WO2002044197A2 - Peptides de fixation des recepteurs des cytokines - Google Patents

Peptides de fixation des recepteurs des cytokines Download PDF

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Publication number
WO2002044197A2
WO2002044197A2 PCT/CA2001/001701 CA0101701W WO0244197A2 WO 2002044197 A2 WO2002044197 A2 WO 2002044197A2 CA 0101701 W CA0101701 W CA 0101701W WO 0244197 A2 WO0244197 A2 WO 0244197A2
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ifn
receptor binding
interferon
leu
seq
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PCT/CA2001/001701
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WO2002044197A3 (fr
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Eleanor N. Fish
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Fish Eleanor N
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates generally to cytokine receptor binding peptide constructs comprising at least one cytokine receptor binding domain inco ⁇ orated into a molecular scaffold that maintains the binding domain in a configuration suitable for binding the cytokine receptor and to nucleic acid sequences encoding the same. More particularly, the invention relates to interferon receptor binding peptide constructs. In another embodiment, the invention relates to the general field of cytokine mimetics and more particularly to an interferon mimetic. Background of the Invention
  • Cytokines are small, secreted proteins, produced by cells in response to different stimuli. They exert widespread effects on the immune system and target tissues and organs. Cytokines are usually not stored, but quickly made and released in response to stimuli. They bind to their specific cognate receptors. Cytokines are essential for immunoregulation, both positive amplification of the immune response and negative immunosuppression to keep the response from getting out of control.
  • cytokines regulate the proliferation and differentiation of target cells, exhibit anti-viral and anti-tumor activities and immune modulating properties.
  • cytokines are in clinical use in chemotherapies against solid tumors and hematological malignancies and viral infections [1-9].
  • cytokines or cytokine antagonists have therapeutic potential in autoimmune diseases, e.g. interferon (IFN-beta) in multiple sclerosis (M.S.) [10].
  • IFN-beta interferon
  • M.S. multiple sclerosis
  • IFNs interferons
  • Type I IFNs include IFN-alpha, -beta and -omega (IFN- ⁇ , - ⁇ , and - ⁇ ) and Type II IFN or IFN- gamma (IFN- ⁇ ) [reviewed in 12].
  • Type I IFNs mediate diverse biological effects including the largely cell type-independent antiviral and anti-proliferative responses, and several cell type-restricted responses of immunological relevance such as the activation of natural killer cells, the regulation of CD8+ memory T-cells and the stimulation of hematopoietic cell differentiation [13-16], These phenotypic changes are thought to be the cumulative consequence of changes in the activity of certain proteins that are products of Type I IFN-stimulated genes (ISGs) [17]. As a result, much effort has been focused on trying to understand the mechanism of Type I IFN signal transduction and regulation of gene expression.
  • IFN receptors are ubiquitous and more specifically, are up-regulated in metabolically active cells such as cancer cells and infected tissues.
  • the present inventor also determined that peptides comprising portions of these domains also possess biological activity (ability to bind to the IFN receptor) such as those comprising amino acid residues 29-35, 78- 95, 123-140 or 130-140. Further the present inventor identified seven preferred interferon receptor binding peptides which have an amino acid sequence selected from the group consisting of: CYS-LEU-LYS-ASP-ARG-HIS-ASP (SEQ. ID NO. 1); ASP-GLU-SER-LEU-LEU-GLU-LYS-PHE-TYR-THR-GLU-LEU-TYR-GLN-GLN- LEU-ASN-ASP (SEQ. ID NO.
  • ASN-GLU-THR-ILE-VAL-GLU-ASN-LEU-LEU -ALA-ASN-VAL-TYR-HIS-GLN-ILE-ASN-HIS SEQ. ID NO. 3
  • TYR-LEU-THR- GLU-LYS-LYS-TYR-SER-PRO-CYS-ALA SEQ. ID NO. 4
  • TYR-PHE-GLN- ARG-ILE-THR-LEU-TYR-LEU-THR-GLU-LYS-LYS-TYR-SER-PRO-CYS-ALA SEQ. ID NO. 5
  • TYR-PHE-GLN-ARG-ILE-THR-LEU-TYR SEQ. ID NO. 6
  • GLU-LEU-TYR-GLN-GLN-LEU-ASN-ASP SEQ. ID NO. 7
  • cytokines It is not unusual for cytokines to have more than one receptor interactive domain. All cytokines exert their functional effects through cell surface expressed receptor complexes. These are comprised of transmembrane subunit components. Generally, cytokine receptor systems associated with helix bundle cytokines consist of at least 2 types of subunits. For a number of different cytokines, separate sites for recognition of he different receptor subunits have been mapped in the cytokines. For IL-5 [21], GM-CSF [22], and growth hormone [23], the presence of distinct receptor interaction sites for the different receptor subunits is consistent with a sequential receptor-subunit-binding scheme. The cytokine initially binds with low affinity to one receptor subunit, followed by the interaction with the second subunit, thereby creating a high affinity receptor complex that mediates signal transduction. Structure of Cytokines
  • cytokines including IFNs are constructed from a common helix bundle structural framework. Rapid advances have been made in relating the structure and function of a growing number of four-helix bundle cytokines.
  • IL-5 and GM-CSF are closely related four-helix bundle growth factor proteins.
  • GM-CSF has a core structure composed of a left-handed, antiparallel four- helix bundle with up-up-down-down connectivity similar to the fold first described for growth hormone [24].
  • IL-5 comprises a core of two four-helix bundles, each of which is composed of a D helix from one polypeptide chain of the IL-5 dimer and three helices from the second chain [25].
  • the helix bundle motifs in IL-5 are similar to those of growth hormone, GM-CSF and, indeed, to those of many other cytokines including IL-2, IL-3, IL-4, M-CSF and G-CSF.
  • a similar fold can be predicted for other cytokines, including Epo, IL-6, IL-7, IL-10, IL- 12 and prolactin, based on gene structure and homology modelling.
  • all known four-helix bundle cytokines contain common ⁇ -sheet elements formed by the strand connecting the A and B helices and the strand connecting helices C and D [reviewed in 26].
  • the helical bundle structure of IFN conforms with other cytokines. Structure of Immunoglobulins
  • Immunoglobulins in general are comprised of two polypeptide chains of different lengths, the light (L) and heavy (H) chains. Two of each of these chains associate to form one antibody molecule.
  • the functions of an antibody can be assigned to structural domains, termed the Fab (antigen-binding fragment) and Fc (constant or crystallizable fragment) [27].
  • the Fab is responsible for molecule- specific functions, such as antigen recognition, while the Fc carries out the activities of antibodies in general, such as complement activation and receptor binding.
  • the crystal structures of a number of Fabs revealed a characteristic protein folding pattern which consisted of two anti-parallel-sheets tied together by an internal disulphide bond [28]. Two of these 'immunoglobulin fold' units associate together non-covalently to form a double-cylindrical barrel structure.
  • the hypervariable stretches described above occur in inter-strand loops, all at one end of the barrel. Thus, although distant in the primary sequences ofthe H and L chains, all six of these stretches come together at one end ofthe antibody to form a single binding surface. As the presence of haptens in the structures identified this surface as the antigen- binding site, the stretches became known as complementarity-determining regions (CDRs).
  • the first two CDRs of each of the heavy and light chains form the periphery of the site, establishing the general features of the shape.
  • the details, and usually most of the specificity determining interactions, are provided by the third CDRs L3 and, especially, H3). These loops usually form the center of the binding site. Shorter H3 and L3 loops tend to fold down, creating shallow or flat sites, while longer loops usually result in deeper, smaller pockets.
  • the primary advantages of this approach has been the relatively small size of the specific binding molecule compared to an intact antibody and, secondly, the potential to over express large amounts of protein product without the use of animals.
  • the antibody domain genes can be expressed fused to the genes encoding surface proteins of bacteriophage. If a large library of such engineered phage is assembled, an Fv of desired specificity can be isolated in most instances by a selection process. The affinity can be improved further by an affinity maturation process [30]. Libraries of such phage with a large number of different Fv fusions are now available and have been used successfully in antibody evolution experiments. Thus, the production of large amounts of high affinity Fv products with a desired specificity is possible. Indeed, with regard to the scFv described below, this recombinant product may be expresed in relatively large amounts and reproducibly folds appropriately [31]. Summary of the Invention
  • the inventors have determined that the presence of multiple interferon binding domains in the interferon molecule and, the conservation of the highly structured helical bundle configuration amongst the cytokines, including IFNs, that potentially represents a stable structure that 'presents' the receptor interactive domains of the different cytokines, enable their interaction with the corresponding receptor contact site(s).
  • the present inventors have determined that the configuration ofthe interferon receptor binding domains, or critical parts thereof, in the native IFN and the subsequent interaction with the IFN receptor, is important in effecting biological outcomes or responses and thus is important to the design of an IFN mimetic.
  • the present inventors have determined that the ability of the recombinant scFv molecules to be reproducibly made in relatively large amounts in a predictable configuration, make them excellent candidates for scaffolds onto which peptide interactive domains of cytokines may be inserted.
  • the present invention provides a cytokine receptor binding peptide construct comprising at least one cytokine receptor binding domain inco ⁇ orated in a suitable molecular scaffold such that the scaffold maintains the binding domain in a configuration suitable for binding to the cytokine receptor.
  • scaffold is defined as any structure that can be manipulated for the pu ⁇ ose of optimizing the spatial positioning of inserted peptides.
  • Suitable scaffolds for use in the present invention may include, but are not limited to polypeptides, proteins, carbohydrates, glycoproteins, synthetic and natural polymers, antibody or any other molecule or substance that may be apparent to one skilled in the art.
  • the scaffold is a scFv molecule, most preferably a C219scFv molecule.
  • the present inventors have now found that the binding domains of cytokines can be inco ⁇ orated into scFv peptide molecules (as the molecular scaffold) in a stable and reproducible configuration.
  • These smaller hybrid molecules that contain the effective portions ofthe cytokine i.e. cytokine receptor binding domains
  • the present invention provides a novel cytokine receptor binding peptide construct comprising at least one cytokine receptor binding domain inco ⁇ orated into an scFv peptide molecule in a manner that maintains the binding domain in a configuration suitable for binding the cytokine receptor.
  • the cytokine peptide comprising the cytokine receptor binding domain is inco ⁇ orated into the scFv peptide at one of the complimentarity determining regions (CDRs, i.e., CDR1, CDR2 or CDR3).
  • CDRs complimentarity determining regions
  • the cytokine is interferon, preferably a Type I human interferon.
  • the present invention provides the insertion of one or more interferon peptides comprising an interferon receptor binding domain into strategic locations along a scaffold, facilitating interaction of one or more of said peptides with the corresponding active site of the Type 1 human interferon receptor.
  • one of the said interferon peptides is inserted at one of the CDR sites of scFv, thereby altering the respective CDR of scFv.
  • the interferon peptides inco ⁇ orated into the suitable scaffold comprise at least an interferon receptor binding domain selected from the group of IFN peptides comprising the IFN amino acid residues: 10- 35 (interferon receptor recognition site I), 78-107 (interferon receptor recognition site 2) and 123- 166 (interferon recognition site III) or IFN receptor binding portions thereof.
  • interferon peptides inco ⁇ orated into the scaffold of the invention comprise at least one of the following interferon amino acid residue domains: amino acid residues 29-35 (IFN receptor recognition peptide I (IRRPl)), 78- 95 (IFN receptor recognition peptide II (IRRP2)), a 79-96 or 89-95 (both from IFN recognition site II), 123-140 (interferon receptor recognition peptide III (IRRP3) 130- 140 (from IFN recognition site III), 123-156 (from IFN recognition site III) or 123- 129 or 129-140 (from IFN recognition site III).
  • IFN receptor recognition peptide I IRRPl
  • 78- 95 IFN receptor recognition peptide II
  • IRRP3 interferon receptor recognition peptide III
  • IRRP3 interferon receptor recognition peptide III
  • the interferon receptor binding peptides inco ⁇ orated into the scaffold comprise the amino acid sequence selected from the group consisting of: CYS-LEU-LYS-ASP-ARG-HIS-ASP (SEQ. ID NO. 1); ASP-GLU-SER-LEU-LEU-GLU-LYS-PHE-TYR-THR-GLU- LEU-TYR-GLN-GLN-LEU-ASN-ASP (SEQ. ID NO. 2); ASN-GLU-THR-ILE- VAL-GLU-ASN-LEU-LEU-ALA-ASN-VAL-TYR-HIS-GLN-ILE-ASN-HIS (SEQ. ID NO.
  • TYR-LEU-THR-GLU-LYS-LYS-TYR-SER-PRO-CYS-ALA SEQ. ID NO. 4
  • TYR-PHE-GLN-ARG-ILE-THR-LEU-TYR-LEU-THR-GLU-LYS-LYS- TYR-SER-PRO-CYS-ALA SEQ. ID NO. 5
  • TYR-PHE-GLN-ARG-ILE-THR-LEU- TYR SEQ. ID NO. 6
  • GLU-LEU-TYR-GLN-GLN-LEU-ASN-ASP SEQ. ID NO.
  • the interferon receptor binding peptide construct of the invention comprises at least 2 and preferably 3 IFN receptor binding domains, one from each ofthe three IFN receptor recognition sites or IFN receptor binding portions thereof and each replacing a different CDR in the scFv scaffold preferably in a configuration which enables the IFN receptor binding domains to interact with their corresponding active sites in the interferon receptor.
  • the IFN receptor recognition sites or binding portions thereof are inserted in the same chain of the scFv scaffold, in another embodiment, they are not all inserted on the same chain.
  • a Type I human IFN receptor binding peptide of domain 1 is inco ⁇ orated at CDR3 and alters CDR3
  • Type I human IFN receptor binding peptide of domain 2 is inco ⁇ orated at CDR2 and alters CDR2
  • Type II human IFN receptor binding peptide of domain 3 is inco ⁇ orated at CDR1 and alters CDR1.
  • only one or two IFN receptor recognition sites or binding portions thereof are inco ⁇ orated into the scaffold.
  • the invention provides an IFN receptor binding peptide construct comprising the amino acid sequence of SEQ. ID No. 22 (pIFNscFv - C219 scaffold with inserted interferon peptides SEQ. ID. NO. 1 at CDR1 of the variable light chain of scFv and SEQ. ID. NO. 21 at CDR3 of variable light chain of scFv.
  • the construct has the amino acid sequence encoded by SEQ.ID.NO.29 or a different nucleic acid sequence coding for the same amino acid sequence due to the degeneracy ofthe genetic code.
  • a Type I human IFN receptor binding peptide of domain 1 is inco ⁇ orated at CDR1, preferably the VH chain of scFv, and alters CDR1;
  • Type I human IFN receptor binding peptide of domain 2 is inco ⁇ orated at CDR2 and alters CDR2, preferably in the VL chain of scFv, and
  • Type I human IFN receptor binding peptide of domain 3 is inco ⁇ orated at CDR3 and alters CDR3, preferably in the VH chain of scFv.
  • three IFN receptor recognition sites or binding portions thereof are inco ⁇ orated into the scaffold.
  • the invention provides an IFN receptor binding peptide construct encoded by SEQ. ID. NO. 32 or a different nucleotide sequence encoding for the same amino acid sequence construct as that encoded for by SEQ. ID. NO. 32 due to the degeneracy of the genetic code.
  • the IFN receptor binding peptide construct has the amino acid sequence of SEQ. ID. NO. 33.
  • the invention provides for nucleic acid molecules encoding the cytokine, preferably IFN, receptor binding peptides of the invention.
  • the invention provides suitable expression vectors which inco ⁇ orate the nucleic acid molecules of the invention.
  • the expression vector is plasmid, pSJF2.
  • the invention provides a nucleic acid sequence encoding an interferon receptor binding peptide construct of the invention and preferably comprising the nucleic acid sequence of SEQ. ID. NO.29 or SEQ. ID. NO. 32.
  • An embodiment of this aspect of the invention provides the protein expressed from the plasmid of the nucleic acid sequence provided in SEQ. ID NO. 29 or SEQ. ID. NO. 32.
  • the invention provides for a cell genetically modified, either through transformation, transfection or other means to express the IFNscFv , preferably a partial (less than three IFN receptor recognition sites or receptor binding portions thereof) or complete (three IFN receptor recognition sites or binding protions thereof) If scFv, protein ofthe invention.
  • the invention provides for a pharmaceutical composition comprising one of the peptides of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises an interferon receptor binding peptide construct ofthe invention and a pharmaceutically acceptable carrier.
  • the invention provides a peptide that mimics the effect of IFN and which can be used in medical therapies for cancers, hematological malignancies, viral infections and autoimmune diseases.
  • the invention provides a peptide that can be used to detect modulators of IFN action.
  • the IFN mimetics of the invention can be used in screening assays to compare the activity and/or interaction with another molecule or potential IFN modulator. This can be done by observing the effect ofthe modulator in the presence of the peptide mimetic of the invention and its substrate (e.g. the IFN receptor).
  • the peptides of the invention can also be used in the diagnosis of IFN activity related disorders, by comparing the effect of the peptide or modulators of the peptide to the invention on IFN activity in a person with the disorder.
  • the interferon receptor binding peptides and peptide constructs of the invention can be used as a carrier for other therapeutic agents.
  • another therapeutic agent could be attached to the scaffold, to provide a bolus of, for example, interferon and another chemotherapeutic agent, or IFN and ribavirin, or IFN and an anti-inflammatory agent.
  • FIG. 1 illustrates the growth inhibitory activities of variant IFN-alphas in T98G cells.
  • FIG. 2 shows five charts illustrating receptor binding characteristics of variant IFN-alphas on T98G cells.
  • FIG. 3 shows four charts illustrating receptor binding characteristics of variant IFN-alphas on T98G cells.
  • FIG. 4 shows secondary structure characteristics of different IFN-alpha species according to amino acid sequence analyses.
  • FIG. 5 is a representation of a model for the tertiary structure of Type 1 IFNs.
  • FIG. 6A is a Ni column purification of IFNscfv from concentrated periplasmic extract dialysed against lOmM Tris pH 8 buffer containing 100 mM NaCl.
  • FIG. 6B is a Western blot showing the size ofthe IFNscfv
  • FIG. 7 is a silver stain of Q-column purified protein from Ni column pooled fractions dialysed against 50mM Tris pH 8.
  • FIG. 8A is a consensus interferon alpha carbon trace, wherein the interferon receptor binding regions are highlighted.
  • FIG 8B is a consensus C219scFv carbon trace, wherein the CDRs in variable light chain regions are highlighted.
  • FIG. 9A is a bar graph illustrating the effect of pIFNscFv on Daudi cell growth. The data semonstrate that addition of C219scFv alone to Daudi cells does not affect their growth. However, addition of the partial mimetic (pIFNscFv) appears to inhibit the growth-regulatory effects of endogenously produced IFN in Daudi cells.
  • FIG. 9 B is a bar groaph illustrating that pIFNscFv can partially rescue Daudi cells from the growth inhibitory effects of IFN-Con. The data demonstrate that at high doses of IFN, that exhibit maximal growth inhibitory activity, competition with the partial mimetic results in some ablation of the IFN-induced growth inhibitory effects at the highest doses of competitor mimetic.
  • FIG. 10 shows the nucleotide sequence that were removed from the C219scFv cDNA and IFN nucleotide sequences that were placed in the CDRs.
  • FIG.H Plasmid map for the cIFNscFv construct in pSJF2 vector.
  • the construct was directionally cloned from BsiWI to BanHI.
  • the unique restriction sites introduced into the construct are indicated. Numbers beside the restriction sites refer to nucleotide numbers within the construct.
  • FIGJ2 Anti-c-Myc Western immunoblot (12% SDS-PAGE) of whole cell lysates to screen for presence of the c-Myc tagges cIFNscFv mimetic.
  • An IPTG inducible band which corresponds to the expected molecular weight ofthe cIFNscFv mimetic, is present in lane 4.
  • the produce is about 37 kDa. Two bands are seen on the immunoblot. The higher band corresponds to unprocessed protein i.e., signal sequence is still present.
  • FIGJ3 Nucleic acid and amino acid sequence of cIFNscFv
  • FIG. 14 IFN- ⁇ primary amino acid sequences and sequence alignment.
  • FIG. 15 Nucleic and amino acid sequence of C219scFv.
  • SEQ. ID. NO 18 is from nucleic acid sequence 76 - 831).
  • SEQ. ID. NO. 19 is from amino acid sequence 26-277.
  • the present invention is directed to cytokine receptor binding peptide constructs, wherein the receptor binding domain ofthe cytokine is inco ⁇ orated into a scaffold such that the scaffold maintains the binding domain in a configuration suitable for binding to the cytokine receptor.
  • the scaffold is preferably an inert (preferably immunologically inert) biological scaffold that preferably optimizes the spatial positioning of inserted peptides.
  • Suitable scaffolds for use in the present invention may include, but are not limited to polypeptides, proteins, carbohydrates, glycoproteins, synthetic and natural polymers, or any other molecule or substence that may be apparent to one skilled in the art.
  • the scaffold is an immunoglobulin or fragment thereof.
  • the scaffold comprises the variable antigen binding domain of an immunoglobulin.
  • the scaffold is a single chain Fv peptide (scFv), such as C219scFv.
  • the scaffold is preferably easily and predictably reproducible, with a predictable spatial configuration.
  • Bioly active proteins have an optimum active configuration that is composed of discrete and unique strategic domains along the polypeptide. These critical structural domains determine such parameters as receptor binding and effector functions. Characterization of these strategic domains, that includes defining their spatial configuration and effector functions, clarify the sequence of events comprising and initiated by receptor binding and that lead to specific biological responses. Defining the spatial configuration of the strategic domains can help in designing peptides which may mimic all or some of the functions of the biologically active proteins.
  • a receptor may be either activated by a ligand/agonist or inhibited/desensitized by a receptor antagonist, resulting in a variety of biological effects.
  • the peptides of the present invention are inco ⁇ orated into a biological scaffold. Their position within the scaffold will determine the degree of interaction with the receptor active sites, resulting in various degrees of stimulation.
  • a therapeutic agent For a therapeutic agent to be optimally active, it must be delivered to the specific site of action intact and must interact with the target tissues.
  • the cells express abundant Type I IFN receptors, that is, IFN-alpha/beta receptor expression at the cell surface is upregulated.
  • A Ala - alanine
  • C Cys - cysteine
  • D Asp- aspartic acid
  • E Glu - glutamic acid
  • F Phe - phenylalanine
  • G Gly - glycine
  • H His - histidine
  • I He - isoleucine
  • K Lys - lysine
  • L Leu - leucine
  • M Met - methionine
  • N Asn - asparagine
  • P Pro - proline
  • Q Gin - glutamine
  • R Arg - arginine
  • S Ser - serine
  • T Thr - threonine
  • V Val - valine
  • W Val - valine
  • the peptide sequence of a number of human Type I interferons is shown in Table I and Figure 14.
  • the present inventor previously identified three human Type I interferon receptor binding domains, from amino acid residues 10-35 (domain 1), 78- 107 (domain 2) and 123-166 (domain 3) and more particularly from amino acid residues 29-35, 78-95 and 123-140, or 130-140 or 123-156.
  • Peptides comprising an amino acid sequence of anyone of these regions or biologically active fragments thereof, in a suitable configuration for binding to the receptor are encompassed within the scope of the present invention.
  • Peptides comprising an amino acid sequence of anyone of these regions includes peptides which may comprise additional amino acid residues at the flanking regions of the sequences, but that do not inhibit the binding activity of the receptor binding domains.
  • the peptides per se that are inco ⁇ orated into the scaffold ofthe invention are also encompassed within the scope ofthe present invention.
  • Cytokine receptor binding domain refers to any peptide that can bind to the corresponding cytokine receptor but may include other regions or additional amino acid residues that do not inhibit the binding activity of the peptide.
  • Interferon receptor binding domain shall have a corresponding meaning.
  • the term "biologically active fragment thereof as used herein means any fragment that can bind the respective cytokine receptor.
  • the present invention provides a novel cytokine receptor binding peptide construct comprising at least one cytokine receptor binding domain inco ⁇ orated into an scFv peptide molecule, such as scFv, preferably having the amino acid sequence of SEQ. ID. NO. 18 (or Figure 15) in a manner that maintains the binding domain in a configuration suitable for binding the cytokine receptor.
  • the cytokine peptide comprising the cytokine receptor binding domain is inco ⁇ orated into the scFv peptide at one of the complimentarity determining regions (CDRs, i.e., CDR1, CDR2 or CDR3) although other sites of inco ⁇ oration may also be suitable.
  • CDRs complimentarity determining regions
  • the scFv peptide would be altered at the site of inco ⁇ oration.
  • the cytokine binding domain can be inco ⁇ orated into the scFv peptide by replacing all or part of a region of scFv (e.g. all or part of CDR1, CDR2 and/or CDR3) or by adding the sequence to the scFv at a particular site.
  • the cytokine is interferon, preferably a Type I human interferon.
  • the present invention provides the insertion of one or more interferon peptides which bind the IFN receptor into strategic locations along a scaffold, facilitating interaction of one or more of said peptides with the corresponding active site of the Type 1 human interferon receptor.
  • one of the said interferon peptides is inserted at one of the CDRs of scFv, altering the respective CDR of scFv.
  • the interferon peptides inco ⁇ orated into the suitable scaffold comprise at least an interferon receptor binding domain selected from IFN amino acid residues: 10-35 (interferon receptor recognition site I), 78-107 (interferon receptor recognition site 2) and 123- 166 (interferon recognition site III) or IFN receptor binding portions thereof.
  • the interferon peptides inco ⁇ orated into the scaffold of the invention comprise at least one of the following amino acid residue domains: amino acid residues 29-35 (IRRP1), 78-95 (IRRP2) (or in other embodiments 89-95 or 79-96) and 123-140 (IRRP3) (or in other embodiments 130-140 or 123-156).
  • the interferon receptor binding peptides inco ⁇ orated into the scaffold comprise the amino acid sequence selected from the group consisting of: CYS-LEU-LYS-ASP-ARG-HIS-ASP (SEQ. ID NO. 1); ASP-GLU-SER-LEU-LEU-GLU-LYS-PHE-TYR-THR-GLU-LEU-TYR-GLN-GLN- LEU-ASN-ASP (SEQ. ID NO. 2); ASN-GLU-THR-ILE-VAL-GLU-ASN-LEU- LEU-ALA-ASN-VAL-TYR-HIS-GLN-ILE-ASN-HIS (SEQ. ID NO.
  • TYR-LEU- THR-GLU-LYS-LYS-TYR-SER-PRO-CYS-ALA SEQ. ID NO. 4
  • TYR-PHE- GLN-ARG-ILE-THR-LEU-TYR-LEU-THR-GLU-LYS-LYS-TYR-SER-PRO-CYS- ALA SEQ. ID NO. 5
  • TYR-PHE-GLN-ARG-ILE-THR-LEU-TYR SEQ. ID NO. 6
  • GLU-LEU-TYR-GLN-GLN-LEU-ASN-ASP SEQ. ID NO.
  • the interferon receptor binding peptide construct of the invention comprises at least 2 and preferably 3 IFN receptor binding domains, one from each of the three IFN receptor recognition sites or respective IFN receptor binding portions thereof and each replacing a different CDR in the scFv scaffold.
  • Optimum configurations for instance IRRP1, IRRP2, or IRRP3 could be inserted at anyone of the CDR regions depending on whether a partial mimetic or construct is being made (ie., one with one or two IFN receptor binding peptide insertions, such as SEQ. ID. NO. 22, pIFNscFv) or whether a full mimetic, (i.e. with an IRRP inserted at each of the CDR sites, such as SEQ. ID. NO. 33 or Figure 13, cIFNscFV).
  • Optimum insertion sites and structures may change depending on factors such as steric hinderance.
  • the molecular structures of the cytokines, such IFNs , and scaffold, such as scFv can be obtained by conventional techniques in the art. Known structures can be obtained from various databases, such the Protein Data Bank at http ://www.rscb . or g/pdb .
  • the protein structures of the scaffold and the cytokine, such as IFN, and respective IFN receptor binding domains or amino acid sequences comprising such domains or IFN receptor binding portions thereof, can be used determine optimum insertion sites to obtain IFN receptor binding using known molecular modeling factors, such as bond lengths or bond stress.
  • the IFN receptor binding domains or portions thereof and their respective nucleic acid coding sequences can be inco ⁇ orated in various permutations into the CDR regions of the scaffold or coding regions therefore.
  • Optimum configurations can be predicted using techniques known in the art, such as computer modelling and confirmed by subsequent construction, expression and activity assays.
  • Type I human IFN receptor binding peptide of domain 1 (comprising amino acid residues of IFN receptor recognition site I or IFN receptor binding portion thereof), is inco ⁇ orated at CDR1, preferably at VH of svFv and alters CDR
  • Type I human IFN receptor binding peptide of domain 1 (comprising amino acid residues of IFN receptor recognition site I or IFN receptor binding portion thereof), is inco ⁇ orated at CDR3, preferably of VL of scFv and alters CDR3, Type I human IFN receptor binding other suitable configurations are possible.
  • the invention provides an IFN receptor binding peptide construct comprising the amino acid sequence of SEQJD No.22 (pIFNscFv - C219 scaffold with inserted interferon peptides) or SEQ.ID No.33 (cIFNscFv-219 scaffold with inserted interferon peptides).
  • the invention provides a peptide encoded by the nucleic acid sequence of SEQ. ID. NO. 29 or 32 ( Figure 13) or a degenerate sequence encoding the same amino acid molecule.
  • interferon receptor binding peptides and constructs of the invention can be used as a carrier for other therapeutic agents.
  • another therapeutic agent could be attached to the scaffold, to provide a bolus of, for example, interferon and another chemotherapeutic agent, or IFN and an ribavirin, or IFN and anti-inflammatory agent.
  • proteins ofthe present invention may also include truncations of the proteins, and analogs, and homologs of the proteins and truncations thereof as described herein.
  • the truncated proteins included in the invention are those that can still bind the respective cytokine receptor.
  • Analogs of the proteins having the amino acid sequences of the invention may include, but are not limited to an amino acid sequence containing one or more amino acid substitutions, insertions, and/or deletions.
  • Amino acid substitutions may be of a conserved or non-conserved nature. conserveed amino acid substitutions involve replacing one or more amino acids of the proteins of the invention with amino acids of similar charge, size, and/or hydrophobicity characteristics. When only conserved substitutions are made the resulting analog should be functionally equivalent.
  • Non-conserved substitutions involve replacing one or more amino acids ofthe amino acid sequence with one or more amino acids which possess dissimilar charge, size, and/or hydrophobicity characteristics.
  • amino acid insertions may be introduced into the amino acid sequences shown in SEQ. ID. NO 22 or 33 ( Figure 13).
  • Amino acid insertions may consist of single amino acid residues or sequential amino acids ranging from 2 to 15 amino acids in length.
  • amino acid insertions may be used to destroy target sequences so that the protein is no longer active. This procedure may be used in vivo to inhibit or compete with the activity of a protein of the invention or a particular cytokine.
  • Deletions may consist of the removal of one or more amino acids, or discrete portions from the amino acid sequence shown in SEQ. ID. NO.22 or 33 ( Figure 13).
  • the deleted amino acids may or may not be contiguous.
  • the lower limit length ofthe resulting analog with a deletion mutation is about 10 amino acids, preferably 100 amino acids.
  • Analogs of the proteins of the invention may be prepared by introducing mutations in the nucleotide sequence encoding the protein. Mutations in nucleotide sequences constructed for expression of analogs of a protein of the invention must preserve the reading frame of the coding sequences. Furthermore, the mutations will preferably not create complementary regions that could hybridize to produce secondary mRNA structures, such as loops or hai ⁇ ins, which could adversely affect translation ofthe mRNA.
  • Mutations may be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.
  • oligonucleotide-directed site specific mutagenesis procedures may be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required.
  • Deletion or truncation of a protein of the invention may also be constructed by utilizing convenient restriction endonuclease sites adjacent to the desired deletion. Subsequent to restriction, overhangs may be filled in, and the DNA religated. Exemplary methods of making the alterations set forth above are disclosed by Sambrook et al [32].
  • the proteins of the invention also include homologs of the amino acid sequence of the invention, especially that of SEQ. ID. NO. 22 or 33 ( Figure 13) and/or truncations thereof as described herein.
  • Such homologs are proteins whose amino acid sequences are encoded by nucleic acid sequences that hybridize under stringent hybridization conditions (see discussion of stringent hybridization conditions herein) with a probe used to obtain a protein of the invention.
  • Homologs of a protein ofthe invention will have the same regions which are characteristic ofthe protein.
  • a homologous protein includes a protein with an amino acid sequence having at least 76%, preferably 80-90% identity with the amino acid sequence as shown in SEQ. ID. NO. 22.
  • the invention also contemplates isoforms ofthe proteins ofthe invention.
  • An isoform contains the same number and kinds of amino acids as a protein of the invention, but the isoform has a different molecular structure.
  • the isoforms contemplated by the present invention are those having the same properties as a protein ofthe invention as described herein.
  • the present invention also includes a protein of the invention conjugated with a selected protein, or a selectable marker protein (see below) to produce fusion proteins.
  • the proteins ofthe invention may be prepared using recombinant DNA methods. These proteins may be purified and/or isolated to various degrees using techniques known in the art. Accordingly, nucleic acid molecules of the present invention having a sequence that encodes a protein of the invention may be inco ⁇ orated according to procedures known in the art into an appropriate expression vector which ensures good expression ofthe protein.
  • Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), so long as the vector is compatible with the host cell used.
  • vectors suitable for transformation of a host cell means that the expression vectors contain a nucleic acid molecule ofthe invention and regulatory sequences, selected on the basis of the host cells to be used for expression, which are operatively linked to the nucleic acid molecule. "Operatively linked” is intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the nucleic acid.
  • the invention therefore contemplates a recombinant expression vector of the invention containing a nucleic acid molecule of the invention, or a fragment thereof, and the necessary regulatory sequences for the transcription and translation of the inserted protein-sequence.
  • Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, or viral genes (For example, see the regulatory sequences described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990) [33]. Selection of appropriate regulatory sequences is dependent on the host cell chosen, and may be readily accomplished by one of ordinary skill in the art.
  • regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be inco ⁇ orated into the expression vector. It will also be appreciated that the necessary regulatory sequences may be supplied by the native protein and/or its flanking regions.
  • the recombinant expression vectors of the invention may also contain a selectable marker gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention.
  • selectable marker genes are genes encoding a protein which confers resistance to certain drugs, such as G418 and hygromycin.
  • markers which can be used are: green fluorescent protein (GFP), ⁇ -galactosidase, chloramphenicol acetyltransferase, or firefly luciferase.
  • selectable marker gene Transcription of the selectable marker gene is monitored by changes in the concentration of the selectable marker protein such as b-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. If the selectable marker gene encodes a protein conferring antibiotic resistance such as neomycin resistance transformant cells can be selected with G418. Cells that have inco ⁇ orated the selectable marker gene will survive, while the other cells die. This makes it possible to visualize and assay for expression of recombinant expression vectors of the invention and in particular to determine the effect of a mutation on expression and phenotype. It will be appreciated that selectable markers can be introduced on a separate vector from the nucleic acid of interest.
  • the recombinant expression vectors may also contain genes that encode a fusion moiety which provides increased expression of the recombinant protein; increased solubility of the recombinant protein; and aid in the purification of a target recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety subsequent to purification ofthe fusion protein.
  • Recombinant expression vectors can be introduced into host cells to produce a transformed host cell.
  • the term "transformed host cell” is intended to include prokaryotic and eukaryotic cells which have been transformed or transfected with a recombinant expression vector of the invention.
  • the terms "transformed with”, “transfected with”, “transformation” and “transfection” are intended to encompass introduction of nucleic acid (e.g. a vector) into a cell by one of many possible techniques known in the art.
  • Prokaryotic cells can be transformed with nucleic acid by, for example, electroporation or calcium chloride mediated transformation.
  • Nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co precipitation, DEAE-dextran- mediated transfection, lipofectin, electroporation or microinjection. Suitable methods for transforming and transfecting host cells can be found in Sambrook et al. [32] and other such laboratory textbooks.
  • Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells.
  • the proteins of the invention may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells or mammalian cells.
  • Other suitable host cells can be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1991) [33].
  • the proteins of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis [34] or synthesis in homogenous solution [35].
  • the invention provides for nucleic acid molecules encoding the cytokine, preferably IFN, receptor binding peptide constructs ofthe invention.
  • the invention provides suitable expression vectors which inco ⁇ orate the nucleic acid molecules of the invention.
  • the expression vector is plasmid, pSJF2.
  • the invention provides a nucleic acid sequence encoding the interferon receptor binding peptide construct of SEQ. ID. NO.29 or SEQ. ID NO.32.
  • An embodiment of this aspect of the invention provides the protein expressed from the plasmid ofthe nucleic acid sequence provided in SEQ. ID NO. 29 or SEQ. ID. NO.33 ( Figure 13).
  • the invention provides for a cell genetically modified, either through transformation, transfection or other means to express the IFNscFv protein of the invention.
  • the invention comprise the nucleic acid sequence of Figure 15 or Figure 11 with the novel restriction sites
  • the present invention provides an isolated nucleic acid molecule comprising a sequence encoding an interferon receptor binding protein.
  • isolated refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized.
  • nucleic acid is intended to include DNA and RNA and can be either double stranded or single stranded.
  • an isolated nucleic acid molecule having a sequence which encodes an interferon receptor binding protein having an amino acid sequence as shown in SEQ ID NO. 22 or SEQ. ID. NO.33 ( Figure 13).
  • the isolated nucleic acid molecule comprises
  • nucleic acid molecule differing from any ofthe nucleic acids of (a) to (c) in codon sequences due to the degeneracy ofthe genetic code;
  • the invention provides a nucleic acid molecule comprising the coding region of a human Type I interferon receptor binding protein , such as SEQ ID NO. 22 or 33 or biologically active fragment thereof.
  • T can also be U.
  • the invention further encompasses, nucleic acid molecules which are complementary in sequence to nucleic acid molecules of the invention, fragments of the nucleic acid molecules of the invention, preferably at least 15 bases, and more preferably of at least 20 to 30 bases, and which will hybridize to the nucleic acid molecules of the invention under stringent hybridization conditions.
  • Appropriate stringency conditions which promote DNA hybridization are known to those skilled in the art, or may be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6 [36].
  • the following may be employed: 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C.
  • SSC sodium chloride/sodium citrate
  • the stringency may be selected based on the conditions used in the wash step.
  • the salt concentration in the wash step can be selected from a high stringency of about 0.2 x SSC at 50°C.
  • the temperature in the wash step can be at high stringency conditions, such as at about 65 °C.
  • the fragments can be used as primers to amplify (using techniques known in the art, such as the polymerase chain reaction) or to detect the nucleic acid sequence of the invention (especially if the primer is labeled).
  • the invention further encompasses nucleic acid molecules that differ from any of the nucleic acid molecules of the invention in codon sequences due to the degeneracy of the genetic code.
  • the invention also encompasses nucleic acid sequences or molecules that are analogs of the nucleic acid sequences and molecules described herein.
  • a nucleic acid sequence which is an analog means a nucleic acid sequence which has been modified as compared to the sequences described herein, such as sequences of (a), (b), (c), (d), or (e), above wherein the modification does not alter the utility ofthe sequences described herein.
  • the modified sequence or analog may have improved properties over the sequence shown in (a), (b),(c), (d) or (e).
  • One example of a modification to prepare an analog is to replace one of the naturally occurring bases (i.e.
  • adenine, guanine, cytosine or thymidine of the sequence with a modified base such as such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6- aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8 amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, th
  • a modification is to include modified phosphorous or oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages in the nucleic acid molecules of the invention.
  • the nucleic acid sequences may contain phosphorothioates, phosphotriesters, methyl phosphonates, and phosphorodithioates .
  • a further example of an analog of a nucleic acid molecule ofthe invention is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polyamide backbone which is similar to that found in peptides [37].
  • PNA analogs have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro. PNAs also bind stronger to a complimentary DNA sequence due to the lack of charge repulsion between the PNA strand and the DNA strand.
  • Other nucleic acid analogs may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones.
  • the nucleotides may have mo ⁇ holino backbone structures (U.S. Pat. No. 5,034,506).
  • the analogs may also contain groups such as reporter groups, a group for improving the pharmacokinetic or pharmacodynamic properties of nucleic acid sequence.
  • the invention includes nucleic acid molecules encoding truncations of proteins of the invention, and analogs and homologs of proteins of the invention and truncations thereof, as described above.
  • the invention further includes biologically active fragments of the nucleic acid molecules of the invention. Such fragments would include, but is not necessarily limited to any nucleic acid molecules which are beneficial in the modulation or simulation ofthe interferon receptor binding protein ofthe invention, or in the identification or production of such agents.
  • the invention includes nucleic acid molecules comprising nucleic acid sequences having substantial sequence homology with the nucleic acid sequences as shown in SEQ. ID. NO. 29 or SEQ. ID. NO.32 ( Figure 13) and fragments thereof.
  • sequences having substantial sequence homology means those nucleic acid sequences that have slight or inconsequential sequence variations from these sequences, i.e., the sequences function in substantially the same manner to produce functionally equivalent proteins. The variations may be attributable to local mutations or structural modifications.
  • Nucleic acid sequences having substantial homology include nucleic acid sequences having at least 85%, preferably 90-95% identity with the nucleic acid sequences ofthe invention.
  • An isolated nucleic acid molecule ofthe invention that is DNA can be formed and isolated by selectively amplifying a nucleic acid encoding an interferon receptor binding domain of the invention ofthe invention using the polymerase chain reaction (PCR) methods and cDNA or genomic DNA. It is possible to design synthetic oligonucleotide primers from the nucleic acid sequences of the invention for use in PCR. A nucleic acid can be amplified from cDNA using these oligonucleotide primers and standard PCR amplification techniques. The primers can be designed so that suitable restriction sites are inco ⁇ orated into the amplified product at its end to enable it to be inserted into the suitable site ofthe scaffold (scFv) peptide.
  • scFv scaffold
  • the nucleic acid sequence encoding the scaffold can also be amplified in a manner to ensure that suitable restriction sites are present at the site of insertion of the interferon receptor binding domain(s).
  • the amplified nucleic acid sequence of the interferon receptor binding domain can be restricted using the appropriate enzymes and then spliced into the analogous restricted site ofthe scaffold.
  • cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guanidinium-thiocyanate extraction procedure of Chirgwin et al., Biochemistry, 18, 5294 5299 (1979) [38]. cDNA is then synthesized from the mRNA using reverse transcriptase (for example, Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St. Russia, FL).
  • reverse transcriptase for example, Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St. Russia, FL).
  • RNA can be isolated by cloning a cDNA encoding a novel protein of the invention into an appropriate vector which allows for transcription of the cDNA to produce an RNA molecule which encodes a protein of the invention.
  • a cDNA can be cloned downstream of a bacteriophage promoter, (e.g., a T7 promoter) in a vector, cDNA can be transcribed in vitro with T7 polymerase, and the resultant RNA can be isolated by standard techniques.
  • a bacteriophage promoter e.g., a T7 promoter
  • a nucleic acid molecule of the invention may also be chemically synthesized using standard techniques.
  • Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071).
  • Determination and confirmation of whether a particular nucleic acid molecule encodes a novel protein of the invention may be accomplished by expressing the cDNA in an appropriate host cell by standard techniques, and testing the activity of the protein using the methods as described herein.
  • a cDNA having the activity of a novel protein of the invention so isolated can be sequenced by standard techniques, such as dideoxynucleotide chain termination or Maxam-Gilbert chemical sequencing or by automated DNA sequencing, to determine the nucleic acid sequence and the predicted amino acid sequence ofthe encoded protein.
  • the sequence of a nucleic acid molecule of the invention may be inverted relative to its normal presentation for transcription to produce an antisense nucleic acid molecule.
  • antisense nucleic acid molecule is a nucleotide sequence that is complementary to its target.
  • an antisense sequence is constructed by inverting a region preceding or targeting the initiation codon or an unconserved region.
  • the antisense sequence targets all or part of the mRNA or cDNA of the nucleic acid sequences of the invention.
  • the nucleic acid sequences contained in the nucleic acid molecules of the invention or a fragment thereof, preferably a nucleic acid sequence shown in SEQ. ID. NO. 29 or 32 ( Figure 13) may be inverted relative to its normal presentation for transcription to produce antisense nucleic acid molecules.
  • the antisense nucleic acid molecules of the invention or a fragment thereof may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability ofthe duplex formed with mRNA or the native gene e.g. phosphorothioate derivatives and acridine substituted nucleotides.
  • the antisense sequences may be produced biologically using an expression vector introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense sequences are produced under the control of a high efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced.
  • the invention also provides nucleic acids encoding fusion proteins comprising a novel protein ofthe invention and a selected protein, or a selectable marker protein
  • primers for use in a PCR are selected so that they will hybridize to different strands of the desired sequence and at relative positions along the sequence such that an extension product synthesized from one primer when it is separated from its template can serve as a template for extension of the other primer into a nucleic acid of defined length.
  • Primers which may be used in the invention are oligonucleotides, i.e., molecules containing two or more deoxyribonucleotides of the nucleic acid molecule of the invention which occur naturally as in a purified restriction endonuclease digest or are produced synthetically using techniques known in the art such as for example phosphotriester and phosphodiester methods [See 39] or automated techniques [See for example 40].
  • the primers are capable of acting as a point of initiation of synthesis when placed under conditions which permit the synthesis of a primer extension product which is complementary to a DNA sequence of the invention, i.e., in the presence of nucleotide substrates, an agent for polymerization such as DNA polymerase and at suitable temperature and pH.
  • the primers are sequences that do not form secondary structures by base pairing with other copies of the primer or sequences that form a hair pin configuration.
  • the primer preferably contains between about 7 and 25 nucleotides.
  • the primers may be labelled with detectable markers which allow for detection of the amplified products.
  • Suitable detectable markers are radioactive markers such as P-32, S-35, 1-125, and H-3, luminescent markers such as chemiluminescent markers, preferably luminol, and fluorescent markers, preferably dansyl chloride, fluorcein-5-isothiocyanate, and 4-fluor-7-nitrobenz-2-axa-l,3 diazole, enzyme markers such as horseradish peroxidase, alkaline phosphatase, ⁇ - galactosidase, acetylcholinesterase, or biotin.
  • the primers may contain non-complementary sequences provided that a sufficient amount of the primer contains a sequence which is complementary to a nucleic acid molecule of the invention or oligonucleotide fragment thereof, which is to be amplified. Restriction site linkers may also be inco ⁇ orated into the primers allowing for digestion ofthe amplified products with the appropriate restriction enzymes facilitating cloning and sequencing of the amplified product.
  • a method of determining the presence of a nucleic acid molecule having a sequence encoding a protein of the invention comprising treating the sample with primers which are capable of amplifying the nucleic acid molecule or a predetermined oligonucleotide fragment thereof in a polymerase chain reaction to form amplified sequences, under conditions which permit the formation of amplified sequences and, assaying for amplified sequences.
  • Polymerase chain reaction refers to a process for amplifying a target nucleic acid sequence as generally described in Innis et al, Academic Press, 1990 [41] in Mullis el al., U.S. Pat. No. 4,863,195 and Mullis, U.S. Patent No. 4,683,202.
  • Conditions for amplifying a nucleic acid template are described in M.A. Innis and D.H. Gelfand, PCR Protocols, A Guide to Methods and Applications M.A. Innis, D.H. Gelfand, JJ. Sninsky and TJ. White eds, pp3-12, Academic Press 1989 [42].
  • the amplified products can be isolated and distinguished based on their respective sizes using techniques known in the art. For example, after amplification, a DNA sample can be separated on an agarose gel and visualized, after staining with ethidium bromide, under ultra violet (uv) light. DNA may be amplified to a desired level and a further extension reaction may be performed to inco ⁇ orate nucleotide derivatives having detectable markers such as radioactive labelled or biotin labelled nucleoside triphosphates. The primers may also be labelled with detectable markers as discussed above. The detectable markers may be analyzed by restriction enzyme digestion and electrophoretic separation or other techniques known in the art.
  • Conditions which may be employed in the methods of the invention using PCR are those which permit hybridization and amplification reactions to proceed in the presence of DNA in a sample and appropriate complementary hybridization primers. Conditions suitable for a polymerase chain reaction are generally known in the art. For example, see [42].
  • the PCR utilizes polymerase obtained from the thermophilic bacterium Thermus aquatics (Taq polymerase, GeneAmp Kit, Perkin Elmer Cetus) or other thermostable polymerase.
  • Interferon is known to affect a wide variety of cellular functions, related to cell growth control (such as in cancer and various neoplasias), the regulation of immune responses and more specifically, the induction of antiviral responses.
  • the peptides of the present invention can be be used to mimic all or some ofthe effects of native interferons and thus can be used in the treatment of a number of medical conditions in place of interferon.
  • the peptides of the present invention have fewer side effects than those of the native cytokine.
  • medical conditions in which the peptides of the present invention can be used include without limtiation to cancers, hematological malignancies, hepatitis B or C infections, multiple sclerosis, different arthritides.
  • nucleic acid sequences of the present invention and constructs, that encode and can express the peptides of the invention under suitable conditions can also be used in the treatment and/or therapy of medical conditions as previously stated.
  • the invention provides a use of the modulating or simulating agents of the invention (which comprise the peptides, nucleic acid molecules, expression vectors and cells expressing the peptides of the invention) for the treatment of a condition wherein IFN treatment is or maybe indicated. Methods of such treatment are also encompassed within the scope ofthe invention. Accordingly, the present invention provides a method of treating or preventing a disease. The invention further comprises uses of the peptides and nucleic acid sequences disclosed herein for the preparation of a medicament for treating a condition wherein IFN treatment is indicated, more preferably human Type I IFN.
  • the invention provides a method for treating such a condition comprising administering an agent that modulates or simulates IFN expression or activity to an animal, preferably a mammal, more preferably a human, in need thereof.
  • agents stimulate or simulate IFN activity.
  • agents that activate or simulate IFN activity would include without limitations, IFN, the gene encoding for IFN with suitable promoters, such promoters preferably being tissue specific promoters and therapeutically effective fragments of the nucleic acid and amino acid sequences of the invention.
  • the invention provides a method for treating a disease or condition wherein IFN treatment is indicated by administering to a patient in need thereof an agent which simulates IFN activity.
  • Agents that activate, or stimulate IFN can be formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in vivo.
  • biologically compatible form suitable for administration in vivo means a form of the substance to be administered in which therapeutic effects outweigh any toxic effects.
  • the substances may be administered to animals in need thereof.
  • Animals, as used herein refers to any animal susceptible to a medical condition that can be modulated or treated with IFN preferably mammals, such as dogs, cats, mice, bovine, horses and humans, preferably humans.
  • an "effective amount" of pharmaceutical compositions of the present invention is defined as an amount of the pharmaceutical composition, at dosages and for periods of time necessary to achieve the desired result.
  • a therapeutically active amount of a substance may vary according to factors such as disease state, age, sex, and weight ofthe recipient, and the ability ofthe substance to elicit a desired response in the recipient. Dosage procedures may be adjusted to provide an optimum Therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies ofthe therapeutic situation.
  • An active substance may be administered in a convenient manner such as by injection (subcutaneous, intravenous, topical, intratumoral etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active substance may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound.
  • compositions described herein can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle or carrier .
  • Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences [44].
  • the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
  • Recombinant nucleic acid molecules comprising a sense, an antisense sequence or oligonucleotide fragment thereof, may be directly introduced into cells or tissues in vivo using delivery vehicles known in the art such as retroviral vectors, adenoviral vectors and DNA virus vectors. They may also be introduced into cells in vivo using physical techniques known in the art such as micro injection and electroporation or chemical methods such as coprecipitation and inco ⁇ oration of DNA into liposomes. Recombinant molecules may also be delivered in the form of an aerosol or by lavage.
  • the peptide, the peptide mimetics ofthe invention can be used to screen for modulators of the IFN activity.
  • the mimetic ofthe invention and its substrate e.g. Ifn receptor
  • the substrate e.g. Ifn receptor
  • the effect on mimetic/substrate binding can be detected.
  • detection systems known in the art such as, assay detection systems, such as colormetric assays or radiolabelling asays. Agonists, antagonists and other modulators can be screened using this or other methods known in the art.
  • the peptide mimetic can be used to diagnose IFN related disorders by obtaining sample (such as blood or tissue sample) from a patient, exposing it it it a peptide mimetic and/or a substrate and comparing effect (e.g. effect on receptor binding) with those of otheres with a known IFN disease state (normal, abnormal or both).
  • sample such as blood or tissue sample
  • the peptide mimetic can be administered to a patient and a patient sample taken afterward for subsequent testing by assay or other means of IFN mimetic/subtrate interaction determination.
  • This model invokes the presence of a generic binding through that allows recognition of conserved structural elements among different cytokines.
  • the present inventor's data support such a model, at least for the different IFN-alpha molecular species and IFN-beta, since they have identified two conserved elements in the Type 1 IFNs that effect receptor recognition.
  • a third structural element, that is an exposed recognition epitope, confers specificity of cytokine function, including species specificity.
  • IFN-o-2 IXDKF ⁇ ELY QQL D LEACY IQGVGVIETP LMKEDSLAV
  • IFN-alN-4 IJUDKFC ELY QQLND LEACY MQEER GETP LMNADSEJV
  • the foregoing table illustrates the amino acid sequence alignment of the different Type 1 IFNs.
  • the designation of the various IFNs is shown in the left hand column and the sequence of IFN-beta is aligned with the other IFNs, commencing with residue 4, to achieve the greatest homology.
  • the critical domains comprising residues 29-35, 78-95 and 123-140 are boxed.
  • the letter codes for the amino acids are as follows: A, ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, He; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gin; R, Arg; S, Ser; T, Thr; V, Val; W, T ⁇ ; and Y, Tyr.
  • the preferred binding domain peptide regions of the invention comprise peptides of specific amino acid sequences. These sequences are:
  • IFN-alpha2a and the various derivatives were provided by I.C.L Pharmaceuticals Division ofthe UK; IFN-alphaCon.subJ was supplied by Amgen of the USA and IFN-alpha. sub J N.delta.4 was supplied by Schering Plough Co ⁇ ofthe USA.
  • IFN-alpha2a, (4-155)IFN-alpha2a, 4-155(S98)IFN-alpha2a and 4- 155(L98)IFN-alpha2a had specific activities of 2.times.l0.sup.8 U/mg protein; (A30,32,33)IFN-alpha2a was inactive in antiviral assays and (ESML)IFN-alpha2a had a specific activity of 7.5.timesJ0.sup.6 U/mg protein; IFN-alpha Con.subJ had a specific activity of 3.0.timesJ0.sup.9 U/mg protein; and IFN-alpha.sub.l N.delta.4 had a specific activity of 7J.timesJ0.sup.6 U/mg protein.
  • the cell culture used in this example comprised T98G cells which were derived from a human glioblastoma multiforma tumor and which express in culture a number of normal and transformed growth characteristics. These cells may be routinely subcultured as monolayers, in modified minimum essential medium
  • alpha-MEM fetal calf serum
  • FCS 10% fetal calf serum
  • IFNs were removed and 10.sup.4 PFU EMCV was added to individual wells in 100 .mu.l alpha-MEM, 2% FCS. After 24 hours, the cells were ethanol (95%) fixed and the extent of EMCV infection was determined by spectrophotometric estimation of viral CPE. The fixed cells were crystal violet (0.P/o in 2% ethanol) stained and destained (0.5M NaCl in 50% ethanol), and the inhibition of virus infection was estimated from absorbance measurements at 570 nm using a Microplate (trade mark) Reader MR600 and a calibration of absorbance against cell numbers. IFN titers were determined using a 50%> cytopathic end-point and converted to international units using an NIH IFN-alpha standard (Ga 23-901-527).
  • T98G cells were seeded in 96-well Microtest II tissue culture plates at a density of 5. times JO. sup.3 /ml and either innoculated with two-fold serial dilutions of different molecular species of IFN-alpha or left untreated. After incubation, at 37.degree. C. for 96 hours, the cells were ethanol fixed (95%), crystal violet (0.1% in 2% ethanol) stained and destained (0.5M NaCl in 50% ethanol), then growth inhibition was estimated from absorbance measurements of destained cells at 570 nm (using a Microplate Reader MR600 and a-calibration of absorbance against cell numbers).
  • Free .sup.125 I was separated from IFN-bound .sup.125 I on a 12 ml Sephadex (trade mark) G-75 column, equilibrated in PBS containing 1 mg/ml BSA. lodination caused no detectable loss of antiviral activity. Fractions containing maximum antiviral activity were pooled and contained 95% TCA (10%) precipitable radioactivity. Sub-confluent T98G cell monolayers were incubated at +4. degree. C. in alpha- MEM containing 2% FCS and indicated concentrations of .sup.125 I-LFN-alpha. After 2 hours, the binding medium was aspirated and the cultures were washed twice with ice-cold PBS.
  • the cells were solubilized in 0.5M NaOH and radioactivity counted in a Beckman (trade mark) 5500 *-counter. Specificity of binding was determined in parallel binding assays containing a 100-fold excess of unlabeled growth factor. For competitive experiments, specified amounts of unlabeled competitor were included in the reaction mixture together with radiolabelled ligand.
  • panel A illustrates the results using .sup.125 I-IFN-alphaCon.subJ
  • panel B illustrates the results using .sup.125 I-4-155(S98)IFN-alpha2a
  • panel C illustrates the results using .sup.125 I-IFN-alpha.subJ N.delta.4.
  • Inset into panels A, B and C are the corresponding Scatchard plots.
  • the competitive displacement profiles are shown in panels D, E and F using 10 ng/ml of .sup.125 I-IFN-alphaCon.subJ, 3.7 ng/ml of .sup.125 I-4-155(S98)IFN-alpha2a and 300 ng/ml of .sup.125 I-IFN- alpha.sub.l N.delta.4 respectively, with no unlabeled competitor (100% bound) or the indicated concentrations of IFNs.
  • the values shown were obtained by subtracting non-specific counts/min bound from total counts/min bound. Non-specific binding was determined in the presence of a 100-fold excess of unlabeled IFN.
  • the points represent the mean of triplicate cultures and exhibited a S.E. of +/-3%.
  • panel A illustrates the results using .sup.125 I-(4-155)IFN-alpha2a
  • panel B illustrates the results using .sup.125 I-4-155(L98)LFN-alpha2a.
  • FIG. 3 panel A illustrates the results using .sup.125 I-(4-155)IFN-alpha2a
  • panel B illustrates the results using .sup.125 I-4-155(L98)LFN-alpha2a.
  • the competitive displacement profiles are shown in panels C and D using 20 ng/ml of .sup.125 I-(4-155)IFN- alpha2a and 8 ng/ml of .sup.125 I-4-155(L98)IFN-alpha2a, with no unlabeled competitor (100% bound) or the indicated concentrations of IFNs.
  • the values shown were obtained by subtracting non-specific counts/min bound from total counts/min bound. Non-specific binding was determined in the presence of a 100-fold excess of unlabele
  • FIGS. 2 and 3 illustrate the steady state receptor binding characteristics ofthe different IFN-alpha molecular species on T98G cells at +4.degree. C. Specific binding to sub-confluent T98G monolayers is resolved into a biphasic Scatchard plot. This IFN binding heterogeneity has been shown to result from negatively cooperative site- site interactions between the ligand receptors. Analysis of the IFN-alpha2a binding data reveals both high and low affinity binding components, with K.sub.d s of 2- 3JimesJ0.sup.-l l M and 2-5.timesJ0.sup.-9 M, respectively.
  • IFN-alpha.sub.l N.delta.4 has a weaker affinity for the IFN-alpha receptor on T98G cells than IFN-alphaCon.subJ.
  • Substitution of the cysteine residue at position 98 in IFN-alpha2a with a serine, does not affect the polarity or charge distribution of the side chain at this position (CH.sub.2 — SH to CH.sub.2 --OH), yet substitution with a leucine residue does introduce an aliphatic side chain and hence alter the polarity (CH.sub.2 --SH to CH-- (CH.sub.3).sub.2). This alteration in side chain polarity at this residue position is not reflected in altered affinity characteristics for the IFN-alpha receptor (FIG.
  • the inventor Since the amino acid sequence dictates the native conformation of a protein, the inventor has ascribed protein structure for the different IFN-alphas and IFN-beta. Receptor recognition epitopes are characteristically hydrophilic and located on the surface ofthe binding molecule. Generally, sites for molecular recognition in proteins are located in loops or turns, whereas alpha-helices are involved in maintaining the structural integrity ofthe protein. Close examination ofthe hydrophilicity and surface probability plots of IFN-alpha2a shows that, in those regions that are critical for the active configuration of IFN-alpha, namely 10-35, 78-107 and 123-166, altering the cysteine at 98 has no effect on these determinants (FIG. 4), and indeed, does not affect biological activity (FIG. 1).
  • FIG. 4 illustrates predicted secondary structure characteristics of different IFN-alpha species according to amino acid sequence analyses. Hydrophilicity (H) and surface probability (S) profiles are depicted for each of the IFN-alphas and IFN-beta whose designations are on the left hand side of each pair. Amino acid residue position is indicated along the horizontal axes of the graphs. The critical domains comprising residues 29-35, 78-95 and 123-140 are boxed. In IFN-alpha2a, in the carboxy-terminal domain there are essentially 3 hydrophilic residue clusters that are likely located on the surface of the molecule (FIG. 4).
  • IFN-alpha2a variants (A30- 32,33)IFN-alpha2a and (E5,S27,M31,L59)IFN-alpha2a, that have lost biological activity and receptor binding characteristics, no longer present this cluster of residues near the surface of the molecule, (FIG. 4). This region constitutes a loop structure.
  • IFN-alpha.sub.l N.delta.4 the amino acid residues that immediately precede the critical 29-35 cluster are different to those in IFN-alpha2a, and thus affect the presentation of this receptor binding epitope somewhat, according to the different predictive algorithms the inventor has employed.
  • the data in FIG. 4 suggest that the cluster of hydrophilic residues that do constitute this receptor recognition epitope will be located near the surface of the molecule in IFN-alpha.sub.1 N.delta.4.
  • substitution of the lysine residue at position 31 by a methionine residue affects the configuration of this receptor recognition epitope, thereby affecting the biological effectiveness of IFN-alpha.sub.l N.delta.4.
  • the Type 1 IFNs share conserved receptor recognition epitopes in the 29-35 and 123-140 regions. Some variance is seen in the human and murine IFN-beta in the 29-35 region, although the presentation of this epitope as a loop structure is conserved.
  • the third strategic region with respect to the active configuration of IFN-alpha spans residues 78-107.
  • a hydrophilic cluster of amino acid residues that are likely located on the surface constitute residues 83-95 (FIG. 4). These residues probably present as a contiguous helical bundle and a loop structure.
  • Several amino acid residues around position 78 also appear to be located at the surface as part of the helical bundle.
  • the inventor has shown that substitution ofthe cysteine at position 98 with either a serine (S) or a leucine (L) does not affect the receptor binding characteristics of IFN-alpha2a, hence the inventor infers that those residues beyond 95, in the previously defined domain 78-107, are likely not critical for receptor recognition in IFN-alpha, since they appear not to be located at the surface of the molecule.
  • the alpha-helical structure allows the appropriate presentation of the recognition epitope that comprises residues 88-95. Of note is the variance in this region between the human IFN-alphas and the murine IFN-alphas, and the human IFN-alphas had human IFN-beta.
  • IFN-alphaAD(BgI II) shows demonstrable biological activity on murine cells and support the hypothesis that this region in the Type I IFNs determines species specificity.
  • the hybrid IFN-alphaAD(PvuII) resembles the human IFN-alphas in this region (FIG. 4) and differs from IFN-alphaAD(BgI II) at just three residue positions, two of which reside in this critical domain: 69 (S/T), 80(T/D) and 86(Y/C).
  • IFN-alphaAD(Pvu II) demonstrates considerably reduced antiviral activity on murine cells compared with IFN-alphaAD(BgI II) yet comparable activity to IFN- alpha 2a, on human cells.
  • Type 1 IFNs Sequence homology among the different Type 1 IFNs in conserved regions would suggest evolutionary significance. It is noteworthy that the amino-and carboxy- terminal domains that have been identified as critical, are highly conserved among the different molecular subtypes of Type 1 IFNs. Within the 29-35 and 123-140 regions are structural motifs that are consistent with receptor binding domains: loop structures that are predominantly hydrophilic and located at the surface of the molecule. Some variation in sequence homology is apparent in the 78-95 region. The critical epitopes for Type I IFN receptor recognition are associated with the residue clusters 29-35 and 130-140, for all species of Type I IFNs. These epitopes constitute the receptor binding domains and are likely located in close spacial proximity to one another in the folded IFN.
  • the specificity of action of a particular Type I IFN is conferred by the recognition epitope 78-95.
  • the basis for the specificity of interaction ofthe 78-95 domain and its putative cognate binding molecule is unknown.
  • Studies with human growth hormone have shown that receptor binding involves both receptor recognition, by an epitope on the growth hormone, and dimerization of receptors, facilitated through the interaction of a separate epitope on the growth hormone.
  • the 78-95 epitope in HuIFN-alpha may interact with another Type 1 receptor, effecting dimerization.
  • IFN-receptor complexes of 80 kDa and 140-160 kDa can be separated by SDS-PAGE.
  • the molecular weight of the predicted IFN-alpha receptor protein is 63 kDa and that of the majority of IFN-alphas is 20 kDa, thus, monomer (receptor-IFN) and dimerized-(receptor-IFN-receptor) complexes, may represent the 80 kDa and 40-160 kDa moieties that have been detected.
  • FIG. 5 illustrates a model for the tertiary structure of Type 1 IFNs.
  • This model inco ⁇ orates a helical bundle core, composed of the five helices A to E.
  • the loop structures that constitute the proposed receptor recognition epitopes, residues 29-35 and 130-140, are shown as heavily shaded, broad lines and are aligned such that they dock in the receptor groove as shown.
  • the third region implicated in the active conformation of the Type 1 IFNs, 78-95 is not buried in the receptor groove and is configured to allow binding to its cognate epitope on another Type 1 IFN receptor.
  • the shaded areas in helices C and D represent residues that are critical for maintaining the correct structural presentation of the corresponding contiguous recognition epitopes.
  • the Type I IFNs are comprised predominantly of alpha-helical bundles that are packed together.
  • the receptor recognition site is comprised ofthe AB loop, 29-35 and the D helix and DE loop, 123-140. These are aligned in such a way as to permit the IFN to bind to its receptor, in the receptor groove, such that the third epitope, 78- 95, is exposed and not buried in the receptor groove.
  • the initial interaction ofthe IFN molecule with the Type IFN receptor would account for the abundant, low affinity receptor binding component, extrapolated from the Scatchard analyses ofthe different binding isotherms. The higher affinity component could be invoked once the IFN molecule is bound to its receptor.
  • the heterogeneity of binding observed for IFN- alpha2a is absent in IFN-alpha.sub.l N.delta.4, and is explained by the alteration of the 29-35 and 78-95 epitopes in IFN-alpha.sub.l N.delta.4, as compared with IFN- alpha2a. This may lead to a reduction in signaling potential ofthe receptor-bound IFN and hence a reduction in biological potency.
  • the proliferative state of a cell will determine whether the high affinity binding component is invoked on IFN-alpha2a binding to its receptor.
  • Non-proliferating cells express fewer Type I IFN receptors and will not exhibit the characteristic heterogeneity of binding seen with proliferating cells.
  • non-proliferating cells do possess both the 80 kDa and 140-160 kDa IFN-binding complexes.
  • the data indicate that non-proliferating cells lack the high affinity component of IFN-alpha binding, that is not associated with IFN- receptor dimerization, yet may represent a secondary binding molecule.
  • a comprehensive binding model therefore, that would account for heterogeneity of binding distinct from receptor dimerization, would invoke the interaction of the IFN- bound receptor complex with a putative secondary binding molecule.
  • the possibility that other accessory molecules are required for the full complement of IFN-receptor interactions, is supported by observations of high molecular weight complexes containing the IFN-alpha-receptor complex.
  • the genetic transfer of the human IFN-alpha receptor into mouse cells led to transfectants that exhibited a poor sensitivity to selected Type 1 human IFNs. These results infer that the transfected protein may not be sufficient for the complete binding activities of the IFNs. Indeed, in the receptor systems described for interleukin-6 and nerve growth factor, accessory proteins are required for the high affinity binding component of the receptor-ligand interaction. In the absence of experimental data, it cannot be discounted that the 78-95 epitope in Type 1 IFNs may interact with a species-specific secondary binding molecule.
  • RNA binding specificity is conferred by short stretches of variant amino acid residues in two ribonucleoproteins that otherwise share extensive sequence homology.
  • Figure 8A illustrates a consensus interferon alpha carbon trace showing the tertiary structure of interferon alpha and three IFN binding domains
  • Figure 8B is a consensus C219scFv carbon trace, wherein the CDRs in the variable light chain region are highlighted.
  • Protein structures of Consensus interferon and C219scfv were superimposed and rotated in three dimensions to determine how the IFN sequences would be placed in the scFv sequence.
  • C219 was developed as a murine monoclonal antibody specific for Chinese hamster P-glycoprotein [47].
  • a single chain variant was later developed [48], which variant was engineered by the inventor to result in the scaffold ofthe present invention.
  • the goal was to reproduce as closely as possible the three-dimensional arrangement seen in the interferon structure.
  • the computational approach allows quick, inexpensive experimentation with different arrangements of loops.
  • the software suite Insightll has the capability for testing various insertion sites on the framework both visually and with energy minimization protocols.
  • the software was run on the OCI Research Unix Network, consisting of mainly Silicon Graphics and Sun workstations.
  • the interferon structure was available to the inventor and used as a guide. Various likely locations for the insertion were sampled in comparison with the relative disposition of the epitope sequences on the native interferon molecule.
  • the advantage of using the scFv scaffold, as opposed to a single-domain framework, for example, is that a number of loop regions were available as potential insertion sites.
  • the constructs of the invention were prepared. Restriction sites were engineered into the CDR loops of variable light chain of scFv using a Stratagene mutagenesis kit. An Accl site was introduced into CDR1 of scFv using an Accl sense primer of SEQ. ID NO. 23 and Accl antisense primer of SEQ. ID NO. 24. These primers were designed with a mutation at nucleotides 20 and 21 in the sense primer, and at nucleotides 21 and 22 in the antisense primer, as shown. The mutation serves to introduce the Accl restriction site, which is engineered into the sequence in order to insert the nucleic acid sequence of SEQ. ID NO. 28, which translates to the interferon-receptor binding peptide of amino acid sequence SEQ. ID NO. 21.
  • Hpal site was introduced into the CDR3 of scFv using an Hpal sense primer of SEQ. ID NO. 25, and Hpal antisense primer of SEQ. ID NO. 26. These primers were designed with a mutation at nucleotides 20 and 22 of the sense primer, and nucleotides 19 and 21 ofthe antisense primer, as shown.
  • the mutation serves to introduce the Hpal restriction site, which is engineered into the sequence in order to insert the nucleic acid sequence of SEQ. ID NO. 27, which translates to the interferon-receptor binding peptide of amino acid sequence SEQ. ID NO. 1.
  • cDNAs corresponding to conserved interferon-receptor binding peptides (SEQ. ID NO. 1, SEQ. ID NO. 20, SEQ. ID NO. 21) were synthesized by known methods. DNA was purified by polyacrylamide gel electrophoresis (PAGE) using known methods. Strands of DNA were annealed to produce double stranded coding sequence.
  • DNA sequences corresponding to interferon-receptor binding peptides (SEQ. ID NO. 1, SEQ. ID NO. 20, SEQ. ID NO. 21) were ligated into the engineered scFv sequence using the appropriate restriction sites. Flanking sequences were also added to increase the stability ofthe protein secondary structure.
  • the CDR1 insert (SEQ. ID NO. 21) was screened by redigestion with Accl and Kpnl. Presence of the CDR3 insert (SEQ. ID NO. 1) was determined by length of the construct using PCR technology and primers as provided in SEQ. ID NO. 30 and SEQ. ID NO. 31.
  • the scFv containing the insert of SEQ. ID NO. 1 should be 93 base pairs in length. Without the insert, the PCR product will be 63 base pairs. To verify that the appropriate inserts were accomplished, sequencing was performed to detect any mutations.
  • TGI E. coli cells were transformed with pSJF2 plasmid containing pIFNscFv (scaffold with inserted interferon-receptor binding sequences) (complete sequence of plasmid containing pIFNscFv is provided in SEQ. ID NO. 22 (for amino acid sequence) and SEQ ID NO. 29 (for nucleic acid sequence) and plated on LB-amp plates. Resulting colonies were picked and grown in 25mL LB containing ampicillin.
  • the periplasmic cell fraction was isolated and screened for the presence of pIFNscFv using an anti-c-Myc antibody and Western blot technology. One positive clone was grown up in large cultures and periplasmic fraction isolated. This fraction was concentrated using an Amicon system under nitrogen gas with a lOkDa cutoff membrane.
  • the concentrated periplasmic fraction was dialysed against lOmM Tris pH 8.0, 300mM NaCl and loaded onto a Ni-agarose column. Protein was eluted using lOOmM imidazole in dialysis buffer. Fractions were analyzed on Western blot using anti-c-Myc antibody for presence of pIFNscFv. The results are shown in Figure 6B.
  • an antiproliferative assay was conducted. Two fold serial dilutions of pIFNscFv in growth meida were added to individual wells of a 96-well tissue culture plate (Sarstedt). IFN-Con was added to each well being treated at indicated amounts (See Figures 9A and 9B). 3 xlO 3 Daudi Cells were introduced to each well. The cultures were then incubated at 37°C at 5% CO 2 humidification for 96 hours. At end of incubation period, an MTT assay (Boehringer Mannheim) was performed in accordance with manufacturer instructions for all culture conditions. Relative to control of untreated cells, the percentage of growth inhibition owas calculated.
  • FIGs 9 A and 9B The results demonstrate that addition of C219scFv alone to Daudi cells does not affect their growth. However, addition of the pIFNscFv appears to inhibit the growth-regulatory effects of endogenously produced IFN in Daudi cells (Fig. 9A).
  • Figure 9B demonstrates that at high doses of IFN, that exhibit maximal growth inhibitory activity, competition with the partial mimetic, pIFNscFv, results in soma ablation of the IFN-induced growth inhibitory effects at the highest doses of competitor mimetic.
  • Example 8 Preparation of Complete IFNscFV mimetic (cIFNscFV)
  • the scaffold needs to accommodate three IRRPs as opposed to two as in the pIFNscFv construct.
  • the construct encoded by the nucleotide sequence of Figure 13 (SEQ. ID. NO. 32) encoding the amino acid sequence also shown in Figure 13 (SEQ. ID. NO. 33) was constructed.
  • amino acid 362 (Ser 362) in Figure 13 is not present in theTGl E.Coli expressed cIFNscFv constructed in this example. However, it is present in the original c219scFv protein.
  • the co-ordinates for Ser362 Gly363 Ser364 are not reported in the B chain. However, in the D chain, these coordinates are reported.
  • the B chain was used in the present structural modeling studies and so.
  • the Ser362 was not include in the present cIFNscFv cDNA sequnce but the Gly363 and Ser362 were included since they comprise a unique BanHI cloning site. .
  • amino acid 362 can made with or be reinserted into the protein.
  • Figure 11 is a plasmid map showing the restirictions introduced into the construct of the scFv scaffold.
  • Figure 11 is a plasmid map of C219 scFv-IFN (cIFNscFv) in pSJF2 vector. The construct was directionally cloned from BsiWI to BanHI. The numbers beside the restriction sites refer to nucleotide numbers within the construct.
  • Figure 15 includes the nucleic acid (SEQ. ID. NO. 18) and amino acid (SEQ. ID. NO.
  • C219scFv begins at amino acid position 26 of the Figure.
  • the sequence 5' to the scFv protein codes for the OmpA sequence.
  • Amino Acid 278-288 is the c-Myc tag, the His tag follows.
  • the scFV coding region was then changed as indicated in Figure 10.
  • the nucleotide sequence (SEQ. ID. NO. 42) encoding for the CDR3 loop of the VH domain (SEQ. ID. NO. 45) was replaced with a nucleotide sequence (SEQ. ID. NO. 36) comprising encoding sequence for IFN residues 122-157 (SEQ. ID. NO. 39) that includes IRRP3 sequence.
  • a poly glycine (G ) tract was added to the C-terminal ofthe 122-157 IFN sequence to allow a flexible link between the IFN and antibody sequences.
  • a poly-glycine (G 3 ) linker was added to the C- terminal of IRRP1 and a 15 A (GGGGS) 3 linker was added to the N-terminal of IRRP1.
  • the cIFNscFv vector construct (pSJF2-cIFNscFv), was espressed in TGI cells with and without IPTG as indicated in Figure 12.
  • An anti-c-Myc Western immunoblot of whole cell lysates was used to screen for the presence of the c-Myc tagged cIFNscFv mimetic.
  • An IPTG band which corresponds to the expected molecular weight of the cIFNscFv mimetic is present in lane 4. The band indicates that the product is about 37kDa. Two bands can actually be seen on the immunoblot, the higher band corresponds tounprocessed protein, i.e., signal sequence is still present.
  • TTC CAG GAC SEQ. ID. NO. 52
  • GGA GGT GGC (SEQ. ID. NO. 34) Phe Tyr Thr Glu Leu Tyr Gin Gin Leu Asn Asp Leu Glu Gly
  • FIG. 1 A first figure.
  • Binding isotherms 3.5x10 5 T98G cells were incubated for 2 hr at +4°C with the indicated concentrations of 1 5 I-IFN-. ⁇ Com, (A), 125 I-4-155(S98)IFN-_ 2a , (B), or 1 5 I-IFN- ⁇ lN ⁇ 4 (C). Inset into A, B and C are the corresponding Scatchard plots.
  • 3.5xl0 5 T98G cells were incubated at +4°C for 2 hr with 20 ng/ml 125 I-(4-155)IFN- ⁇ 2a, (C), or 8 ng/ml 1 5 I-4-155(L98)IFN-- ⁇ 2a, (D), containing no unlabeled competitor (100%) bound) or the indicated concentrations of IFNs.
  • Model for the tertiary structure of Type I IFNs This model inco ⁇ orates a helical bundle core, composed of the 5 helices A-E.
  • the loop structures that constitute the proposed receptor recognition epitopes, residues 29-35 and 130-140, shown here as heavily shaded, broad lines, are aligned such that they dock in the receptor groove as shown.
  • the third region implicated in the active conformation ofthe Type I IFNs, 78- 95, is not buried in the receptor groove and is configured to allow binding to its cognate epitope on another Type 1 IFN receptor.
  • the shaded areas in helices C and D represent residues that are critical for maintaining the correct structural presentation ofthe corresponding contiguous recognition epitopes (see text).
  • FIG. 6 Coomassie stain (A) and corresponding Western blot (B) of periplasmic extract purified on a Ni-agarose column. Purified C219scFv was used as a positive control. The Load fio-thru lane indicates protein that is not binding to the column. 6mM imidazole washes elute protein species that have bound non-specifically to the column. The anti-c-Myc Western blot shows the expected IFNscFv band at approximately 40kDa. Fractions collected during elution with buffer containing lOOmM Imidazole are numbered 1 through 10. 16 ⁇ L from 2mL fractions were resolved in 12% SDS-polyacrylamide gels.
  • FIG. 8A is a consensus interferon alpha carbon trace showing the tertiary structure of interferon alpha and three IFN binding domains.
  • FIG. 8B is a consensus C219scFv carbon trace, wherein the CDRs in the variable light chain region are highlighted.

Abstract

L'invention concerne des constructions peptidiques fixant les récepteurs des cytokines et leurs procédés de production. Ces constructions sont de préférence des constructions peptidiques fixant les récepteurs des interférons comprenant au moins un interféron, un domaine de fixation des récepteurs introduit dans une plate-forme structurale appropriée permettant de maintenir le domaine de fixation dans une configuration adaptée à la fixation du récepteur des interférons, et un interféron. Les constructions peptidiques fixant les interférons comprennent de préférence trois domaines fixant les interférons, un des domaines fixant les interférons des interférons humains du type 1 (régions d'acides aminés 10-35, 78-107 et 123-166, de préférence 29-35, 78-95 et 130-140, respectivement), la plate-forme structurale permettant de maintenir les trois domaines dans une configuration adaptée à la fixation sur leurs sites de fixation sur le récepteur des interférons de type I. La construction résultante présente de préférence une activité biologique comparable à celle des interférons naturels.
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