2022-10-13 | エフ・ホフマン-ラ・ロシュ・アクチェンゲゼルシャフト | Antibody that binds to GPRC5D |
WO2021018925A1 (en) | 2019-07-31 | 2021-02-04 | F. Hoffmann-La Roche Ag | Antibodies binding to gprc5d |
CA3148930A1 (en) | 2019-08-02 | 2021-02-11 | Fundacio Clinic Per A La Recerca Biomedica | Car t-cells against bcma for the treatment of multiple myeloma |
KR102509648B1 (en) | 2019-08-06 | 2023-03-15 | 아프리노이아 테라퓨틱스 리미티드 | Antibodies that bind to pathological Tau species and uses thereof |
WO2021023860A1 (en) | 2019-08-07 | 2021-02-11 | Db Biotech, As | Improved horseradish peroxidase polypeptides |
EP4013506A1 (en) | 2019-08-12 | 2022-06-22 | Aptevo Research and Development LLC | 4-1bb and ox40 binding proteins and related compositions and methods, antibodies against 4-1bb, antibodies against ox40 |
AU2020328507A1 (en) | 2019-08-12 | 2022-03-17 | Purinomia Biotech, Inc. | Methods and compositions for promoting and potentiating T-cell mediated immune responses through ADCC targeting of CD39 expressing cells |
WO2021028752A1 (en) | 2019-08-15 | 2021-02-18 | Janssen Biotech, Inc. | Anti-tfn antibodies for treating type i diabetes |
TW202122420A (en) | 2019-08-30 | 2021-06-16 | 美商艾吉納斯公司 | Anti-cd96 antibodies and methods of use thereof |
US20220332799A1 (en) | 2019-09-04 | 2022-10-20 | Deutsches Zentrum Für Neurodegenerative Erkrankungen E.V. (Dzne) | Herv inhibitors for use in treating tauopathies |
KR20220088847A (en) | 2019-09-04 | 2022-06-28 | 주식회사 와이바이오로직스 | Anti-VSIG4 antibodies or antigen-binding fragments and uses thereof |
CN114761424A (en) | 2019-09-05 | 2022-07-15 | 波赛达治疗公司 | Allogeneic cell compositions and methods of use |
CA3146616A1 (en) | 2019-09-12 | 2021-03-18 | Matthew Dominic CASCINO | Compositions and methods of treating lupus nephritis |
AU2020351122A1 (en) | 2019-09-16 | 2022-02-17 | Surface Oncology, Inc. | Anti-CD39 antibody compositions and methods |
JP2022548881A (en) | 2019-09-18 | 2022-11-22 | ノバルティス アーゲー | ENTPD2 Antibodies, Combination Therapy and Methods of Using Antibodies and Combination Therapy |
AU2020349509A1 (en) | 2019-09-18 | 2022-03-31 | Genentech, Inc. | Anti-KLK7 antibodies, anti-KLK5 antibodies, multispecific anti-KLK5/KLK7 antibodies, and methods of use |
TW202124446A (en) | 2019-09-18 | 2021-07-01 | 瑞士商諾華公司 | Combination therapies with entpd2 antibodies |
CR20220149A (en) | 2019-09-20 | 2022-05-23 | Genentech Inc | Dosing for anti-tryptase antibodies |
CA3151078A1 (en) | 2019-09-25 | 2021-04-01 | Surface Oncology, Inc. | Anti-il-27 antibodies and uses thereof |
WO2021062323A1 (en) | 2019-09-26 | 2021-04-01 | Stcube & Co. | Antibodies specific to glycosylated ctla-4 and methods of use thereof |
CN114555116A (en) | 2019-09-27 | 2022-05-27 | 豪夫迈·罗氏有限公司 | Administration for anti-TIGIT and anti-PD-L1 antagonist antibody therapy |
EP4036116A4 (en) | 2019-09-27 | 2024-01-24 | Nanjing Genscript Biotech Co Ltd | Anti-vhh domain antibodies and use thereof |
CN114746119A (en) | 2019-09-27 | 2022-07-12 | 詹森生物科技公司 | anti-CEACAM antibodies and uses thereof |
CN114829404A (en) | 2019-10-09 | 2022-07-29 | 斯特库比公司 | Antibodies specific for glycosylated LAG3 and methods of use thereof |
AU2020365836A1 (en) | 2019-10-18 | 2022-04-28 | F. Hoffmann-La Roche Ag | Methods of using anti-CD79b immunoconjugates to treat diffuse large B-cell lymphoma |
EP4048285A1 (en) | 2019-10-21 | 2022-08-31 | Novartis AG | Tim-3 inhibitors and uses thereof |
BR112022007376A2 (en) | 2019-10-21 | 2022-07-05 | Novartis Ag | COMBINATION THERAPIES WITH VENETOCLAX AND TIM-3 INHIBITORS |
EP3812008A1 (en) | 2019-10-23 | 2021-04-28 | Gamamabs Pharma | Amh-competitive antagonist antibody |
US11459389B2 (en) | 2019-10-24 | 2022-10-04 | Massachusetts Institute Of Technology | Monoclonal antibodies that bind human CD161 |
CA3155922A1 (en) | 2019-11-06 | 2021-05-14 | Huang Huang | Diagnostic and therapeutic methods for treatment of hematologic cancers |
EP4061406A1 (en) | 2019-11-20 | 2022-09-28 | Bavarian Nordic A/S | Recombinant mva viruses for intratumoral and/or intravenous administration for treating cancer |
GB201917480D0 (en) | 2019-11-29 | 2020-01-15 | Univ Oxford Innovation Ltd | Antibodies |
CR20230210A (en) | 2019-12-13 | 2023-06-14 | Genentech Inc | Anti-ly6g6d antibodies and methods of use |
IL293827A (en) | 2019-12-13 | 2022-08-01 | Alector Llc | Anti-mertk antibodies and methods of use thereof |
CN114828965A (en) | 2019-12-18 | 2022-07-29 | 豪夫迈·罗氏有限公司 | Antibodies that bind to HLA-A2/MAGE-A4 |
EP4077376A2 (en) | 2019-12-19 | 2022-10-26 | Quidel Corporation | Monoclonal antibody fusions |
CN115135672A (en) | 2019-12-20 | 2022-09-30 | 波赛达治疗公司 | anti-MUC 1 compositions and methods of use |
IL293834A (en) | 2019-12-20 | 2022-08-01 | Novartis Ag | Combination of anti tim-3 antibody mbg453 and anti tgf-beta antibody nis793, with or without decitabine or the anti pd-1 antibody spartalizumab, for treating myelofibrosis and myelodysplastic syndrome |
GB201919061D0 (en) | 2019-12-20 | 2020-02-05 | Ucb Biopharma Sprl | Multi-specific antibody |
GB201919058D0 (en) | 2019-12-20 | 2020-02-05 | Ucb Biopharma Sprl | Multi-specific antibodies |
GB201919062D0 (en) | 2019-12-20 | 2020-02-05 | Ucb Biopharma Sprl | Antibody |
AU2019479791A1 (en) | 2019-12-27 | 2022-07-14 | Chugai Seiyaku Kabushiki Kaisha | Anti-CTLA-4 antibody and use thereof |
TW202138388A (en) | 2019-12-30 | 2021-10-16 | 美商西根公司 | Methods of treating cancer with nonfucosylated anti-cd70 antibodies |
WO2021138407A2 (en) | 2020-01-03 | 2021-07-08 | Marengo Therapeutics, Inc. | Multifunctional molecules that bind to cd33 and uses thereof |
EP4087607A1 (en) | 2020-01-06 | 2022-11-16 | Vaccinex, Inc. | Anti-ccr8 antibodies and uses thereof |
CN110818795B (en) | 2020-01-10 | 2020-04-24 | 上海复宏汉霖生物技术股份有限公司 | anti-TIGIT antibodies and methods of use |
US20230050148A1 (en) | 2020-01-11 | 2023-02-16 | Scholar Rock, Inc. | Tgf-beta inhibitors and use thereof |
WO2021142427A1 (en) | 2020-01-11 | 2021-07-15 | Scholar Rock, Inc. | TGFβ INHIBITORS AND USE THEREOF |
TW202140553A (en) | 2020-01-13 | 2021-11-01 | 美商威特拉公司 | Antibody molecules to c5ar1 and uses thereof |
CN115315273A (en) | 2020-01-14 | 2022-11-08 | 辛德凯因股份有限公司 | IL-2 orthologs and methods of use thereof |
KR20220128389A (en) | 2020-01-17 | 2022-09-20 | 노파르티스 아게 | A combination comprising a TIM-3 inhibitor and a hypomethylating agent for use in treating myelodysplastic syndrome or chronic myelomonocytic leukemia |
WO2021194481A1 (en) | 2020-03-24 | 2021-09-30 | Genentech, Inc. | Dosing for treatment with anti-tigit and anti-pd-l1 antagonist antibodies |
WO2022050954A1 (en) | 2020-09-04 | 2022-03-10 | Genentech, Inc. | Dosing for treatment with anti-tigit and anti-pd-l1 antagonist antibodies |
KR20220137698A (en) | 2020-02-05 | 2022-10-12 | 라리마 테라퓨틱스, 인코포레이티드 | TAT peptide binding protein and uses thereof |
CA3167299A1 (en) | 2020-02-10 | 2021-08-19 | Shanghai Escugen Biotechnology Co., Ltd. | Cldn18.2 antibody and use thereof |
CA3167349A1 (en) | 2020-02-10 | 2021-08-19 | Qing Zhou | Claudin 18.2 antibody and use thereof |
TW202144395A (en) | 2020-02-12 | 2021-12-01 | 日商中外製藥股份有限公司 | Anti-CD137 antigen-binding molecule for use in cancer treatment |
US20230151109A1 (en) | 2020-02-13 | 2023-05-18 | UCB Biopharma SRL | Bispecific antibodies against cd9 |
US20230096030A1 (en) | 2020-02-13 | 2023-03-30 | UCB Biopharma SRL | Bispecific antibodies against cd9 and cd7 |
US20230125234A1 (en) | 2020-02-13 | 2023-04-27 | UCB Biopharma SRL | Anti cd44-ctla4 bispecific antibodies |
EP4103608A1 (en) | 2020-02-13 | 2022-12-21 | UCB Biopharma SRL | Bispecific antibodies against cd9 and cd137 |
US20230192900A1 (en) | 2020-02-13 | 2023-06-22 | UCB Biopharma SRL | Bispecific antibodies binding hvem and cd9 |
CN115087488A (en) | 2020-02-14 | 2022-09-20 | 震动疗法股份有限公司 | Antibodies and fusion proteins binding to CCR8 and uses thereof |
CA3166155A1 (en) | 2020-02-18 | 2021-08-26 | Spencer LIANG | Pilra antibodies and methods of use thereof |
WO2021170067A1 (en) | 2020-02-28 | 2021-09-02 | 上海复宏汉霖生物技术股份有限公司 | Anti-cd137 construct and use thereof |
WO2021170071A1 (en) | 2020-02-28 | 2021-09-02 | Shanghai Henlius Biotech, Inc. | Anti-cd137 constructs, multispecific antibody and uses thereof |
WO2021176424A1 (en) | 2020-03-06 | 2021-09-10 | Ona Therapeutics, S.L. | Anti-cd36 antibodies and their use to treat cancer |
CN115485295A (en) | 2020-03-10 | 2022-12-16 | 麻省理工学院 | Compositions and methods for immunotherapy of NPM1 c-positive cancers |
WO2021183795A1 (en) | 2020-03-11 | 2021-09-16 | Poseida Therapeutics, Inc. | Chimeric stimulatory receptors and methods of use in t cell activation and differentiation |
MX2022011289A (en) | 2020-03-11 | 2022-12-08 | Fundacio Inst De Recerca Contra La Leucemia Josep Carreras | Cd22 targeting-moiety for the treatment of b-cell acute lymphoblastic leukemia (b-all). |
IL296256A (en) | 2020-03-13 | 2022-11-01 | Genentech Inc | Anti-interleukin-33 antibodies and uses thereof |
IL296427A (en) | 2020-03-19 | 2022-11-01 | Genentech Inc | Isoform-selective anti-tgf-beta antibodies and methods of use |
BR112022018847A2 (en) | 2020-03-24 | 2022-11-22 | Genentech Inc | ANTIBODIES, NUCLEIC ACID, HOST CELL, CONJUGATES, PHARMACEUTICAL COMPOSITION, LONG-ACTION DELIVERY DEVICE FOR OCULAR DELIVERY, METHOD FOR TREAT A DISORDER AND USE OF THE ANTIBODY |
CN115397850A (en) | 2020-03-30 | 2022-11-25 | 豪夫迈·罗氏有限公司 | Antibodies that bind to VEGF and PDGF-B and methods of use thereof |
WO2021202473A2 (en) | 2020-03-30 | 2021-10-07 | Danisco Us Inc | Engineered antibodies |
JP2023519962A (en) | 2020-03-31 | 2023-05-15 | アレクトル エルエルシー | ANTI-MERTK ANTIBODY AND METHOD OF USE THEREOF |
EP4126934A1 (en) | 2020-04-01 | 2023-02-08 | University of Rochester | Monoclonal antibodies against the hemagglutinin (ha) and neuraminidase (na) of influenza h3n2 viruses |
WO2021203024A1 (en) | 2020-04-03 | 2021-10-07 | Visterra, Inc. | Antibody molecule-drug conjugates and uses thereof |
WO2021202959A1 (en) | 2020-04-03 | 2021-10-07 | Genentech, Inc. | Therapeutic and diagnostic methods for cancer |
EP4136459A1 (en) | 2020-04-13 | 2023-02-22 | Abbott Laboratories | Methods, complexes and kits for detecting or determining an amount of a ss-coronavirus antibody in a sample |
MX2022012956A (en) | 2020-04-14 | 2023-03-27 | Poseida Therapeutics Inc | Compositions and methods for use in the treatment of cancer. |
IL294451A (en) | 2020-04-15 | 2022-09-01 | Hoffmann La Roche | Immunoconjugates |
CA3180321A1 (en) | 2020-04-24 | 2021-10-28 | Marengo Therapeutics, Inc. | Multifunctional molecules that bind to t cell related cancer cells and uses thereof |
TW202206111A (en) | 2020-04-24 | 2022-02-16 | 美商建南德克公司 | Methods of using anti-cd79b immunoconjugates |
CN115885050A (en) | 2020-04-28 | 2023-03-31 | 基因泰克公司 | Methods and compositions for non-small cell lung cancer immunotherapy |
TW202216757A (en) | 2020-04-28 | 2022-05-01 | 美國洛克菲勒大學 | Neutralizing anti-sars-cov-2 antibodies and methods of use thereof |
CR20220598A (en) | 2020-04-30 | 2023-01-17 | Genentech Inc | Kras specific antibodies and uses thereof |
US20230242647A1 (en) | 2020-05-01 | 2023-08-03 | Novartis Ag | Engineered immunoglobulins |
CN115461363A (en) | 2020-05-01 | 2022-12-09 | 诺华股份有限公司 | Immunoglobulin variants |
AU2021267995A1 (en) | 2020-05-03 | 2022-12-08 | Levena (Suzhou) Biopharma Co., Ltd. | Antibody-drug conjugates (ADCs) comprising an anti-Trop-2 antibody, compositions comprising such ADCs, as well as methods of making and using the same |
WO2021226290A1 (en) | 2020-05-05 | 2021-11-11 | 10X Genomics, Inc. | Methods for identification of antigen-binding molecules |
IL297977A (en) | 2020-05-17 | 2023-01-01 | Astrazeneca Uk Ltd | Sars-cov-2 antibodies and methods of selecting and using the same |
WO2021239666A1 (en) | 2020-05-26 | 2021-12-02 | Diaccurate | Therapeutic methods |
CN113993900B (en) | 2020-05-27 | 2023-08-04 | 舒泰神(北京)生物制药股份有限公司 | Antibodies specifically recognizing nerve growth factor and uses thereof |
MX2022015376A (en) | 2020-06-02 | 2023-04-14 | Dynamicure Biotechnology Llc | Anti-cd93 constructs and uses thereof. |
CN116529260A (en) | 2020-06-02 | 2023-08-01 | 当康生物技术有限责任公司 | anti-CD 93 constructs and uses thereof |
EP4158058A1 (en) | 2020-06-02 | 2023-04-05 | 10X Genomics, Inc. | Enrichment of nucleic acid sequences |
EP4161653A1 (en) | 2020-06-03 | 2023-04-12 | Bionecure Therapeutics, Inc. | Trophoblast cell-surface antigen-2 (trop-2) antibodies |
WO2021249990A2 (en) | 2020-06-08 | 2021-12-16 | Hoffmann-La Roche Inc. | Anti-hbv antibodies and methods of use |
GB202008651D0 (en) | 2020-06-09 | 2020-07-22 | Univ Newcastle | Method of identifying complement modulators |
JP2023529206A (en) | 2020-06-12 | 2023-07-07 | ジェネンテック, インコーポレイテッド | Methods and compositions for cancer immunotherapy |
CA3181820A1 (en) | 2020-06-16 | 2021-12-23 | Genentech, Inc. | Methods and compositions for treating triple-negative breast cancer |
BR112022025801A2 (en) | 2020-06-18 | 2023-10-03 | Hoffmann La Roche | METHODS FOR TREATING A PATIENT AND FOR TREATING A PATIENT WITH ADVANCED ESCC, KIT, ANTIBODY, USE OF AN ANTIBODY, AND USE OF A BINDING ANTAGONIST |
KR20230042273A (en) | 2020-06-24 | 2023-03-28 | 비스테라, 인크. | Antibody Molecules to APRIL and Uses Thereof |
US20230256114A1 (en) | 2020-07-07 | 2023-08-17 | Bionecure Therapeutics, Inc. | Novel maytansinoids as adc payloads and their use for the treatment of cancer |
BR112023000701A2 (en) | 2020-07-17 | 2023-02-07 | Pfizer | THERAPEUTIC ANTIBODIES AND THEIR USES |
KR20230038735A (en) | 2020-07-17 | 2023-03-21 | 제넨테크, 인크. | Anti-NOTCH2 Antibodies and Methods of Use |
EP4182343A2 (en) | 2020-07-20 | 2023-05-24 | AstraZeneca UK Limited | Sars-cov-2 proteins, anti-sars-cov-2 antibodies, and methods of using the same |
KR20230040331A (en) | 2020-07-21 | 2023-03-22 | 알로젠 테라퓨틱스 인코포레이티드 | Chimeric Antigen Receptors with Enhanced Signal Transduction and Activity and Uses Thereof |
TW202216215A (en) | 2020-07-21 | 2022-05-01 | 美商建南德克公司 | Antibody-conjugated chemical inducers of degradation of brm and methods thereof |
WO2022026592A2 (en) | 2020-07-28 | 2022-02-03 | Celltas Bio, Inc. | Antibody molecules to coronavirus and uses thereof |
GB2597532A (en) | 2020-07-28 | 2022-02-02 | Femtogenix Ltd | Cytotoxic compounds |
TW202221029A (en) | 2020-07-29 | 2022-06-01 | 美商當康生物科技有限公司 | Anti-cd93 constructs and uses thereof |
KR20230042301A (en) | 2020-08-04 | 2023-03-28 | 애벗트 라보라토리이즈 | Improved methods and kits for detecting SARS-COV-2 proteins in samples |
WO2022040345A1 (en) | 2020-08-18 | 2022-02-24 | Cephalon, Inc. | Anti-par-2 antibodies and methods of use thereof |
EP4204450A2 (en) | 2020-08-26 | 2023-07-05 | Marengo Therapeutics, Inc. | Multifunctional molecules that bind to calreticulin and uses thereof |
AU2021333779A1 (en) | 2020-08-26 | 2023-04-13 | Marengo Therapeutics, Inc. | Methods of detecting TRBC1 or TRBC2 |
JP2023539645A (en) | 2020-08-26 | 2023-09-15 | マレンゴ・セラピューティクス,インコーポレーテッド | Antibody molecules that bind to NKP30 and uses thereof |
EP4204021A1 (en) | 2020-08-31 | 2023-07-05 | Advanced Accelerator Applications International S.A. | Method of treating psma-expressing cancers |
WO2022043558A1 (en) | 2020-08-31 | 2022-03-03 | Advanced Accelerator Applications International Sa | Method of treating psma-expressing cancers |
CR20230146A (en) | 2020-09-11 | 2023-06-07 | Medimmune Ltd | THERAPEUTIC B7-H4 BINDING MOLECULES |
AU2021342349A1 (en) | 2020-09-12 | 2023-05-25 | Medimmune Limited | A scoring method for an anti-b7h4 antibody-drug conjugate therapy |
EP3981789A1 (en) | 2020-10-12 | 2022-04-13 | Commissariat À L'Énergie Atomique Et Aux Énergies Alternatives | Anti-lilrb antibodies and uses thereof |
CA3198456A1 (en) | 2020-10-14 | 2022-04-21 | Five Prime Therapeutics, Inc. | Anti-c-c chemokine receptor 8 (ccr8) antibodies and methods of use thereof |
WO2022081436A1 (en) | 2020-10-15 | 2022-04-21 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Antibody specific for sars-cov-2 receptor binding domain and therapeutic methods |
CA3198049A1 (en) | 2020-10-15 | 2022-04-21 | UCB Biopharma SRL | Binding molecules that multimerise cd45 |
KR20230091871A (en) | 2020-10-20 | 2023-06-23 | 에프. 호프만-라 로슈 아게 | Combination therapy of a PD-1 axis binding antagonist and a LRRK2 inhibitor |
WO2022087274A1 (en) | 2020-10-21 | 2022-04-28 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Antibodies that neutralize type-i interferon (ifn) activity |
WO2022090181A1 (en) | 2020-10-28 | 2022-05-05 | F. Hoffmann-La Roche Ag | Improved antigen binding receptors |
US20220153858A1 (en) | 2020-11-04 | 2022-05-19 | Genentech, Inc. | Subcutaneous dosing of anti-cd20/anti-cd3 bispecific antibodies |
WO2022098870A1 (en) | 2020-11-04 | 2022-05-12 | The Rockefeller University | Neutralizing anti-sars-cov-2 antibodies |
MX2023005132A (en) | 2020-11-04 | 2023-05-25 | Genentech Inc | Dosing for treatment with anti-cd20/anti-cd3 bispecific antibodies. |
WO2022098648A2 (en) | 2020-11-04 | 2022-05-12 | Genentech, Inc. | Dosing for treatment with anti-cd20/anti-cd3 bispecific antibodies and anti-cd79b antibody drug conjugates |
EP4251647A1 (en) | 2020-11-24 | 2023-10-04 | Bio-Techne Corporation | Anti-severe acute respiratory syndrome coronavirus antibodies |
WO2022119841A1 (en) | 2020-12-01 | 2022-06-09 | Abbott Laboratories | Use of one or more biomarkers to determine traumatic brain injury (tbi) in a subject having received a head computerized tomography scan that is negative for a tbi |
WO2023102384A1 (en) | 2021-11-30 | 2023-06-08 | Abbott Laboratories | Use of one or more biomarkers to determine traumatic brain injury (tbi) in a subject having received a head computerized tomography scan that is negative for a tbi |
JP2023551907A (en) | 2020-12-01 | 2023-12-13 | アプティーボ リサーチ アンド デベロップメント エルエルシー | Tumor-associated antigens and CD3 binding proteins, related compositions, and methods |
EP4255930A1 (en) | 2020-12-02 | 2023-10-11 | Alector LLC | Methods of use of anti-sortilin antibodies |
US20220168293A1 (en) | 2020-12-02 | 2022-06-02 | Pfizer Inc. | Time to resolution of axitinib-related adverse events |
EP4255466A1 (en) | 2020-12-04 | 2023-10-11 | Visterra, Inc. | Methods of using interleukin-2 agents |
CN116670166A (en) | 2020-12-07 | 2023-08-29 | Ucb生物制药有限责任公司 | Multispecific antibodies and antibody combinations |
JP2023551983A (en) | 2020-12-07 | 2023-12-13 | ユーシービー バイオファルマ エスアールエル | Antibodies against interleukin-22 |
CA3204702A1 (en) | 2020-12-17 | 2022-06-23 | F. Hoffmann-La Roche Ag | Anti-hla-g antibodies and use thereof |
WO2022132904A1 (en) | 2020-12-17 | 2022-06-23 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Human monoclonal antibodies targeting sars-cov-2 |
CN117043181A (en) | 2020-12-18 | 2023-11-10 | 基尼科萨制药有限公司 | Protein compositions and methods of making and using the same |
WO2022147147A1 (en) | 2020-12-30 | 2022-07-07 | Abbott Laboratories | Methods for determining sars-cov-2 antigen and anti-sars-cov-2 antibody in a sample |
JP2024502832A (en) | 2020-12-31 | 2024-01-23 | アラマー バイオサイエンシーズ, インコーポレイテッド | Binding agent molecules with high affinity and/or specificity and methods for their production and use |
WO2022148853A1 (en) | 2021-01-11 | 2022-07-14 | F. Hoffmann-La Roche Ag | Immunoconjugates |
TW202237135A (en) | 2021-01-13 | 2022-10-01 | 紀念斯隆凱特琳癌症中心 | Antibody-pyrrolobenzodiazepine derivative conjugate |
AU2022208361A1 (en) | 2021-01-13 | 2023-07-27 | Daiichi Sankyo Company, Limited | Anti-dll3 antibody-drug conjugate |
WO2022155324A1 (en) | 2021-01-15 | 2022-07-21 | The Rockefeller University | Neutralizing anti-sars-cov-2 antibodies |
CA3208934A1 (en) | 2021-01-20 | 2022-07-28 | Visterra, Inc. | Interleukin-2 mutants and uses thereof |
EP4284510A1 (en) | 2021-01-29 | 2023-12-06 | Novartis AG | Dosage regimes for anti-cd73 and anti-entpd2 antibodies and uses thereof |
AU2022220611A1 (en) | 2021-02-09 | 2023-08-24 | University Of Georgia Research Foundation, Inc. | Human monoclonal antibodies against pneumococcal antigens |
JP2024506315A (en) | 2021-02-09 | 2024-02-13 | ザ ユナイテッド ステイツ オブ アメリカ アズ リプリゼンテッド バイ ザ セクレタリー、デパートメント オブ ヘルス アンド ヒューマン サービシーズ | Antibodies that target the coronavirus spike protein |
WO2022182872A2 (en) | 2021-02-24 | 2022-09-01 | Alladapt Immunotherapeutics, Inc. | Compositions and methods for identification of cross-reactive allergenic proteins and treatment of allergies |
KR20230157986A (en) | 2021-03-02 | 2023-11-17 | 체게에르페 다이어그노스틱스 게엠베하 | Treating and/or Reducing the Occurrence of Migraine |
KR20230156727A (en) | 2021-03-03 | 2023-11-14 | 피에르 파브르 메디카먼트 | Anti-VSIG4 antibody or antigen-binding fragment thereof and uses |
JP2024512305A (en) | 2021-03-03 | 2024-03-19 | ザ ユナイテッド ステイツ オブ アメリカ アズ リプリゼンテッド バイ ザ セクレタリー、デパートメント オブ ヘルス アンド ヒューマン サービシーズ | La protein as a novel regulator of osteoclast production |
JP2024509169A (en) | 2021-03-03 | 2024-02-29 | ソレント・セラピューティクス・インコーポレイテッド | Antibody-drug conjugates including anti-BCMA antibodies |
EP4301472A1 (en) | 2021-03-05 | 2024-01-10 | Dynamicure Biotechnology LLC | Anti-vista constructs and uses thereof |
WO2022192647A1 (en) | 2021-03-12 | 2022-09-15 | Genentech, Inc. | Anti-klk7 antibodies, anti-klk5 antibodies, multispecific anti-klk5/klk7 antibodies, and methods of use |
IL305802A (en) | 2021-03-12 | 2023-11-01 | Janssen Biotech Inc | Safe and effective method of treating psoriatic arthritis with anti-il23 specific antibody |
BR112023018400A2 (en) | 2021-03-12 | 2023-12-12 | Janssen Biotech Inc | METHOD FOR TREATMENT OF PSORIATIC ARTHRITIS PATIENTS WITH INADEQUATE RESPONSE TO TNF THERAPY WITH SPECIFIC ANTI-IL23 ANTIBODY |
KR20230156373A (en) | 2021-03-15 | 2023-11-14 | 제넨테크, 인크. | Therapeutic compositions and methods of treating lupus nephritis |
BR112023018331A2 (en) | 2021-03-18 | 2023-12-12 | Medimmune Ltd | THERAPEUTIC BINDING MOLECULE THAT BINDS TO CCR9 |
CN116981696A (en) | 2021-03-18 | 2023-10-31 | 艾莱克特有限责任公司 | anti-TMEM 106B antibodies and methods of use thereof |
WO2022195551A1 (en) | 2021-03-18 | 2022-09-22 | Novartis Ag | Biomarkers for cancer and methods of use thereof |
WO2022197877A1 (en) | 2021-03-19 | 2022-09-22 | Genentech, Inc. | Methods and compositions for time delayed bio-orthogonal release of cytotoxic agents |
EP4314063A1 (en) | 2021-03-23 | 2024-02-07 | Alector LLC | Anti-tmem106b antibodies for treating and preventing coronavirus infections |
TW202300648A (en) | 2021-03-25 | 2023-01-01 | 美商當康生物科技有限公司 | Anti-igfbp7 constructs and uses thereof |
WO2022204581A2 (en) | 2021-03-26 | 2022-09-29 | Scholar Rock, Inc. | Tgf-beta inhibitors and use thereof |
EP4067381A1 (en) | 2021-04-01 | 2022-10-05 | Julius-Maximilians-Universität Würzburg | Novel tnfr2 binding molecules |
TW202304979A (en) | 2021-04-07 | 2023-02-01 | 瑞士商諾華公司 | USES OF ANTI-TGFβ ANTIBODIES AND OTHER THERAPEUTIC AGENTS FOR THE TREATMENT OF PROLIFERATIVE DISEASES |
WO2022216993A2 (en) | 2021-04-08 | 2022-10-13 | Marengo Therapeutics, Inc. | Multifuntional molecules binding to tcr and uses thereof |
AR125344A1 (en) | 2021-04-15 | 2023-07-05 | Chugai Pharmaceutical Co Ltd | ANTI-C1S ANTIBODY |
CA3217803A1 (en) | 2021-04-30 | 2022-11-03 | F. Hoffmann-La Roche Ag | Dosing for treatment with anti-cd20/anti-cd3 bispecific antibody |
CN117321078A (en) | 2021-04-30 | 2023-12-29 | 豪夫迈·罗氏有限公司 | Administration for combination therapy with anti-CD 20/anti-CD 3 bispecific antibody and anti-CD 79B antibody drug conjugates |
EP4334355A1 (en) | 2021-05-03 | 2024-03-13 | UCB Biopharma SRL | Antibodies |
WO2022235867A2 (en) | 2021-05-06 | 2022-11-10 | The Rockefeller University | Neutralizing anti-sars- cov-2 antibodies and methods of use thereof |
US20220389089A1 (en) | 2021-05-07 | 2022-12-08 | Surface Oncology, Inc. | Anti-il-27 antibodies and uses thereof |
WO2022241446A1 (en) | 2021-05-12 | 2022-11-17 | Genentech, Inc. | Methods of using anti-cd79b immunoconjugates to treat diffuse large b-cell lymphoma |
US20220381796A1 (en) | 2021-05-18 | 2022-12-01 | Abbott Laboratories | Methods of evaluating brain injury in a pediatric subject |
CN113278071B (en) | 2021-05-27 | 2021-12-21 | 江苏荃信生物医药股份有限公司 | Anti-human interferon alpha receptor1 monoclonal antibody and application thereof |
CA3220629A1 (en) | 2021-05-28 | 2022-12-01 | Tobin J. CAMMETT | Methods for detecting cm-tma biomarkers |
WO2022256313A1 (en) | 2021-06-01 | 2022-12-08 | 10X Genomics, Inc. | Validation of a unique molecular identifier associated with a nucleic acid sequence of interest |
WO2022256723A2 (en) | 2021-06-03 | 2022-12-08 | Scholar Rock, Inc. | Tgf-beta inhibitors and therapeutic use thereof |
WO2022255440A1 (en) | 2021-06-04 | 2022-12-08 | Chugai Seiyaku Kabushiki Kaisha | Anti-ddr2 antibodies and uses thereof |
WO2022261018A1 (en) | 2021-06-07 | 2022-12-15 | Providence Health & Services - Oregon | Cxcr5, pd-1, and icos expressing tumor reactive cd4 t cells and their use |
WO2022261183A2 (en) | 2021-06-08 | 2022-12-15 | Dana-Farber Cancer Institute, Inc. | Compositions and methods for treating and/or identifying an agent for treating intestinal cancers |
CA3216220A1 (en) | 2021-06-09 | 2022-12-15 | F. Hoffmann-La Roche Ag | Combination of a particular braf inhibitor (paradox breaker) and a pd-1 axis binding antagonist for use in the treatment of cancer |
IL309349A (en) | 2021-06-14 | 2024-02-01 | argenx BV | Anti-il-9 antibodies and methods of use thereof |
AU2022293389A1 (en) | 2021-06-14 | 2024-01-04 | Abbott Laboratories | Methods of diagnosing or aiding in diagnosis of brain injury caused by acoustic energy, electromagnetic energy, an over pressurization wave, and/or blast wind |
WO2022266221A1 (en) | 2021-06-16 | 2022-12-22 | Alector Llc | Monovalent anti-mertk antibodies and methods of use thereof |
CN117642426A (en) | 2021-06-16 | 2024-03-01 | 艾莱克特有限责任公司 | Bispecific anti-MerTK and anti-PDL 1 antibodies and methods of use thereof |
WO2022266660A1 (en) | 2021-06-17 | 2022-12-22 | Amberstone Biosciences, Inc. | Anti-cd3 constructs and uses thereof |
WO2022271867A1 (en) | 2021-06-23 | 2022-12-29 | Scholar Rock, Inc. | A myostatin pathway inhibitor in combination with a glp-1 pathway activator for use in treating metabolic disorders |
KR20240024255A (en) | 2021-06-25 | 2024-02-23 | 추가이 세이야쿠 가부시키가이샤 | Use of anti-CTLA-4 antibodies |
TW202317627A (en) | 2021-06-25 | 2023-05-01 | 日商中外製藥股份有限公司 | Anti-ctla-4 antibodies |
KR20240025597A (en) | 2021-06-29 | 2024-02-27 | 씨젠 인크. | Methods of treating cancer with a combination of afucosylated anti-CD70 antibody and CD47 antagonist |
TW202317633A (en) | 2021-07-08 | 2023-05-01 | 美商舒泰神(加州)生物科技有限公司 | Antibodies specifically recognizing tnfr2 and uses thereof |
AU2022308201A1 (en) | 2021-07-09 | 2024-02-22 | Janssen Biotech, Inc. | Manufacturing methods for producing anti-tnf antibody compositions |
AU2022306144A1 (en) | 2021-07-09 | 2024-02-22 | Janssen Biotech, Inc. | Manufacturing methods for producing anti-tnf antibody compositions |
IL309987A (en) | 2021-07-09 | 2024-03-01 | Janssen Biotech Inc | Manufacturing methods for producing anti-il12/il23 antibody compositions |
WO2023285878A1 (en) | 2021-07-13 | 2023-01-19 | Aviation-Ophthalmology | Methods for detecting, treating, and preventing gpr68-mediated ocular diseases, disorders, and conditions |
CA3225252A1 (en) | 2021-07-14 | 2023-01-19 | Jordan JARJOUR | Engineered t cell receptors fused to binding domains from antibodies |
WO2023284714A1 (en) | 2021-07-14 | 2023-01-19 | 舒泰神(北京)生物制药股份有限公司 | Antibody that specifically recognizes cd40 and application thereof |
CA3219606A1 (en) | 2021-07-22 | 2023-01-26 | F. Hoffmann-La Roche Ag | Heterodimeric fc domain antibodies |
WO2023004386A1 (en) | 2021-07-22 | 2023-01-26 | Genentech, Inc. | Brain targeting compositions and methods of use thereof |
WO2023007472A1 (en) | 2021-07-30 | 2023-02-02 | ONA Therapeutics S.L. | Anti-cd36 antibodies and their use to treat cancer |
CN117794953A (en) | 2021-08-03 | 2024-03-29 | 豪夫迈·罗氏有限公司 | Bispecific antibodies and methods of use |
US11807685B2 (en) | 2021-08-05 | 2023-11-07 | The Uab Research Foundation | Anti-CD47 antibody and uses thereof |
WO2023012343A1 (en) | 2021-08-06 | 2023-02-09 | Institut Du Cancer De Montpellier | Methods for the treatment of cancer |
CA3228576A1 (en) | 2021-08-10 | 2023-02-16 | Byomass Inc. | Anti-gdf15 antibodies, compositions and uses thereof |
WO2023019239A1 (en) | 2021-08-13 | 2023-02-16 | Genentech, Inc. | Dosing for anti-tryptase antibodies |
GB202111905D0 (en) | 2021-08-19 | 2021-10-06 | UCB Biopharma SRL | Antibodies |
WO2023026205A1 (en) | 2021-08-24 | 2023-03-02 | Cgrp Diagnostics Gmbh | Preventative treatment of migraine |
CA3228822A1 (en) | 2021-08-26 | 2023-03-02 | Jan Tkac | Glycoprotein biomarkers for diagnosing cancer |
TW202325727A (en) | 2021-08-30 | 2023-07-01 | 美商建南德克公司 | Anti-polyubiquitin multispecific antibodies |
TW202323289A (en) | 2021-08-31 | 2023-06-16 | 日商大正製藥股份有限公司 | Anti-growth hormone antibody |
CA3230038A1 (en) | 2021-08-31 | 2023-03-09 | Hongwei Zhang | Methods and systems of diagnosing brain injury |
CA3230815A1 (en) | 2021-09-03 | 2023-03-09 | University Of Bern | Compositions and methods for treating long ot syndrome |
CN113603775B (en) | 2021-09-03 | 2022-05-20 | 江苏荃信生物医药股份有限公司 | Anti-human interleukin-33 monoclonal antibody and application thereof |
CN113683694B (en) | 2021-09-03 | 2022-05-13 | 江苏荃信生物医药股份有限公司 | Anti-human TSLP monoclonal antibody and application thereof |
AU2022346688A1 (en) | 2021-09-14 | 2024-04-04 | Glycanostics S.R.O | Use of lectins to determine mammaglobin-a glycoforms in breast cancer |
AU2022345881A1 (en) | 2021-09-20 | 2024-03-21 | Alnylam Pharmaceuticals, Inc. | Inhibin subunit beta e (inhbe) modulator compositions and methods of use thereof |
WO2023044483A2 (en) | 2021-09-20 | 2023-03-23 | Voyager Therapeutics, Inc. | Compositions and methods for the treatment of her2 positive cancer |
TW202321308A (en) | 2021-09-30 | 2023-06-01 | 美商建南德克公司 | Methods for treatment of hematologic cancers using anti-tigit antibodies, anti-cd38 antibodies, and pd-1 axis binding antagonists |
WO2023056069A1 (en) | 2021-09-30 | 2023-04-06 | Angiex, Inc. | Degrader-antibody conjugates and methods of using same |
WO2023056268A1 (en) | 2021-09-30 | 2023-04-06 | Abbott Laboratories | Methods and systems of diagnosing brain injury |
WO2023060088A1 (en) | 2021-10-04 | 2023-04-13 | Poseida Therapeutics, Inc. | Transposon compositions and methods of use thereof |
WO2023057882A1 (en) | 2021-10-05 | 2023-04-13 | Pfizer Inc. | Combinations of azalactam compounds with a pd-1 axis binding antagonist for the treatment of cancer |
TW202323810A (en) | 2021-10-08 | 2023-06-16 | 日商中外製藥股份有限公司 | Method for preparing prefilled syringe formulation |
WO2023062048A1 (en) | 2021-10-14 | 2023-04-20 | F. Hoffmann-La Roche Ag | Alternative pd1-il7v immunoconjugates for the treatment of cancer |
WO2023062050A1 (en) | 2021-10-14 | 2023-04-20 | F. Hoffmann-La Roche Ag | New interleukin-7 immunoconjugates |
WO2023069421A1 (en) | 2021-10-18 | 2023-04-27 | Byomass Inc. | Anti-activin a antibodies, compositions and uses thereof |
WO2023069919A1 (en) | 2021-10-19 | 2023-04-27 | Alector Llc | Anti-cd300lb antibodies and methods of use thereof |
WO2023073615A1 (en) | 2021-10-29 | 2023-05-04 | Janssen Biotech, Inc. | Methods of treating crohn's disease with anti-il23 specific antibody |
EP4177266A1 (en) | 2021-11-05 | 2023-05-10 | Katholieke Universiteit Leuven | Neutralizing anti-sars-cov-2 human antibodies |
WO2023086824A1 (en) | 2021-11-10 | 2023-05-19 | 10X Genomics, Inc. | Methods for identification of antigen-binding molecules |
WO2023086807A1 (en) | 2021-11-10 | 2023-05-19 | Genentech, Inc. | Anti-interleukin-33 antibodies and uses thereof |
WO2023084488A1 (en) | 2021-11-15 | 2023-05-19 | Janssen Biotech, Inc. | Methods of treating crohn's disease with anti-il23 specific antibody |
TW202337494A (en) | 2021-11-16 | 2023-10-01 | 美商建南德克公司 | Methods and compositions for treating systemic lupus erythematosus (sle) with mosunetuzumab |
WO2023092004A1 (en) | 2021-11-17 | 2023-05-25 | Voyager Therapeutics, Inc. | Compositions and methods for the treatment of tau-related disorders |
US20230159633A1 (en) | 2021-11-23 | 2023-05-25 | Janssen Biotech, Inc. | Method of Treating Ulcerative Colitis with Anti-IL23 Specific Antibody |
US20230348614A1 (en) | 2021-11-24 | 2023-11-02 | Visterra, Inc. | Engineered antibody molecules to cd138 and uses thereof |
WO2023097119A2 (en) | 2021-11-29 | 2023-06-01 | Dana-Farber Cancer Institute, Inc. | Methods and compositions to modulate riok2 |
US20240041981A1 (en) | 2021-12-01 | 2024-02-08 | Visterra, Inc. | Methods of using interleukin-2 agents |
WO2023114978A1 (en) | 2021-12-17 | 2023-06-22 | Abbott Laboratories | Systems and methods for determining uch-l1, gfap, and other biomarkers in blood samples |
WO2023122213A1 (en) | 2021-12-22 | 2023-06-29 | Byomass Inc. | Targeting gdf15-gfral pathway cross-reference to related applications |
WO2023118508A1 (en) | 2021-12-23 | 2023-06-29 | Bavarian Nordic A/S | Recombinant mva viruses for intraperitoneal administration for treating cancer |
WO2023129942A1 (en) | 2021-12-28 | 2023-07-06 | Abbott Laboratories | Use of biomarkers to determine sub-acute traumatic brain injury (tbi) in a subject having received a head computerized tomography (ct) scan that is negative for a tbi or no head ct scan |
WO2023141445A1 (en) | 2022-01-19 | 2023-07-27 | Genentech, Inc. | Anti-notch2 antibodies and conjugates and methods of use |
WO2023141576A1 (en) | 2022-01-21 | 2023-07-27 | Poseida Therapeutics, Inc. | Compositions and methods for delivery of nucleic acids |
WO2023147399A1 (en) | 2022-01-27 | 2023-08-03 | The Rockefeller University | Broadly neutralizing anti-sars-cov-2 antibodies targeting the n-terminal domain of the spike protein and methods of use thereof |
WO2023147107A1 (en) | 2022-01-31 | 2023-08-03 | Byomass Inc. | Myeloproliferative conditions |
WO2023150652A1 (en) | 2022-02-04 | 2023-08-10 | Abbott Laboratories | Lateral flow methods, assays, and devices for detecting the presence or measuring the amount of ubiquitin carboxy-terminal hydrolase l1 and/or glial fibrillary acidic protein in a sample |
WO2023150778A1 (en) | 2022-02-07 | 2023-08-10 | Visterra, Inc. | Anti-idiotype antibody molecules and uses thereof |
WO2023154824A1 (en) | 2022-02-10 | 2023-08-17 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Human monoclonal antibodies that broadly target coronaviruses |
TW202348252A (en) | 2022-02-16 | 2023-12-16 | 英商梅迪繆思有限公司 | Combination therapies for treatment of cancer with therapeutic binding molecules |
WO2023169896A1 (en) | 2022-03-09 | 2023-09-14 | Astrazeneca Ab | BINDING MOLECULES AGAINST FRα |
WO2023170216A1 (en) | 2022-03-11 | 2023-09-14 | Astrazeneca Ab | A SCORING METHOD FOR AN ANTI-FRα ANTIBODY-DRUG CONJUGATE THERAPY |
WO2023175614A1 (en) | 2022-03-15 | 2023-09-21 | Yeda Research And Development Co. Ltd. | Anti glucocorticoid-induced tnfr-related (gitr) protein antibodies and uses thereof |
TW202346365A (en) | 2022-03-23 | 2023-12-01 | 瑞士商赫孚孟拉羅股份公司 | Combination treatment of an anti-cd20/anti-cd3 bispecific antibody and chemotherapy |
WO2023180511A1 (en) | 2022-03-25 | 2023-09-28 | F. Hoffmann-La Roche Ag | Improved chimeric receptors |
US20230312703A1 (en) | 2022-03-30 | 2023-10-05 | Janssen Biotech, Inc. | Method of Treating Psoriasis with IL-23 Specific Antibody |
WO2023192436A1 (en) | 2022-03-31 | 2023-10-05 | Alexion Pharmaceuticals, Inc. | Singleplex or multiplexed assay for complement markers in fresh biological samples |
WO2023192478A1 (en) | 2022-04-01 | 2023-10-05 | Bristol-Myers Squibb Company | Combination therapy with anti-il-8 antibodies and anti-pd-1 antibodies for treating cancer |
US20230416361A1 (en) | 2022-04-06 | 2023-12-28 | Mirobio Limited | Engineered cd200r antibodies and uses thereof |
GB202205200D0 (en) | 2022-04-08 | 2022-05-25 | Ucb Biopharma Sprl | Combination with chemotherapy |
GB202205203D0 (en) | 2022-04-08 | 2022-05-25 | UCB Biopharma SRL | Combination with inhibitor |
WO2023198727A1 (en) | 2022-04-13 | 2023-10-19 | F. Hoffmann-La Roche Ag | Pharmaceutical compositions of anti-cd20/anti-cd3 bispecific antibodies and methods of use |
TW202400637A (en) | 2022-04-25 | 2024-01-01 | 美商威特拉公司 | Antibody molecules to april and uses thereof |
WO2023209177A1 (en) | 2022-04-29 | 2023-11-02 | Astrazeneca Uk Limited | Sars-cov-2 antibodies and methods of using the same |
WO2023215737A1 (en) | 2022-05-03 | 2023-11-09 | Genentech, Inc. | Anti-ly6e antibodies, immunoconjugates, and uses thereof |
WO2023220695A2 (en) | 2022-05-13 | 2023-11-16 | Voyager Therapeutics, Inc. | Compositions and methods for the treatment of her2 positive cancer |
US20230374122A1 (en) | 2022-05-18 | 2023-11-23 | Janssen Biotech, Inc. | Method for Evaluating and Treating Psoriatic Arthritis with IL23 Antibody |
WO2023235699A1 (en) | 2022-05-31 | 2023-12-07 | Jounce Therapeutics, Inc. | Antibodies to lilrb4 and uses thereof |
WO2023240124A1 (en) | 2022-06-07 | 2023-12-14 | Regeneron Pharmaceuticals, Inc. | Pseudotyped viral particles for targeting tcr-expressing cells |
WO2023240058A2 (en) | 2022-06-07 | 2023-12-14 | Genentech, Inc. | Prognostic and therapeutic methods for cancer |
WO2023239803A1 (en) | 2022-06-08 | 2023-12-14 | Angiex, Inc. | Anti-tm4sf1 antibody-drug conjugates comprising cleavable linkers and methods of using same |
WO2023250402A2 (en) | 2022-06-22 | 2023-12-28 | Antlera Therapeutics Inc. | Tetravalent fzd and wnt co-receptor binding antibody molecules and uses thereof |
EP4296279A1 (en) | 2022-06-23 | 2023-12-27 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Anti-transthyretin (ttr) binding proteins and uses thereof |
WO2024006876A1 (en) | 2022-06-29 | 2024-01-04 | Abbott Laboratories | Magnetic point-of-care systems and assays for determining gfap in biological samples |
WO2024013727A1 (en) | 2022-07-15 | 2024-01-18 | Janssen Biotech, Inc. | Material and methods for improved bioengineered pairing of antigen-binding variable regions |
WO2024015953A1 (en) | 2022-07-15 | 2024-01-18 | Danisco Us Inc. | Methods for producing monoclonal antibodies |
WO2024020407A1 (en) | 2022-07-19 | 2024-01-25 | Staidson Biopharma Inc. | Antibodies specifically recognizing b- and t-lymphocyte attenuator (btla) and uses thereof |
WO2024020564A1 (en) | 2022-07-22 | 2024-01-25 | Genentech, Inc. | Anti-steap1 antigen-binding molecules and uses thereof |
WO2024026471A1 (en) | 2022-07-29 | 2024-02-01 | Alector Llc | Cd98hc antigen-binding domains and uses therefor |
WO2024026472A2 (en) | 2022-07-29 | 2024-02-01 | Alector Llc | Transferrin receptor antigen-binding domains and uses therefor |
WO2024026447A1 (en) | 2022-07-29 | 2024-02-01 | Alector Llc | Anti-gpnmb antibodies and methods of use thereof |
WO2024030829A1 (en) | 2022-08-01 | 2024-02-08 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Monoclonal antibodies that bind to the underside of influenza viral neuraminidase |
WO2024030976A2 (en) | 2022-08-03 | 2024-02-08 | Voyager Therapeutics, Inc. | Compositions and methods for crossing the blood brain barrier |
WO2024050354A1 (en) | 2022-08-31 | 2024-03-07 | Washington University | Alphavirus antigen binding antibodies and uses thereof |
WO2024050524A1 (en) | 2022-09-01 | 2024-03-07 | University Of Georgia Research Foundation, Inc. | Compositions and methods for directing apolipoprotein l1 to induce mammalian cell death |
WO2024049949A1 (en) | 2022-09-01 | 2024-03-07 | Genentech, Inc. | Therapeutic and diagnostic methods for bladder cancer |
WO2024050526A1 (en) | 2022-09-02 | 2024-03-07 | Biomarin Pharmaceutical Inc. | Compositions and methods for treating long qt syndrome |
WO2024054436A1 (en) | 2022-09-06 | 2024-03-14 | Alexion Pharmaceuticals, Inc. | Diagnostic and prognostic biomarker profiles in patients with hematopoietic stem cell transplant-associated thrombotic microangiopathy (hsct-tma) |
WO2024054929A1 (en) | 2022-09-07 | 2024-03-14 | Dynamicure Biotechnology Llc | Anti-vista constructs and uses thereof |
WO2024054822A1 (en) | 2022-09-07 | 2024-03-14 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Engineered sars-cov-2 antibodies with increased neutralization breadth |
WO2024059708A1 (en) | 2022-09-15 | 2024-03-21 | Abbott Laboratories | Biomarkers and methods for differentiating between mild and supermild traumatic brain injury |
WO2024062038A1 (en) | 2022-09-21 | 2024-03-28 | Elthera Ag | Novel binding molecules binding to l1cam |
Citations (35)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3853833A (en) * | 1971-04-27 | 1974-12-10 | Hormone Res Foundation | Synthetic human growth-promoting and lactogenic hormones and method of producing same |
US3853832A (en) * | 1971-04-27 | 1974-12-10 | Harmone Res Foundation | Synthetic human pituitary growth hormone and method of producing it |
US4446235A (en) * | 1982-03-22 | 1984-05-01 | Genentech, Inc. | Method for cloning human growth hormone varient genes |
US4593002A (en) * | 1982-01-11 | 1986-06-03 | Salk Institute Biotechnology/Industrial Associates, Inc. | Viruses with recombinant surface proteins |
US4655160A (en) * | 1985-09-10 | 1987-04-07 | David R. Ligh | Deck box |
US4670393A (en) * | 1982-03-22 | 1987-06-02 | Genentech, Inc. | DNA vectors encoding a novel human growth hormone-variant protein |
US4673641A (en) * | 1982-12-16 | 1987-06-16 | Molecular Genetics Research And Development Limited Partnership | Co-aggregate purification of proteins |
US4699897A (en) * | 1983-06-04 | 1987-10-13 | Amgen | Biologically active peptides structurally related to regions within growth hormones |
US4880910A (en) * | 1981-09-18 | 1989-11-14 | Genentech, Inc. | Terminal methionyl bovine growth hormone and its use |
US4888286A (en) * | 1984-02-06 | 1989-12-19 | Creative Biomolecules, Inc. | Production of gene and protein analogs through synthetic gene design using double stranded synthetic oligonucleotides |
US5013653A (en) * | 1987-03-20 | 1991-05-07 | Creative Biomolecules, Inc. | Product and process for introduction of a hinge region into a fusion protein to facilitate cleavage |
US5047333A (en) * | 1987-12-22 | 1991-09-10 | Eniricerche S.P.A. | Method for the preparation of natural human growth hormone in pure form |
US5223409A (en) * | 1988-09-02 | 1993-06-29 | Protein Engineering Corp. | Directed evolution of novel binding proteins |
US5350836A (en) * | 1989-10-12 | 1994-09-27 | Ohio University | Growth hormone antagonists |
US5427908A (en) * | 1990-05-01 | 1995-06-27 | Affymax Technologies N.V. | Recombinant library screening methods |
US5432018A (en) * | 1990-06-20 | 1995-07-11 | Affymax Technologies N.V. | Peptide library and screening systems |
US5498538A (en) * | 1990-02-15 | 1996-03-12 | The University Of North Carolina At Chapel Hill | Totally synthetic affinity reagents |
US5514548A (en) * | 1993-02-17 | 1996-05-07 | Morphosys Gesellschaft Fur Proteinoptimerung Mbh | Method for in vivo selection of ligand-binding proteins |
US5516637A (en) * | 1994-06-10 | 1996-05-14 | Dade International Inc. | Method involving display of protein binding pairs on the surface of bacterial pili and bacteriophage |
US5534617A (en) * | 1988-10-28 | 1996-07-09 | Genentech, Inc. | Human growth hormone variants having greater affinity for human growth hormone receptor at site 1 |
US5622699A (en) * | 1995-09-11 | 1997-04-22 | La Jolla Cancer Research Foundation | Method of identifying molecules that home to a selected organ in vivo |
US5627024A (en) * | 1994-08-05 | 1997-05-06 | The Scripps Research Institute | Lambdoid bacteriophage vectors for expression and display of foreign proteins |
US5658727A (en) * | 1991-04-10 | 1997-08-19 | The Scripps Research Institute | Heterodimeric receptor libraries using phagemids |
US5663143A (en) * | 1988-09-02 | 1997-09-02 | Dyax Corp. | Engineered human-derived kunitz domains that inhibit human neutrophil elastase |
US5688666A (en) * | 1988-10-28 | 1997-11-18 | Genentech, Inc. | Growth hormone variants with altered binding properties |
US5702892A (en) * | 1995-05-09 | 1997-12-30 | The United States Of America As Represented By The Department Of Health And Human Services | Phage-display of immunoglobulin heavy chain libraries |
US5712089A (en) * | 1993-12-06 | 1998-01-27 | Bioinvent International Ab | Method of selecting specific bacteriophages |
US5733743A (en) * | 1992-03-24 | 1998-03-31 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
US5747334A (en) * | 1990-02-15 | 1998-05-05 | The University Of North Carolina At Chapel Hill | Random peptide library |
US5750373A (en) * | 1990-12-03 | 1998-05-12 | Genentech, Inc. | Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants |
US5770356A (en) * | 1992-09-04 | 1998-06-23 | The Scripps Research Institute | Phagemids coexpressing a surface receptor and a surface heterologous protein |
US5770434A (en) * | 1990-09-28 | 1998-06-23 | Ixsys Incorporated | Soluble peptides having constrained, secondary conformation in solution and method of making same |
US5780279A (en) * | 1990-12-03 | 1998-07-14 | Genentech, Inc. | Method of selection of proteolytic cleavage sites by directed evolution and phagemid display |
US5811093A (en) * | 1994-04-05 | 1998-09-22 | Exponential Biotherapies, Inc. | Bacteriophage genotypically modified to delay inactivations by the host defense system |
US5955341A (en) * | 1991-04-10 | 1999-09-21 | The Scripps Research Institute | Heterodimeric receptor libraries using phagemids |
Family Cites Families (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA1340288C (en) * | 1988-09-02 | 1998-12-29 | Robert Charles Ladner | Generation and selection of novel binding proteins |
WO1990004788A1 (en) * | 1988-10-28 | 1990-05-03 | Genentech, Inc. | Method for identifying active domains and amino acid residues in polypeptides and hormone variants |
GB9015198D0 (en) * | 1990-07-10 | 1990-08-29 | Brien Caroline J O | Binding substance |
EP0760012A4 (en) * | 1994-06-10 | 1997-07-02 | Symbiotech Inc | Method of detecting compounds utilizing genetically modified lambdoid bacteriophage |
GB9500851D0 (en) * | 1995-01-17 | 1995-03-08 | Bionvent International Ab | Method of selecting specific bacteriophages |
CN1196094A (en) * | 1995-09-07 | 1998-10-14 | 诺沃挪第克公司 | Phage display for detergent enzyme activity |
DE69731084T2 (en) * | 1996-03-20 | 2006-02-23 | Dyax Corp., Cambridge | CLEANING OF TISSUE PLASMINOGENACTIVATOR (tPA) |
US6190856B1 (en) * | 1996-05-22 | 2001-02-20 | The Johns Hopkins University | Methods of detection utilizing modified bacteriophage |
JP2000512981A (en) * | 1996-06-06 | 2000-10-03 | ラ ホヤ ファーマシューティカル カンパニー | aPL immunoreactive peptide, conjugate thereof and method of treatment for aPL antibody-mediated pathology |
DE69731226T2 (en) * | 1996-06-10 | 2006-03-09 | The Scripps Research Institute, La Jolla | USE OF SUBSTRATE SUBTRACTION LIBRARIES FOR THE DISTINCTION OF ENZYME SPECIFICATIONS |
AU3737297A (en) * | 1996-08-05 | 1998-02-25 | Brigham And Women's Hospital | Bacteriophage-mediated gene therapy |
EP0934526B1 (en) * | 1996-10-08 | 2003-01-08 | U-BISys B.V. | Methods and means for selecting peptides and proteins having specific affinity for a target |
-
1991
- 1991-12-03 DE DE69129154T patent/DE69129154T2/en not_active Expired - Lifetime
- 1991-12-03 CA CA002095633A patent/CA2095633C/en not_active Expired - Lifetime
- 1991-12-03 WO PCT/US1991/009133 patent/WO1992009690A2/en active IP Right Grant
- 1991-12-03 AT AT92902109T patent/ATE164395T1/en not_active IP Right Cessation
- 1991-12-03 ES ES92902109T patent/ES2113940T3/en not_active Expired - Lifetime
- 1991-12-03 US US08/050,058 patent/US5750373A/en not_active Expired - Lifetime
- 1991-12-03 CA CA002405246A patent/CA2405246A1/en not_active Abandoned
- 1991-12-03 EP EP92902109A patent/EP0564531B1/en not_active Expired - Lifetime
- 1991-12-03 DK DK92902109T patent/DK0564531T3/en active
-
1995
- 1995-06-05 US US08/463,587 patent/US5821047A/en not_active Expired - Lifetime
- 1995-06-05 US US08/463,667 patent/US5834598A/en not_active Expired - Lifetime
-
1997
- 1997-09-03 US US08/923,854 patent/US6040136A/en not_active Expired - Fee Related
-
1998
- 1998-03-27 GR GR980400652T patent/GR3026468T3/en unknown
-
2005
- 2005-08-08 US US11/199,062 patent/US20060115874A1/en not_active Abandoned
-
2007
- 2007-06-11 US US11/761,180 patent/US20080038717A1/en not_active Abandoned
-
2009
- 2009-07-24 US US12/508,859 patent/US20100035236A1/en not_active Abandoned
Patent Citations (41)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3853832A (en) * | 1971-04-27 | 1974-12-10 | Harmone Res Foundation | Synthetic human pituitary growth hormone and method of producing it |
US3853833A (en) * | 1971-04-27 | 1974-12-10 | Hormone Res Foundation | Synthetic human growth-promoting and lactogenic hormones and method of producing same |
US4880910A (en) * | 1981-09-18 | 1989-11-14 | Genentech, Inc. | Terminal methionyl bovine growth hormone and its use |
US4593002A (en) * | 1982-01-11 | 1986-06-03 | Salk Institute Biotechnology/Industrial Associates, Inc. | Viruses with recombinant surface proteins |
US4446235A (en) * | 1982-03-22 | 1984-05-01 | Genentech, Inc. | Method for cloning human growth hormone varient genes |
US4670393A (en) * | 1982-03-22 | 1987-06-02 | Genentech, Inc. | DNA vectors encoding a novel human growth hormone-variant protein |
US4673641A (en) * | 1982-12-16 | 1987-06-16 | Molecular Genetics Research And Development Limited Partnership | Co-aggregate purification of proteins |
US4699897A (en) * | 1983-06-04 | 1987-10-13 | Amgen | Biologically active peptides structurally related to regions within growth hormones |
US4888286A (en) * | 1984-02-06 | 1989-12-19 | Creative Biomolecules, Inc. | Production of gene and protein analogs through synthetic gene design using double stranded synthetic oligonucleotides |
US4655160A (en) * | 1985-09-10 | 1987-04-07 | David R. Ligh | Deck box |
US5013653A (en) * | 1987-03-20 | 1991-05-07 | Creative Biomolecules, Inc. | Product and process for introduction of a hinge region into a fusion protein to facilitate cleavage |
US5047333A (en) * | 1987-12-22 | 1991-09-10 | Eniricerche S.P.A. | Method for the preparation of natural human growth hormone in pure form |
US5223409A (en) * | 1988-09-02 | 1993-06-29 | Protein Engineering Corp. | Directed evolution of novel binding proteins |
US5663143A (en) * | 1988-09-02 | 1997-09-02 | Dyax Corp. | Engineered human-derived kunitz domains that inhibit human neutrophil elastase |
US5403484A (en) * | 1988-09-02 | 1995-04-04 | Protein Engineering Corporation | Viruses expressing chimeric binding proteins |
US5571698A (en) * | 1988-09-02 | 1996-11-05 | Protein Engineering Corporation | Directed evolution of novel binding proteins |
US5534617A (en) * | 1988-10-28 | 1996-07-09 | Genentech, Inc. | Human growth hormone variants having greater affinity for human growth hormone receptor at site 1 |
US5688666A (en) * | 1988-10-28 | 1997-11-18 | Genentech, Inc. | Growth hormone variants with altered binding properties |
US5350836A (en) * | 1989-10-12 | 1994-09-27 | Ohio University | Growth hormone antagonists |
US5747334A (en) * | 1990-02-15 | 1998-05-05 | The University Of North Carolina At Chapel Hill | Random peptide library |
US5498538A (en) * | 1990-02-15 | 1996-03-12 | The University Of North Carolina At Chapel Hill | Totally synthetic affinity reagents |
US5427908A (en) * | 1990-05-01 | 1995-06-27 | Affymax Technologies N.V. | Recombinant library screening methods |
US5580717A (en) * | 1990-05-01 | 1996-12-03 | Affymax Technologies N.V. | Recombinant library screening methods |
US5723286A (en) * | 1990-06-20 | 1998-03-03 | Affymax Technologies N.V. | Peptide library and screening systems |
US5432018A (en) * | 1990-06-20 | 1995-07-11 | Affymax Technologies N.V. | Peptide library and screening systems |
US5770434A (en) * | 1990-09-28 | 1998-06-23 | Ixsys Incorporated | Soluble peptides having constrained, secondary conformation in solution and method of making same |
US6040136A (en) * | 1990-12-03 | 2000-03-21 | Genentech, Inc. | Enrichment method for variant proteins with altered binding properties |
US5750373A (en) * | 1990-12-03 | 1998-05-12 | Genentech, Inc. | Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants |
US5780279A (en) * | 1990-12-03 | 1998-07-14 | Genentech, Inc. | Method of selection of proteolytic cleavage sites by directed evolution and phagemid display |
US5658727A (en) * | 1991-04-10 | 1997-08-19 | The Scripps Research Institute | Heterodimeric receptor libraries using phagemids |
US5955341A (en) * | 1991-04-10 | 1999-09-21 | The Scripps Research Institute | Heterodimeric receptor libraries using phagemids |
US5759817A (en) * | 1991-04-10 | 1998-06-02 | The Scripps Research Institute | Heterodimeric receptor libraries using phagemids |
US5733743A (en) * | 1992-03-24 | 1998-03-31 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
US5770356A (en) * | 1992-09-04 | 1998-06-23 | The Scripps Research Institute | Phagemids coexpressing a surface receptor and a surface heterologous protein |
US5514548A (en) * | 1993-02-17 | 1996-05-07 | Morphosys Gesellschaft Fur Proteinoptimerung Mbh | Method for in vivo selection of ligand-binding proteins |
US5712089A (en) * | 1993-12-06 | 1998-01-27 | Bioinvent International Ab | Method of selecting specific bacteriophages |
US5811093A (en) * | 1994-04-05 | 1998-09-22 | Exponential Biotherapies, Inc. | Bacteriophage genotypically modified to delay inactivations by the host defense system |
US5516637A (en) * | 1994-06-10 | 1996-05-14 | Dade International Inc. | Method involving display of protein binding pairs on the surface of bacterial pili and bacteriophage |
US5627024A (en) * | 1994-08-05 | 1997-05-06 | The Scripps Research Institute | Lambdoid bacteriophage vectors for expression and display of foreign proteins |
US5702892A (en) * | 1995-05-09 | 1997-12-30 | The United States Of America As Represented By The Department Of Health And Human Services | Phage-display of immunoglobulin heavy chain libraries |
US5622699A (en) * | 1995-09-11 | 1997-04-22 | La Jolla Cancer Research Foundation | Method of identifying molecules that home to a selected organ in vivo |
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US9067988B2 (en) | 2010-12-01 | 2015-06-30 | Alderbio Holdings Llc | Methods of preventing or treating pain using anti-NGF antibodies |
US9078878B2 (en) | 2010-12-01 | 2015-07-14 | Alderbio Holdings Llc | Anti-NGF antibodies that selectively inhibit the association of NGF with TrkA, without affecting the association of NGF with p75 |
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Also Published As
Publication number | Publication date |
---|---|
GR3026468T3 (en) | 1998-06-30 |
US20060115874A1 (en) | 2006-06-01 |
US20080038717A1 (en) | 2008-02-14 |
WO1992009690A3 (en) | 1992-12-10 |
CA2405246A1 (en) | 1992-06-11 |
US5821047A (en) | 1998-10-13 |
ES2113940T3 (en) | 1998-05-16 |
US5750373A (en) | 1998-05-12 |
CA2095633C (en) | 2003-02-04 |
WO1992009690A2 (en) | 1992-06-11 |
DK0564531T3 (en) | 1998-09-28 |
CA2095633A1 (en) | 1992-06-04 |
US5834598A (en) | 1998-11-10 |
EP0564531A1 (en) | 1993-10-13 |
DE69129154D1 (en) | 1998-04-30 |
DE69129154T2 (en) | 1998-08-20 |
ATE164395T1 (en) | 1998-04-15 |
EP0564531B1 (en) | 1998-03-25 |
US6040136A (en) | 2000-03-21 |
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