WO2011100455A1

WO2011100455A1 - Inhibition of antibody responses to foreign proteins

Info

Publication number: WO2011100455A1
Application number: PCT/US2011/024382
Authority: WO
Inventors: David J. Fitzgerald; Ira H. Pastan; Masanori Onda; John O'shea; Craig Thomas; Jian-kang JIANG
Original assignee: The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services
Priority date: 2010-02-12
Filing date: 2011-02-10
Publication date: 2011-08-18

Abstract

The present invention provides methods and compositions for reducing, inhibiting or preventing the development and/or production of neutralizing antibodies against therapeutic foreign proteins by co-administering the therapeutic foreign protein with a Janus kinase 3 (JAK3) inhibitor.

Description

INHIBITION OF ANTIBODY RESPONSES TO FOREIGN PROTEINS CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of US Patent Application No. 61/304,293, filed February 12, 2010 which is incorporated herein by reference. FIELD OF THE INVENTION

[0002] The present invention relates to methods and compositions for suppressing neutralizing antibody responses against therapeutic foreign proteins.

BACKGROUND OF THE INVENTION

[0003] Immunoconjugates have been developed as an alternative therapeutic approach to treat malignancies. Immunoconjugates were originally composed of an antibody chemically conjugated to a plant or a bacterial toxin, a form that is known as an immunotoxin. The antibody binds to the antigen expressed on the target cell and the toxin is internalized causing cell death by arresting protein synthesis and inducing apoptosis (Brinkmann, U., Mol. Med. Today, 2:439- 446 (1996)). More recently, genes encoding the antibody and the toxin have been fused and the immunotoxin expressed as a fusion protein.

[0004] A number of studies have been conducted on immunotoxins which use as the toxic moiety a bacterial toxin known as Pseudomonas exotoxin A ("PE"). Typically, the PE has been truncated or mutated to reduce its non-specific toxicity without destroying its toxicity to cells to which it is targeted by the targeting portion of the immunotoxin. Clinical trials are currently underway testing the use of PE-based immunotoxins as treatments for a variety of cancers.

[0005] Current PE-based immunotoxins are highly immunogenic. Antibody formation limits the number of treatment cycles that can be given to patients with solid tumors who have normal immune systems but is less of a problem in the treatment of hematological malignancies, in which the ability of the immune system to mount a response is often compromised and several treatment cycles can be given before an immune response develops. Since many protocols call for a three week period between administration of immunotoxins, the development of the antibodies during this period effectively means that, for solid tumors, usually only one administration can be made of a PE-based immunotoxin before the patient's antibodies render it ineffective. Neutralizing antibody responses can also hamper the therapeutic treatment of hematological malignancies. [0006] Immunogenicity is also problem with the administration of other therapeutic foreign proteins. Thus, it has been suggested that this problem can be addressed by administering immunosuppressive agents together with the immunotoxin or other therapeutic protein. The results of experiments testing this approach have not been uniform {see Frankel, Clin, Cancer Res. (2004) 10: 13-15,). For example, cyclophosphamide and cyclosporine did not prevent antibody production in patients treated with a mouse monoclonal antibody-ricin A chain conjugate (Oratz, et al, J. Biol. Response Modif., ( 1990) 9: 345-354, and Selvaggi, et al, J. Immunother., (1993) 13: 201-207). CTLA4Ig, however, was able to block anti-immunotoxin immunogenicity in rodents and dogs (Siegall et al. , J. Immunol, (1997) 159: 5168-5173).

Deoxysperguahn was able to reduce the anti-immunotoxin immune response in rodents and dogs (Pai et al, Cancer Res., (1990) 50: 7750-7753) but not substantially in primates (Hubbard et al, Hum. Immunol, (2001) 62: 479-487). Anti-CD4 monoclonal antibodies blocked anti- immunotoxin antibody generation in mice (Jin et al , J. Immunol, (1991) 146: 1806-181 1). Rituximab has been tested in two clinical studies in combination with recombinant Pseudomonas exotoxin immunotoxins. In one study, inhibition of anti-immunotoxin antibody production was observed after administration of rituximab (Saleh et al, Proc. Am. Soc. Clin. Oncol, (2002) 21 : 28a). Another study found that rituximab did not prevent the anti-immunotoxin antibody response (Hassan et al, Clin. Cancer Res., (2004) 10: 16-18).

[0007] The Janus kinases, JAK1 , JAK2, JAK3, and Tyk2, are cytoplasmic protein tyrosine kinases involved in cytokine receptor signal transduction through the STAT (signal transducers and activators of transcription) proteins. Binding of cytokines activates the Janus kinases which phosphorylate and activate the STAT proteins. JAK3, which is associated with the cytokine signaling through the IL-2 (and other ) receptor (s), is important to lymphocyte survival, differentiation, and function. Thus, inhibition of JAK3 has been recognized as potential approach for treatment of autoimmune diseases and to inhibit immune responses in organ transplantation. [0008] CP-690,550, an exemplary JAK 3 inhibitor has been shown to effect immunosuppression in the context of solid organ transplantation and treatment of the

autoimmune disease, rheumatoid arthritis. See, e.g., van Gurp, et al., Transplantation (2009) 87(l):79-86; van Gurp, et al., Am J Transplantation (2008) 8: 171 1-1718; Conklyn, et al., J Leukocyte Biol (2004) 76: 1248- 1255 and Kremer, et al, Arthritis Rheum. (2009) 60(7): 1895- 905. Administration of CP-690,550 to human patients resulted in decreased numbers of CD4+ T-cells and natural killer ("NK") cells. The number of B-cells increased, and CD8+ T cells did not change.

[0009] A need exists to improve administration of therapeutic foreign proteins by reducing the antibody response against the protein in the patient. The present invention addresses this and other needs.

BRIEF SUMMARY OF THE INVENTION

[0010] The present invention provides compositions and methods for reducing, inhibiting and preventing the development and production of neutralizing antibodies that bind to and inactivate a therapeutic foreign protein in a patient. Accordingly, in one aspect, the invention provides methods of reducing, inhibiting or preventing a neutralizing antibody response to a therapeutic foreign protein in a patient in need thereof. In some embodiments, the methods comprise co-administering to the patient the foreign protein and a JAK3 inhibitor {e.g., CP-690,550), thereby reducing, inhibiting or preventing the neutralizing antibody response to the therapeutic foreign protein.

[0011] In another aspect, the invention provides compositions comprising a mixture of a therapeutic protein that elicits neutralizing antibodies against the protein in a human and the JAK3 inhibitor.

[0012] With respect to the embodiments of the methods and compositions, in some embodiments, the patient is a human and the foreign protein is a non-human protein.

[0013] In some embodiments, the foreign protein is a bacterial protein or a protein expressed in a bacterial cell. In some embodiments, the foreign protein is a viral protein or a protein expressed by a virus. In some embodiments, the foreign protein is a plant protein or a protein expressed in a plant cell. In some embodiments, the foreign protein is a fungal protein or a protein expressed in a fungal cell. In some embodiments, the foreign protein is a yeast protein or a protein expressed in a yeast cell.

[0014] In some embodiments, the foreign protein is an antibody.

[0015] In some embodiments, the foreign protein is a protein that is not endogenously expressed by the patient. For example, the patient may not express the foreign protein at all, or may express a mutated form of the foreign protein, e.g., due to a mutation, substitution, deletion, or addition in the gene encoding the foreign protein.

[0016] In some embodiments, the foreign protein is a chimeric molecule comprising a targeting moiety and a cytotoxin moiety. In some embodiments, the chimeric molecule is an immunotoxin comprising an antibody against a cell surface antigen on a tumor cell and a cytotoxin moiety.

[0017] In some embodiments, the cell surface antigen is on a lymphocytic cell. In some embodiments, the cell surface antigen is selected from the group consisting of CD 19, CD21 , CD22, CD25, CD30, CD33, CD79b, transferrin receptor, EGF receptor, mesothelin, cadherin and Lewis Y.

[0018] In some embodiments, the antibody is selected from the group consisting of B3, RFB4, SS I , SS 1P, SS 1P-LR, MN, HN 1 , HN2 and HB21.

[0019] In some embodiments, the cytotoxin moiety is selected from Pseudomonas exotoxin A, diphtheria toxin, cholix toxin, cholera exotoxin, shiga toxin, ricin toxin and pokeweed antiviral protein (PAP). In some embodiments, the cytotoxin moiety is a Pseudomonas exotoxin A. In some embodiments, the Pseudomonas exotoxin A is selected from the group consisting of PE25, PE35, PE38, PE40, Domain III of PE, PE-LR, PE-6X, PE-LR/6X, PE-8X, PE-LR/8X, and variants thereof.

[0020] In some embodiments, the immunotoxin is selected from the group consisting of LMB- 2, LMB-7, LMB-9, BL22, HA22, HA22-LR, HA22-LR/6X, HA22-LR/8X, SS 1P, SS 1P-LR, SS 1P-LR/6X and SS 1 P-LR/8X.

[0021] In some embodiments, the foreign protein and the CP-690,550 are administered concurrently. In some embodiments, the foreign protein and the CP-690,550 are administered sequentially. [0022] In some embodiments, the JAK3 inhibitor is administered in an extended-release formulation.

[0023] In some embodiments, the patient has already produced neutralizing antibodies to the foreign protein. In some embodiments, the patient has not produced neutralizing antibodies to the foreign protein.

[0024] In some embodiments, the patient is human.

[0025] Further embodiments of the invention are described herein, e.g., in the detailed description of the invention.

DEFINITIONS [0026] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0027] A "Janus kinase 3 inhibitor" or "JAK3 inhibitor" is compound capable of inhibiting the activity JAK3 such that signal transduction through STAT proteins is inhibited. In a typical embodiment of the invention, the JAK3 inhibitor reduces, inhibits or prevents a neutralizing antibody immune response against a therapeutic foreign protein (e.g. , an immunotoxin). A number of JAK3 inhibitors are known. Examples include: leflunomide (5-methyl-N-[4- (trifluoromethyl) phenyl]-isoxazole-4-carboxamide, CAS Number 75706-12-6); CP-690,550 ((3R,4R)-4-Methyl-3-(methyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-P-oxo-l - piperidinepropanenitrile, CAS Number: 477600-75-2); PF-956980 (Changelian et al , Blood (2008) 1 1 1 -2155-2157); WHI-P131 (4-[(6,7-dimethoxy-4-quinazolinyl)amino]-phenol CAS

Number 202475-60-3); WHI-P 154 (2-Bromo-4-[(6,7-dimethoxy-4-quinazolinyl)amino]phenol, CAS Number 21 1555-04-3); and PNU 156804 (Mortellaro et al, J. Immunol. ( 1999) 162:7102- 7109). [0028] Other known inhibitors include pyrrolopyrimidine-based inhibitors (Clark et al, Bioorg Med Chem Lett (2007) 17: 1250-1253; pyrimidine-based inhibitors (Chen et al, Bioorg Med Chem Lett (2006) 17:5633-5638), and staurosporine analogs Yang et al.,. Bioorg Med Chem Lett (2007) 17:326-331. See also US Patent 7,491 ,732 and US Patent Application No.

2008/0194603. For a review, see, Vassilev et al, Curr Drug Targets (2006) 7:327-343.

[0029] The term "CP-690,550" refers to a JAK3 inhibitor with the chemical name (3R,4R)-4- Methyl-3-(methyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-P-oxo-l -piperidinepropanenitrile and CAS Registry Number: 477600-75-2. The structure of CP-690,550 is as follows:

[0030] CP-690,550 is commercially available, e.g. , from LC Laboratories (Woburn, MA, on the internet at lclabs.com), and Selleck Chemicals LLC, available through VWR Intl (on the internet at selleckchem.com and vwr.com).

[0031] The term "therapeutic foreign protein" refers to a protein that is a foreign antigen to the patient to whom it is administered. Therefore, the patient oftentimes will mount an immune response with neutralizing antibodies against the foreign protein. For example, foreign protein can be a protein from a species other than the patient {e.g., administering a bacterial, viral or plant or otherwise non-mammalian protein to a mammal, or administering a non-human protein to a human). The foreign protein can also be a protein that is not expressed or not functionally expressed in the patient, e.g. , in the case of replacement protein therapies {e.g., administering Factor VIII to a hemophiliac).

[0032] "Exogenous protein" or "heterologous protein" as used herein refers to a protein not naturally present in a particular tissue or cell, a protein that is the expression product of an exogenous expression construct or transgene, or a protein not naturally present in a given quantity in a particular tissue or cell. As used herein, exogenous proteins and heterologous proteins are foreign proteins to the immune system of a patient. [0033] The term "neutralizing antibody response" refers to the generation of antibodies in a patient that bind to and reduce or diminish the activity for its intended therapeutic purpose of an administered foreign protein. The activity of the foreign protein can be reduced by a detectable amount, e.g., 10%, 25%, 50%, 75%, or 100% (i.e., completely inactivated), e.g., in comparison to the activity of the foreign protein in the absence of or prior to eliciting the neutralizing antibody response. The activity of the foreign protein will depend on the foreign protein (e.g., target antigen binding, target cell killing, protein replacement therapy, etc.), and can be determined by any method known in the art.

[0034] "CD22" refers to a lineage-restricted B cell antigen belonging to the Ig superfamily. It is expressed in 60-70%) of B cell lymphomas and leukemias and is not present on the cell surface in early stages of B cell development or on stem cells. See, e.g. Vaickus et al., Crit. Rev.

Oncol/Hematol. 11 :267-297 (1991 ).

[0035] As used herein, the term "anti-CD22" in reference to an antibody that specifically binds CD22 and includes reference to an antibody which is generated against CD22. In preferred embodiments, the CD22 is a primate CD22, such as human CD22. In one preferred

embodiment, the antibody is generated against human CD22 synthesized by a non-primate mammal after introduction into the animal of cDNA which encodes human CD22.

[0036] "CD25" or "Tac" refers to the alpha chain of the IL-2 receptor (IL2R). It is a type I transmembrane protein present on activated T cells, activated B cells, some thymocytes, myeloid precursors, and oligodendrocytes that associates with CD 122 to form a heterodimer that can act as a high-affinity receptor for IL-2. CD25 expressed in most B-cell neoplasms, some acute nonlymphocytic leukemias, and neuroblastomas.

[0037] As used herein, the term "anti-CD25" in reference to an antibody that specifically binds CD25 and includes reference to an antibody which is generated against CD25. In preferred embodiments, the CD25 is a primate CD25, such as human CD25. In one preferred

embodiment, the antibody is generated against human CD25 synthesized by a non-primate mammal after introduction into the animal of cDNA which encodes human CD25.

[0038] The term "mesothelin" refers to a protein and fragments thereof present on the surface of some human cells and bound by, for example, the Kl antibody. Nucleic acid and amino acid sequences of mesothelin are set forth in, for example, PCT published application WO 97/25,068 and U.S. Patent Nos. 6,083,502 and 6,153,430. See also, Chang, K. & Pastan, I., Int. J. Cancer 57:90 (1994); Chang, K. & Pastan, I., Proc. Nat'l Acad. Set USA 93:136 (1996); Brinkmann U., et al, Int. J. Cancer 71:638 (1997); Chowdhury, P.S., et a!., Mol. Immunol. 34:9 (1997), and U.S. Patent No. 6,809,184. Mesothelin is expressed as a precursor protein of approximately 69 kDa, that then is processed to release a 30 kDa protein, while leaving attached to the cell surface the 40 kDa glycosylphosphatidyl inositol linked cell surface glycoprotein described in the Background. The 40 kDa glycoprotein is the one referred to by the term "mesothelin" herein. The nucleic acid and amino acid sequences of mesothelin have been recorded from several species, e.g., human (NM_005823.4→NP_005814.2; and NM_013404.3→NP_037536.2), mouse (NM_018857.1→NP_061345.1 ), rat (NM_031658.1→NP_1 13846.1 ), bovine

(NM_001 100374.1→NP_001093844).

[0039] "RFB4" refers to a mouse IgGl monoclonal antibody that specifically binds to human CD22. RFB4 is commercially available under the name RFB4 from several sources, such as Southern Biotechnology Associates, Inc. (Birmingham AL; Cat. No. 9360-01), Autogen Bioclear UK Ltd. (Calne, Wilts, UK; Cat. No. AB147), Axxora LLC. (San Diego, CA). RFB4 is highly specific for cells of the B lineage and has no detectable cross-reactivity with other normal cell types. Li et al., Cell. Immunol. 1 18:85-99 (1989). The heavy and light chains of RFB4 have been cloned. See, Mansfield et al., Blood 90:2020-2026 (1997), which is incorporated herein by reference. [0040] As used herein, "antibody" includes reference to an immunoglobulin molecule immunologically reactive with a particular antigen, and includes both polyclonal and monoclonal antibodies. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies), recombinant single chain Fv fragments (scFv), and disulfide stabilized (dsFv) Fv fragments (see, co-owned U.S. Patent No. 5,747,654, which is incorporated herein by reference). The term "antibody" also includes antigen binding forms of antibodies (e.g., Fab', F(ab')₂, Fab, Fv and rlgG. See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Goldsby et al., eds., Kuby, J., Immunology, 4th Ed., W.H. Freeman & Co., New York (2000).

[0041] An antibody immunologically reactive with a particular antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, see, e.g., Huse, et al, Science 246:1275-1281 (1989); Ward, et al, Nature 341:544-546 (1989); and Vaughan, et al, Nature Biotech. 14:309-314 (1996), or by immunizing an animal with the antigen or with DNA encoding the antigen.

[0042] Typically, an immunoglobulin has a heavy and light chain. Each heavy and light chain contains a constant region and a variable region, (the regions are also known as "domains"). Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs". The extent of the framework region and CDRs have been defined. See, Kabat and Wu, supra. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three dimensional space.

[0043] The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDRl , CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a V_L CDRl is the CDRl from the variable domain of the light chain of the antibody in which it is found.

[0044] References to "V_H" or a "VH" refer to the variable region of an immunoglobulin heavy chain, including an Fv, scFv , dsFv or Fab. References to "V_L" or a "VL" refer to the variable region of an immunoglobulin light chain, including of an Fv, scFv , dsFv or Fab.

[0045] The phrase "single chain Fv" or "scFv" refers to an antibody in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form one chain. Typically, a linker peptide is inserted between the two chains to allow for proper folding and creation of an active binding site. [0046] The phrase "disulfide bond" or "cysteine-cysteine disulfide bond" refers to a covalent interaction between two cysteines in which the sulfur atoms of the cysteines are oxidized to form a disulfide bond. The average bond energy of a disulfide bond is about 60 kcal/mol compared to 1 -2 kcal/mol for a hydrogen bond.

[0047] The phrase "disulfide stabilized Fv" or "dsFv" refer to the variable region of an immunoglobulin in which there is a disulfide bond between the light chain and the heavy chain. In the context of this invention, the cysteines which form the disulfide bond are within the framework regions of the antibody chains and serve to stabilize the conformation of the antibody. Typically, the antibody is engineered to introduce cysteines in the framework region at positions where the substitution will not interfere with antigen binding. [0048] The term "linker peptide" includes reference to a peptide within an antibody binding fragment (e.g., Fv fragment) which serves to indirectly bond the variable domain of the heavy chain to the variable domain of the light chain.

[0049] The term "parental antibody" means any antibody of interest which is to be mutated or varied to obtain antibodies or fragments thereof which bind to the same epitope as the parental antibody, but with higher affinity.

[0050] The term "hotspot" means a portion of a nucleotide sequence of a CDR or of a framework region of a variable domain which is a site of particularly high natural variation. Although CDRs are themselves considered to be regions of hypervariability, it has been learned that mutations are not evenly distributed throughout the CDRs. Particular sites, or hotspots, have been identified as these locations which undergo concentrated mutations. The hotspots are characterized by a number of structural features and sequences. These "hotspot motifs" can be used to identify hotspots. Two consensus sequences motifs which are especially well characterized are the tetranucleotide sequence RGYW and the serine sequence AGY, where R is A or G, Y is C or T, and W is A or T. [0051] An "immunoconjugate" is a molecule comprised of a targeting portion, or moiety, such as an antibody or fragment thereof which retains antigen recognition capability, and an effector molecule, such as a therapeutic moiety or a detectable label.

[0052] An "immunotoxin" is an immunoconjugate in which the therapeutic moiety is a cytotoxin. [0053] A "targeting moiety" is the portion of an immunoconjugate intended to target the immunoconjugate to a cell of interest. Typically, the targeting moiety is an antibody, a scFv, a dsFv, an Fab, or an F(ab')₂.

[0054] The term "toxin" or "cytotoxin" includes reference to abrin, ricin, Pseudomonas exotoxin A (or "PE"), diphtheria toxin ("DT"), cholix toxin ("CT"), cholera exotoxin ("CET"), botulinum toxin, pokeweed antiviral protein or modified toxins thereof. For example, PE and DT are highly toxic compounds that typically bring about death through liver toxicity.

Cytotoxins, however, can be modified into a form for use as an immunotoxin by removing the native targeting component of the toxin (e.g. , domain la of PE or the B chain of DT) and replacing it with a different targeting moiety, such as an antibody. See, e.g. , Kreitman, The AAPS Journal (2006) 8(3):E532-551 and the references cited therein. Preferred toxins inhibit protein synthesis, e.g., are ADP-ribosylating agents or ribosomal inactivating agents.

[0055] Pseudomonas exotoxin A ("PE") is an extremely active monomeric protein (molecular weight 66 kD), secreted by Pseudomonas aeruginosa, which inhibits protein synthesis in eukaryotic cells. The native PE sequence (SEQ ID NO.: l ) is set forth in U.S. Patent No.

5,602,095, incorporated herein by reference. The method of action and structure of PE, as well as the modifications resulting in a number of variants of PE, are discussed in some detail in a section devoted to this purpose within.

[0056] Mutations of PE are described herein by reference to the amino acid residue present at a particular position of the 613-amino acid sequence of native PE (SEQ ID NO: l ), followed by the amino acid with which that residue has been replaced in the particular mutation under discussion. Thus, for example, the term "R490A" indicates that the "R" (arginine, in standard single letter code) at position 490 of the referenced molecule is replaced by an "A" (alanine, in standard single letter code), while "K590Q" indicates that the lysine normally present at position 590 has been replaced with a glutamine. The standard single letter code for common amino acids is set forth below.

[0057] As indicated by the preceding paragraph, the term Pseudomonas exotoxin A ("PE") as used herein includes reference to forms of PE which have been modified but which retain cytotoxic function. Thus, the PE molecule can be truncated to provide a fragment of PE which is cytotoxic but which does not bind cells, as in the fragments known as PE38 and PE40, or can have mutations which reduce non-specific binding, as in the version called "PE4E", in which four residues are mutated to glutamic acid. Further, a portion of the PE sequence can be altered to increase toxicity, as in the form called "PE38 DEL", in which the C-terminal sequence of native PE is altered, or the form of PE discussed herein, in which the arginine corresponding to position 490 of the native PE sequence is replaced by alanine, glycine, valine, leucine, or isoleucine. [0058] As used herein, the terms "Cholix toxin" or "CT" and "Cholera exotoxin" or "CET" refer to a toxin expressed by some strains of Vibrio cholerae that do not cause cholera disease. According to the article reporting the discovery of the Cholix toxin (Jorgensen, R. et ah, J Biol Chem. 283(16):10671-10678 (2008)), mature cholix toxin is a 70.7 kD, 634 residue protein, Figure 9C of PCT/US2009/046292. The Jorgensen authors deposited in the NCBI Entrez Protein database a 642-residue sequence which consists of what they termed the full length cholix toxin A chain plus, at the N-terminus an additional 8 residues, consisting of a 6 histidine tag (SEQ ID NO: 10) flanked by methionine residues, presumably introduced to facilitate expression and separation of the protein. The 642-residue sequence is available on-line in the Entrez Protein database under accession number 2Q5T A and can be converted to the 634 amino acid sequence by simply deleting the first 8 amino acids of the deposited sequence. Mature CT has four domains: Domain la (amino acid residues 1 -269), Domain II (amino acid residues 270-386), Domain lb (amino acid residues 387-415), and Domain III (amino acid residues 417-634).

[0059] As used herein, the terms "Cholera exotoxin" or "CET" refer to a toxin expressed by some strains of Vibrio cholerae that do not cause cholera disease and include mature CET and cytotoxic fragments thereof. Mature cholera exotoxin (CET) is a 634 amino acid residue protein whose sequence is set forth as in Figure 9C of PCT/US2009/046292 (SEQ ID NO:2). For convenience of reference, the terms "cholera exotoxin," and "CET" as used herein may refer to the native or mature toxin, but more commonly refer to forms in which the toxin has been modified to reduce non-specific binding, for example, by deletion of domain la, or otherwise improve its utility for use in immunotoxins. A CET protein may be a full-length CET protein or it may be a partial CET protein comprising one or more subdomains of a CET protein and having cytotoxic activity as described herein. Mature CET has four domains: Domain la (amino acid residues 1-269), Domain II (amino acid residues 270-386), Domain lb (amino acid residues 387- 415), and Domain III (amino acid residues 417-634).

[0060] The term "contacting" includes reference to placement in direct physical association.

[0061] An "expression plasmid" comprises a nucleotide sequence encoding a molecule or interest, which is operably linked to a promoter.

[0062] As used herein, "polypeptide", "peptide" and "protein" are used interchangeably and include reference to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms also apply to polymers containing conservative amino acid substitutions such that the protein remains functional.

[0063] The term "residue" or "amino acid residue" or "amino acid" includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively "peptide"). The amino acid can be a naturally occurring amino acid and, unless otherwise limited, can encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.

[0064] The amino acids and analogs referred to herein are described by shorthand designations as follows in Table A:

Table A: Amino Acid Nomenclature

Name 3-letter 1 -letter

Alanine Ala A

Arginine Arg R

Asparagine Asn N

Aspartic Acid Asp D

Cysteine Cys C

Glutamic Acid Glu E

Glutamine Gin Q

Glycine Gly G

Histidine His H

Homoserine Hse -

Isoleucine He I

Leucine Leu L

Lysine Lys K

Methionine Met M

Methionine sulfoxide Met (O) -

Methionine

methylsulfonium Met (S-Me) -

Norleucine Nle - Phenylalanine Phe F

Proline Pro P

Serine Ser S

Threonine Thr T

Tryptophan Trp W

Tyrosine Tyr Y

Valine Val V

[0065] A "conservative substitution", when describing a protein refers to a change in the amino acid composition of the protein that does not substantially alter the protein's activity. Thus, "conservatively modified variations" of a particular amino acid sequence refers to amino acid substitutions of those amino acids that are not critical for protein activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitutions of even critical amino acids do not substantially alter activity. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups in Table B each contain amino acids that are conservative substitutions for one another:

Table B

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

See also, Creighton, PROTEINS, W.H. Freeman and Company, New York ( 1984). [0066] The terms "substantially similar" in the context of a peptide indicates that a peptide comprises a sequence with at least 90%, preferably at least 95% sequence identity to the reference sequence over a comparison window of 10-20 amino acids. Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0067] The terms "conjugating," "joining," "bonding" or "linking" refer to making two polypeptides into one contiguous polypeptide molecule. In the context of the present invention, the terms include reference to joining an antibody moiety to an effector molecule (EM). The linkage can be either by chemical or recombinant means. Chemical means refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule. [0068] As used herein, "recombinant" includes reference to a protein produced using cells that do not have, in their native state, an endogenous copy of the DNA able to express the protein. The cells produce the recombinant protein because they have been genetically altered by the introduction of the appropriate isolated nucleic acid sequence. The term also includes reference to a cell, or nucleic acid, or vector, that has been modified by the introduction of a heterologous nucleic acid or the alteration of a native nucleic acid to a form not native to that cell, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, express mutants of genes that are found within the native form, or express native genes that are otherwise abnormally expressed, underexpressed or not expressed at all. [0069] As used herein, "nucleic acid" or "nucleic acid sequence" includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence includes the complementary sequence thereof as well as conservative variants, i.e., nucleic acids present in wobble positions of codons and variants that, when translated into a protein, result in a conservative substitution of an amino acid.

[0070] As used herein, "encoding" with respect to a specified nucleic acid, includes reference to nucleic acids which comprise the information for translation into the specified protein. The information is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the "universal" genetic code. However, variants of the universal code, such as is present in some plant, animal, and fungal mitochondria, the bacterium Mycoplasma capricolum (Proc. Nat Ί Acad. Sci. USA 82:2306-2309 (1985), or the ciliate Macron cleus, may be used when the nucleic acid is expressed in using the translational machinery of these organisms.

[0071] The phrase "fusing in frame" refers to joining two or more nucleic acid sequences which encode polypeptides so that the joined nucleic acid sequence translates into a single chain protein which comprises the original polypeptide chains.

[0072] As used herein, "expressed" includes reference to translation of a nucleic acid into a protein. Proteins may be expressed and remain intracellular, become a component of the cell surface membrane or be secreted into the extracellular matrix or medium. [0073] By "host cell" is meant a cell which can support the replication or expression of the expression vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells.

[0074] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.

[0075] As used herein, the term "substantially identical," in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least 60%, more preferably 65%, even more preferably 70%, still more preferably 75%, even more preferably 80%, and most preferably 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher nucleotide or amino acid residue identity, when compared and aligned for maximum

correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues. In a most preferred embodiment, the sequences are substantially identical over the entire length of the coding regions.

[0076] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. [0077] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr.,

Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, F.M. Ausubel et ah, eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

[0078] Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25: 3389- 3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the Web at "ncbi.nlm.nih.gov/"). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 1 1 , an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0079] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g. , Karlin & Altschul, iVoc. Nat l Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1 , more preferably less than about 0.01 , and most preferably less than about 0.001 . [0080] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.

[0081] The term "in vivo" includes reference to inside the body of the organism from which the cell was obtained. "Ex vivo" and "in vitro " means outside the body of the organism from which the cell was obtained. [0082] The phrase "malignant cell" or "malignancy" refers to tumors or tumor cells that are invasive and/or able to undergo metastasis, i.e., a cancerous cell.

[0083] As used herein, "mammalian cells" includes reference to cells derived from mammals including humans, rats, mice, guinea pigs, chimpanzees, or macaques. The cells may be cultured in vivo or in vitro. [0084] The term "selectively reactive" refers, with respect to an antigen, the preferential association of an antibody, in whole or part, with a cell or tissue bearing that antigen and not to cells or tissues lacking that antigen. It is, of course, recognized that a certain degree of nonspecific interaction may occur between a molecule and a non-target cell or tissue. Nevertheless, selective reactivity, may be distinguished as mediated through specific recognition of the antigen. Although selectively reactive antibodies bind antigen, they may do so with low affinity. On the other hand, specific binding results in a much stronger association between the antibody and cells bearing the antigen than between the bound antibody and cells lacking the antigen. Specific binding typically results in greater than 2-fold, preferably greater than 5-fold, more preferably greater than 10-fold and most preferably greater than 100-fold increase in amount of bound antibody (per unit time) to a cell or tissue bearing CD22 as compared to a cell or tissue lacking CD22. Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats are appropriate for selecting antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York ( 1 988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0085] The term "immunologically reactive conditions" includes reference to conditions which allow an antibody generated to a particular epitope to bind to that epitope to a detectably greater degree than, and/or to the substantial exclusion of, binding to substantially all other epitopes. Immunologically reactive conditions are dependent upon the format of the antibody binding reaction and typically are those utilized in immunoassay protocols or those conditions encountered in vivo. See Harlow & Lane, supra, for a description of immunoassay formats and conditions. Preferably, the immunologically reactive conditions employed in the methods of the present invention are "physiological conditions" which include reference to conditions (e.g., temperature, osmolarity, pH) that are typical inside a living mammal or a mammalian cell.

While it is recognized that some organs are subject to extreme conditions, the intra-organismal and intracellular environment normally lies around pH 7 (i.e., from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the predominant solvent, and exists at a temperature above 0°C and below 50°C. Osmolarity is within the range that is supportive of cell viability and proliferation. [0086] The terms "effective amount" or "amount effective to" or "therapeutically effective amount" includes reference to a dosage of a therapeutic agent sufficient to produce a desired result, such as inhibiting cell protein synthesis by at least 50%, or killing the cell.

[0087] The terms "systemic administration" and "systemically administered" refer to a method of administering a compound or composition to a mammal so that the compound or composition is delivered to sites in the body, including the targeted site of pharmaceutical action, via the circulatory system. Systemic administration includes, but is not limited to, oral, intranasal, rectal and parenteral (i.e., other than through the alimentary tract, such as intramuscular, intravenous, intra-arterial, transdermal and subcutaneous) administration. [0088] The term "co-administer" refers to the simultaneous presence of two active agents (e.g., a therapeutic foreign protein and CP-690,550) in the blood of an individual. Active agents that are co-administered can be concurrently or sequentially delivered.

[0089] As used herein, the terms "treating" and "treatment" refer to delaying the onset of, retarding or reversing the progress of, or alleviating or preventing either the disease or condition to which the term applies (e.g. , the development and production of antibodies that bind to and render inactive a therapeutic foreign protein), or one or more symptoms of such disease or condition.

[0090] The terms "inhibiting," "reducing," "decreasing" with respect to reducing, inhibiting or preventing a neutralizing antibody response against a therapeutic foreign protein refers to inhibiting the development and production of antibodies that bind to and inactivate the therapeutic foreign protein in a subject by a measurable amount using any method known in the art. The antibody response to the therapeutic foreign protein is inhibited, reduced or decreased if the levels of antibodies that bind to and inactivate the therapeutic foreign protein are at least about 10%, 20%, 30%, 50%, 80%, or 100% reduced in comparison to the levels of antibodies that bind to and inactivate the therapeutic foreign protein prior to or in the absence of the co-administration of CP-690,550. In some embodiments, the levels of antibodies that bind to and inactivate the therapeutic foreign protein are inhibited, reduced or decreased by at least about 1 -fold, 2-fold, 3-fold, 4-fold, or more in comparison to levels of antibodies that bind to and inactivate the therapeutic foreign protein prior to or in the absence of administration of

CP-690,550. [0091] As used herein, the phrase "consisting essentially of refers to the genera or species of active pharmaceutical agents included in a method or composition, as well as any excipients inactive for the intended purpose of the methods or compositions. In some embodiments, the phrase "consisting essentially of expressly excludes the inclusion of one or more additional active agents other than the therapeutic foreign protein and CP-690,550.

[0092] The terms "controlled release," "sustained release," "extended release," and "timed release" are intended to refer interchangeably to any drug-containing formulation in which release of the drug is not immediate, i.e., with a "controlled release" formulation, oral administration does not result in immediate release of the drug into an absorption pool. The terms are used interchangeably with "nonimmediate release" as defined in Remington: The Science and Practice of Pharmacy, 21 ^st Ed., Lippincott Williams & Wilkins (2005). As discussed therein, immediate and nonimmediate release can be defined kinetically by reference to the following equation: k k k

Dosage ^r Absorption ^a _> Target ^e _> Form drug Pool absorption Area elimination release

[0093] The "absorption pool" represents a solution of the drug administered at a particular absorption site, and k_r, k_a and k_e are first-order rate constants for (1) release of the drug from the formulation, (2) absorption, and (3) elimination, respectively. For immediate release dosage forms, the rate constant for drug release k_r is far greater than the absorption rate constant k_a. For controlled release formulations, the opposite is true, i.e., k_r «k_a, such that the rate of release of drug from the dosage form is the rate-limiting step in the delivery of the drug to the target area.

[0094] The terms "sustained release" and "extended release" are used in their conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, for example, 12 hours or more, and that preferably, although not necessarily, results in substantially steady-state blood levels of a drug over an extended time period.

[0095] As used herein, the term "delayed release" refers to a pharmaceutical preparation that passes through the stomach intact and dissolves in the small intestine. [0096] The term "patient," "individual" or "subject" interchangeably refer to any mammal, including a human or non-human primate. The mammal can also be a domesticated mammal (e.g., a canine or feline), an agricultural mammal (e.g., equine, bovine, ovine, porcine), or a laboratory mammal (e.g., murine, rattus, lagomorpha, hamster). BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1 illustrates the interval schedules for immunization and bleeds in a mouse model to evaluate antibody responses against injected therapeutic foreign proteins (e.g., immunotoxins), with or without co-administered JAK3 inhibitor CP-690,550.

[0098] Figure 2 illustrates the presence of antibodies that bind to SS IP as measured by immune complex capture ("ICC") ELISA produced in Balb/c mice co-administered 5 μg SS 1 P and 20 mg/kg/day CP-690,550 (column 1 ), 10 mg/kg/day CP-690,550 (column 2), 5 mg/kg/day CP-690,550 (column 3) or polyethylene glycol (PEG) (column 4). The SS I P was administered intraperitoneal ly (ip) and the CP-690,550 was administered via a subcutaneously implanted Alzet pump. [0099] Figure 3 illustrates the presence of antibodies that bind to SS IP as measured by ICC ELISA produced in Balb/c (column 1 ), JAK3 knock-out (column 2), SCID (column 3) and Athymic nude (column 4) mice administered 5 μg SS IP alone.

[0100] Figure 4 illustrates the presence of antibodies that bind to SS 1 P as measured by direct capture ("DC") ELISA produced in Balb/c mice co-administered 5 μg SS I P and 20 mg/kg/day CP-690,550 (column 1), 10 mg/kg/day CP-690,550 (column 2), 5 mg/kg/day CP-690,550 (column 3) or PEG (column 4). The SS IP was administered intraperitoneally (ip) and the CP-690,550 was administered via a subcutaneously implanted Alzet pump.

[0101] Figure 5 illustrates the presence of antibodies that bind to SS I P as measured by DC ELISA produced in Balb/c (column 1 ), JAK3 knock-out (column 2), SCID (column 3) and Athymic nude (column 4) mice administered 5 μg SS IP alone.

[0102] Figure 6 illustrates the presence of antibodies that bind to KLH produced in Balb/c mice co-administered 5 μg KLH and CP-690,550 (column 1), PEG (column 2) or control vehicle (column 3). The KLH was administered intraperitoneally (ip) and the CP-690,550 was administered via a subcutaneously implanted Alzet pump. [0103] Figure 7 illustrates the presence of antibodies that bind to KLH produced in JAK3 knock-out (column 1), SCID (column 2) and Athymic nude (column 3) mice administered 5 μg KLH alone.

[0104] Figure 8 illustrates the presence of antibodies that bind to HA22 as measured by ICC ELISA produced in Balb/c mice administered HA22 and 20 mg/kg/day CP-690,550 delivered via implanted minipump (column 1), HA22 and PBS delivered via implanted minipump (column 2), and HA22 without implanted minipump (column 3).

[0105] Figure 9 illustrates the presence of antibodies that bind to HA22 as measured by DC ELISA produced in Balb/c mice administered HA22 and 20 mg/kg/day CP-690,550 delivered via implanted minipump (column 1 ), HA22 and PBS delivered via implanted minipump (column 2), and HA22 without implanted minipump (column 3).

[0106] Figure 10 illustrates the presence of antibodies that bind to KLH as measured by DC ELISA produced in Balb/c mice co-administered 5 μg KLH and 20 mg/kg/day CP-690,550 (DKLH 1 ) or 5μg KLH and PBS (DKLH2-DKLH5). [0107] Figure 1 1 shows KLH-specific immunoglobulins after IP injection of KLH (5 ug) with or without CP-690550 treatment.

[0108] Figure 12 shows total KLH-specific immunoglobulins after IP injection of KLH (5 ug) with or without CP-690550 treatment.

[0109] Figure 13 shows total number of splenocytes with or without CP-690550 treatment DETAILED DESCRIPTION

1. Introduction

[0110] The present invention is based, in part, on the discovery that Janus kinase 3 (JAK3) inhibitors (e.g., CP-690,550) suppress a neutralizing antibody response against therapeutic foreign proteins administered to a subject. The ability of CP-690,550 to reduce, inhibit or prevent neutralizing antibodies to administered foreign proteins is surprising in view of the human clinical data showing that patients administered CP-690,550 had increased B cell numbers. Also, other agents known to suppress B cell activity, e.g., antibodies against the B- lymphocyte antigen CD20, were not useful in suppressing a neutralizing antibody response. See, e.g., Hassan, et ah, Clinical Cancer Research (2004) 10: 16-18.

2. JAK3 inhibitors

[0111] CP-690,550 is an exemplary Janus kinase 3 (JAK3) inhibitor with the chemical name (3R,4R)-4-Methyl-3-(methyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-P-oxo-l - piperidinepropanenitrile and CAS Registry Number: 477600-75-2. The structure of CP-690,550 is as follows:

[0112] CP-690,550 or other JAK3 inhibitor can be administered at a dose of about 5-1000 mg, for example, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 50 mg, 100 mg. 500 mg or 1000 mg once or twice daily. In some embodiments, the JAK3 inhibitor is administered orally or subcutaneously. In some embodiments, the JAK3 inhibitor is delivered in a controlled-release, sustained-release or extended-release formulation. In formulations or devices for controlled delivery over an extended period of time, the JAK3 inhibitor can be administered at a dose of about 5-75 mg/kg/day, for example, about 5, 10, 15, 20, 25, 30, 50, or 75 mg/kg/day. Further applicable formulations and routes of administration of JA 3 inhibitors are discussed below. The JAK3 inhibitor may be administered by the same or different route of administration as the therapeutic foreign protein. The JAK3 inhibitor may be administered concurrently with the therapeutic foreign protein. Alternatively, the JAK3 inhibitor may be administered before or after the therapeutic foreign protein.

3. Therapeutic Foreign Proteins

[0113] The present methods and compositions find use in reducing, inhibiting, and/or preventing a neutralizing antibody response against a therapeutically administered foreign protein. Foreign proteins are not endogenously expressed by the patient to whom the foreign protein is administered. Foreign proteins are proteins exogenous or heterologous to the patient. Therefore, the patient's immune system recognizes the foreign protein as a foreign antigen and mounts an immune response against it, including the production of antibodies that bind to the foreign protein and reduce, inhibit or eliminate its inactivity for its intended purpose. a. Proteins not expressed by the patient [0114] Proteins not expressed by the patient can be a protein natively expressed in another species (e.g., administering a plant, fungal, bacterial, viral, or otherwise non-mammalian protein to a mammal) or a protein of the same species that is not endogenously expressed by the patient (e.g., administering a human protein not expressed or not functionally expressed, e.g. , due to a genetic mutation, deletion, addition or substitution, to a human). The foreign protein can also be a protein that is or was endogenously expressed by the patient, but wherein the administered form of the foreign protein is expressed in a non-mammalian cell, e.g. , an avian cell, a plant cell, a bacterial cell, an insect cell, a yeast cell (e.g., insulin).

[0115] Bacterial cytotoxins (described below in greater detail) are foreign proteins to a mammalian patient, including a human patient. For example, as discussed herein, bacterial cytotoxins find use for the targeted killing of target cells in humans, including cancer cells and inappropriately activated immune cells. The human patient's immune system recognizes the bacterial cytotoxin as a foreign antigen and produces antibodies that bind to and inactivate the cytotoxin, thereby neutralizing the bacterial cytotoxin.

[0116] Proteins administered in replacement therapies are foreign antigens. For example, proteins administered to treat a monogenic disease, i.e., an inherited disease or condition that results from inactivation or malfunctioning of a single gene (e.g., due to mutation, substitution, addition or deletion) occurring in all cells in an individual, are foreign proteins to a patient that can elicit neutralizing antibodies. Monogenic diseases can be treated by administering the deficient protein or a nucleic acid encoding the deficient protein. For example, monogenic diseases that lead to hormone, enzyme or blood factor deficiencies can be treated by

administration of the deficient protein, either by administering the protein or a nucleic acid encoding the protein. i. Hormone/Enzyme Deficiencies

[0117] Examples of hormone and enzyme deficiencies (sometimes referred to as monogenic diseases) for which gene therapy clinical trials have been pursued include Hurler's syndrome, Hunter's syndrome, Gaucher's disease, purine nucleoside phosphorylase deficiency, ornithine transcarbamylase deficiency and Fabry disease.

[0118] Further examples of enzyme deficiency diseases that can be treated by administration of a foreign protein include: Lesch-Nyhan which has been treated with expression of

hypoxanthine-guanine phosphoribosyl transferase; phenylketonuria (PKU) which has been treated with expression of phenylalanine hydroxylase; emphysema which has been treated with expression of alpha- 1 -antitrypsin (AAT); Gaucher's disease has been treated with

glucocerebrosidase (Cerezyme); Wolman's Disease (WD) and cholesteryl ester storage disease (CESD) have been treated with lysosomal acid lipase (cholesterase); galatosialidosis (GS) has been treated with β-Galactosidase and Neuraminidase; sialidosis has been treated with neuraminidase; CNS (central nervous system) disease have been treated with

galactosylceramidase (GALC); Fabry Disease has been treated with Agalsidase alpha (Replagal), Agalsidase beta (Fabrazyme) or alpha galactosidase A; Pompe disease has been treated with alpha-glucosidase (MYOZYME); Niemann-Pick Disease type AB has been treated with Acid Sphingomyelinase (rhASM); and Globoid cell leukodystrophy (GLD, Krabbe disease or CNS disease) has been treat with galactosylceramidase (GALC).

[0119] Several mucopolysaccharidosis (MPS) diseases (also known as lysosomal storage diseases) have been successfully treated by gene therapy methods as well. The MPS diseases which can be treated by administration of a foreign protein include MPS I

(Mucopolysaccharidosis Type I or Hurler Syndrome) has been treated with alpha-L-iduronidase (ALDURAZYME); MPS II (mucopolysaccharidosis type II or Hunter Syndrome) has been treated with idursulfase (Elaprase); MPS IIIA (Mucopolysaccharidosis Type IIIA or Sanfilippo Syndrome) has been treated with heparin sulfamidase; MPS IIIB (Mucopolysaccharidosis Type IIIB or Sanfilippo syndrome type IIIB or Sanfilippo Syndrome) has been treated with N- acetylglucosaminidase; MPS IIIC (Mucopolysaccharidosis Type IIIC or Sanfilippo Syndrome) has been treated with alpha-glucosaminide N-acetyltransferase; MPS HID

(Mucopolysaccharidosis Type HID or Sanfilippo Syndrome) has been treated with N- acetylgIucosamine-6-sulfate sulfatase; MPS IV type A (Morquio syndrome) has been treated with N-acetylgalactosamine 6-sulfatase (GALNS or galactose 6-sulfatase); MPS IV type B (Morquio syndrome) has been treated with beta-galactosidase; MPS VI (Mucopolysaccharidosis Type VI or Maroteaux-Lamy syndrome) has been treated with galsulfase (NAGLAZYME) as well as arylsulfatase B, recombinant human arylsulfatase B, rhASB, BM 102 or N-acetylgalactosamine-4-sulfatase) and MPS VII (mucopolysaccharide disease also known as Sly syndrome) has been treated with glucoronidase. Gene therapy clinical trials for MPS I, MPS II, MPS IIIA, MPS VI and MPS VII have been pursued. (See, e.g., Sly, et al , PNAS (2002) 99(9):5760-5762 and the World Wide Web at wiley.co.uk/genmed/clinical/.) [0120] Examples of hormone deficiency diseases that can be treated by administration of foreign protein include heat stress, which has been treated with expression of plasmid growth hormone-releasing hormone treatment; growth hormone deficiency treatment with expression of growth hormone (GH); leptin expression for the treatment of obesity; and insulin for the treatment of diabetes. Other indications that can be treated with hormone gene therapy include fractures. For example, gene therapy clinical trials employing parathyroid hormone to treat tibia fractures have been pursued. (See, e.g., Edelstein, et al., Journal of Gene Medicine (2004) 6:597-602 and the World Wide Web at wiley.co.uk/genmed/clinical/.) ii. Blood Factor Deficiencies

[0121] Blood factor deficiencies and blood disorders which have been successfully treated using gene therapy and/or administration of a foreign protein. (See, e.g., Nienhuis, Blood (2008) 1 1 1 (9):4431 -4444.) Examples of blood factor deficiencies and blood disorders (some of which are referred to as monogenic diseases) for which gene therapy clinical trials are currently being pursued include Haemophilia A and B, Fanconi's anaemia, Leukocyte adherence deficiency and chronic granulomatous disease. (See, e.g., Edelstein, et al, Journal of Gene Medicine (2004) 6:597-602.)

[0122] Further examples of blood factor deficiencies and blood disorders that can be successfully treated by administration of a foreign protein include Fanconi Anemia which has been treated with replacement of one of the seven Fanconi anemia proteins (FANCA,

FANCB/D 1 , FANCC, FANCE, FANCF or FANCG); blood coagulation Factor X deficiency which has been treated with expression of Factor X; Hemophilia A which has been treated with Factor VIII expression; Hemophilia B which has been treated with expression of Factor IX; chronic granulomatous disease (CGD) has been treated with expression of one or more proto- oncogenes including MDS 1 -EV1 1 , PRDM 16 and SETBP 1 ; hemoglobin disorders which have been treated with expressions of globins; sickle cell anemia has been treated with expression of globins; β-thalassemia has been treated with expression of globins; and prevention of clot formation can be achieved with an antibody against anti-glycoprotein Ilb/IIIa receptor on platelets.

[0123] Examples of genes useful in the treatment and therapy of blood factor deficiencies and blood disorders include but are not limited to the seven Fanconi anemia proteins including FANCA, FANCB/D1, FANCC, FANCE, FANCF and FANCG.

[0124] Examples also include coagulation regulating proteins including anticoagulants, antithrombin III, thrombin (activated Factor II or Ila), tissue-type plasminogen activator (tPA), Factor Vila, Factor VIII, Factor IX, Factor X, Factor VIII (b-domain deleted) and anti- glycoprotein Ilb/IIIa receptor can be delivered. Platelet aggregation inhibitors including

REOPRO (abciximab) can be delivered. Chemokines including MIP-1 alpha (macrophage inflammatory protein- 1 alpha) and MIP-3 alpha (macrophage inflammatory protein 3 alpha) can be delivered. Antiangiogenic proteins including thrombospondin can be delivered. Further, Erythropoiesis proteins including erythropoietin (EPO), human erythropoietin, darbepoetin alfa and erythropoietin derivatives can also be delivered. iii. Therapeutic Antibodies

[0125] Therapeutic antibodies are also foreign proteins, especially when the antibodies are produced in a species that is different from the patient, or when the antibodies comprise sequence segments that are derived from a species that is different from the patient (e.g., administering chimeric or humanized antibodies to a human, which have non-human sequence segments). [0126] Examples of therapeutic antibodies include but are not limited to HERCEPTIN™

(Trastuzumab) (Genentech, CA) which is a humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer; REOPRO™ (abciximab) (Centocor) which is an anti-glycoprotein Ilb/IIIa receptor on the platelets for the prevention of clot formation;

ZENAPAX™ (daclizumab) (Roche Pharmaceuticals, Switzerland) which is an

immunosuppressive, humanized anti-CD25 monoclonal antibody for the prevention of acute renal allograft rejection; PANOREX™ which is a murine anti-17-IA cell surface antigen IgG2a antibody (Glaxo Wellcome/Centocor); BEC2 which is a murine anti-idiotype (GD3 epitope); IgG antibody (ImClone System); IMC-C225 which is a chimeric anti-EGFR IgG antibody; VITAXI ™ which is a humanized anti-a5p3 integrin antibody (Applied Molecular

Evolution/Medlmmune); Campath 1FI/LDP-03 which is a humanized anti CD52 IgGl antibody (Leukosite); Smart Ml 95 which is a humanized anti-CD33 IgG antibody (Protein Design Lab/Kanebo); RITUXAN™ which is a chimeric anti-CD20 IgGl antibody (IDEC

Pharm/Genentech, Roche/Zettyaku); LYMPHOCIDE™ which is a humanized anti-CD22 IgG antibody (Immunomedics); ICM3 which is a humanized anti-ICAM3 antibody (ICOS Pharm); IDEC-1 14 which is a primate anti-CD80 antibody (IDEC Pharm/Mitsubishi); ZEVALIN™ which is a radiolabelled murine anti-CD20 antibody (IDEC/Schering AG); IDEC-131 which is a humanized anti-CD40L antibody (IDEC/Eisai); IDEC-151 which is a primatized anti-CD4 antibody (IDEC); IDEC- 152 which is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMART anti-CD3 which is a humanized anti-CD3 IgG (Protein Design Lab); 5G 1.1 which is a humanized anti-complement factor 5 (CS) antibody (Alexion Pharm); D2E7 which is a humanized anti-TNF-a antibody (CATIBASF); CDP870 which is a humanized anti-TNF-a Fab fragment (Celltech); IDEC- 151 which is a primatized anti-CD4 IgG l antibody (IDEC

Pharm/SmithKline Beecham); MDX-CD4 which is a human anti-CD4 IgG antibody

(Medarex/Eisai/Genmab); CDP571 which is a humanized anti-TNF-α IgG4 antibody (Celltech); LDP-02 which is a humanized anti- a 4,7 antibody (LeukoSite/Genentech); OrthoClone OKT4A which is a humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVA™ which is a humanized anti-CD40L IgG antibody (Biogen); ATMTEGREN™ which is a humanized anti- VLA-4 IgG antibody (Elan); and CAT-152 which is a human anti-TGF- 2 antibody (Cambridge Ab Tech). Further therapeutic antibodies are described herein.

b. Chimeric Molecules

[0127] Immunoconjugates for co-administration with the JAK3 inhibitor include, but are not limited to, molecules in which there is a covalent linkage of a cytotoxin molecule to an antibody or other targeting agent. The choice of a particular targeting agent depends on the particular cell to be targeted. With the cytotoxin molecules provided herein, one of skill can readily construct a variety of clones containing functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same cytotoxin and antibody sequence. Thus, the present invention provides nucleic acids encoding antibodies and cytoxin conjugates and fusion proteins thereof. a. Production of Immunoconjugates i. Non-Recombinant Methods

[0128] In a non-recombinant embodiment of the invention, a targeting molecule, such as an antibody, is linked to a cytoxin molecule of the present invention using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used with cytoxin molecules of the present invention.

[0129] The procedure for attaching a cytoxin molecule to an antibody or other targeting molecule ("TM") will vary according to the chemical structure of the TM. Polypeptides typically contain a variety of functional groups; e.g., carboxylic acid (COOH), free amine (-NH₂) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody, for example, to result in the binding of the cytoxin molecule.

[0130] Alternatively, the antibody or other TM is derivatized to expose or to attach additional reactive functional groups. The derivatization may involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford Illinois.

[0131] A "linker", as used herein, is a molecule that is used to join the TM to the cytotoxin molecule. The linker is capable of forming covalent bonds to both the antibody and to the effector molecule. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the effector molecule are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (e.g., through a disulfide linkage to cysteine). However, in a preferred embodiment, the linkers will be joined to the alpha carbon amino and carboxyl groups of the terminal amino acids.

[0132] In some circumstances, it is desirable to free the cytoxin molecule from the TM when the immunoconjugate has reached its target site. Therefore, in these circumstances,

immunoconjugates will comprise linkages which are cleavable in the vicinity of the target site. Cleavage of the linker to release the cytoxin molecule from the TM may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site. When the target site is a tumor, a linker which is cleavable under conditions present at the tumor site (e.g. when exposed to tumor-associated enzymes or acidic pH) may be used. ii. Recombinant Methods

[0133] The nucleic acid sequences of the present invention can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang, et al, Meth. Enzymol. 68:90-99 (1 979); the phosphodiester method of Brown, et al, Meth. Enzymol. 68: 109-1 5 1 ( 1979); the diethylphosphoramidite method of Beaucage, et al, Tetra. Lett. 22: 1 859- 1 862 ( 1 981 ); the solid phase phosphoramidite triester method described by Beaucage & Caruthers, Tetra. Letts.

22(20): 1 859- 1 862 ( 1 98 1 ), e.g., using an automated synthesizer as described in, for example, Needham-VanDevanter, et al. Nucl. Acids Res. 12:61 59-61 68 ( 1984); and, the solid support method of U.S. Patent No. 4,458,066. Chemical synthesis produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill would recognize that while chemical synthesis of DNA is limited to sequences of about 1 00 bases, longer sequences may be obtained by the ligation of shorter sequences.

[0134] In a preferred embodiment, the nucleic acid sequences of this invention are prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1 -3, Cold Spring Harbor Laboratory (1 989)), Berger and Kimmel (eds.), GUIDE TO MOLECULAR CLONING TECHNIQUES, Academic Press, Inc., San Diego CA ( 1 987)), or Ausubel, et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing and Wiley-Interscience, NY (1987). Product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA chemical company (Saint Louis, MO), R&D systems (Minneapolis, MN), Pharmacia LKB Biotechnology (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc.

(Gaithersberg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen, San Diego, CA, and Applied Biosystems (Foster City, CA), as well as many other commercial sources known to one of skill. [0135] Nucleic acids encoding native cytoxin can also be modified to form the

immunoconjugates of the present invention. Modification by site-directed mutagenesis is well known in the art. Nucleic acids encoding cytoxin can be amplified by in vitro methods.

Amplification methods include the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill.

[0136] In a preferred embodiment, immunoconjugates are prepared by inserting the cDNA which encodes an antibody or other TM of choice into a vector which comprises the cDNA encoding a desired cytoxin of the invention. The insertion is made so that the targeting agent (for ease of discussion, the discussion herein will assume the targeting agent is an Fv, although other targeting agents could be substituted with equal effect) and the cytoxin are read in frame, that is in one continuous polypeptide which contains a functional Fv region and a functional cytoxin region. In a particularly preferred embodiment, cDNA encoding a cytoxin of the invention is ligated to a scFv so that the toxin is located at the carboxyl terminus of the scFv. In other preferred embodiments, cDNA encoding a cytoxin of the invention is ligated to a scFv so that the toxin is located at the amino terminus of the scFv.

[0137] Once the nucleic acids encoding a PE, antibody, or an immunoconjugate of the present invention are isolated and cloned, one may express the desired protein in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells. It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for expression of proteins including E. coli, other bacterial hosts, yeast, and various higher eucaryotic cells such as the COS, CHO, HeLa and myeloma cell lines. No attempt to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes will be made. In brief, the expression of natural or synthetic nucleic acids encoding the isolated proteins of the invention will typically be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression cassette. The cassettes can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression cassettes contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding the protein. To obtain high level expression of a cloned gene, it is desirable to construct expression cassettes which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator. For E. coli this includes a promoter such as the T7, trp, lac, or lambda promoters, a ribosome binding site and preferably a transcription termination signal. For eukaryotic cells, the control sequences can include a promoter and preferably an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, and a polyadenylation sequence, and may include splice donor and acceptor sequences. The cassettes of the invention can be transferred into the chosen host cell by well- known methods such as calcium chloride transformation or electroporation for E. coli and calcium phosphate treatment, electroporation or lipofection for mammalian cells. Cells transformed by the cassettes can be selected by resistance to antibiotics conferred by genes contained in the cassettes, such as the amp, gpt, neo and hyg genes.

[0138] One of skill would recognize that modifications can be made to a nucleic acid encoding a polypeptide of the present invention {i.e. , a cytoxin or an immunoconjugate formed from a cytoxin of the invention) without diminishing its biological activity. Some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, termination codons, a methionine added at the amino terminus to provide an initiation, site, additional amino acids placed on either terminus to create conveniently located restriction sites, or additional amino acids (such as poly His) to aid in purification steps.

[0139] In addition to recombinant methods, the immunoconjugates and PEs of the present invention can also be constructed in whole or in part using standard peptide synthesis. Solid phase synthesis of the polypeptides of the present invention of less than about 50 amino acids in length may be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany & Merrifield, THE PEPTIDES:

ANALYSIS, SYNTHESIS, BIOLOGY. VOL. 2: SPECIAL METHODS IN PEPTIDE SYNTHESIS, PART A. pp. 3-284; Merrifield, et al. J. Am. Chem. Soc. 85:2149-2156 (1963), and Stewart, et al, SOLID PHASE PEPTIDE SYNTHESIS, 2ND ED. , Pierce Chem. Co., Rockford, 111. (1984). Proteins of greater length may be synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxyl terminal end {e.g., by the use of the coupling reagent N, N'-dicycylohexylcarbodiimide) are known to those of skill. iii. Purification [0140] Once expressed, the recombinant immunoconjugates and PEs of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like {see, generally, R. Scopes, PROTEIN PURIFICATION, Springer- Verlag, N.Y. (1982)). Substantially pure compositions of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred for pharmaceutical uses. Once purified, partially or to homogeneity as desired, if to be used therapeutically, the polypeptides should be substantially free of endotoxin.

[0141] Methods for expression of single chain antibodies and/or refolding to an appropriate active form, including single chain antibodies, from bacteria such as E. coli have been described and are well-known and are applicable to the antibodies of this invention. See, Buchner, et al, Anal. Biochem. 205:263-270 (1992); Pluckthun, Biotechnology 9:545 ( 1991 ); Huse, et al,

Science 246: 1275 (1989) and Ward, et al , Nature 341 :544 (1989), all incorporated by reference herein.

[0142] Often, functional heterologous proteins from E. coli or other bacteria are isolated from inclusion bodies and require solubilization using strong denaturants, and subsequent refolding. During the solubilization step, as is well-known in the art, a reducing agent must be present to separate disulfide bonds. An exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). Reoxidation of the disulfide bonds can occur in the presence of low molecular weight thiol reagents in reduced and oxidized form, as described in Saxena, et al , Biochemistry 9: 5015-5021 (1970), incorporated by reference herein, and especially as described by Buchner, et al, supra. [0143] Renaturation is typically accomplished by dilution (e.g., 100-fold) of the denatured and reduced protein into refolding buffer. An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M

L-arginine, 8 mM oxidized glutathione, and 2 mM EDTA.

[0144] As a modification to the two chain antibody purification protocol, the heavy and light chain regions are separately solubilized and reduced and then combined in the refolding solution. A preferred yield is obtained when these two proteins are mixed in a molar ratio such that a 5 fold molar excess of one protein over the other is not exceeded. It is desirable to add excess oxidized glutathione or other oxidizing low molecular weight compounds to the refolding solution after the redox-shuffling is completed. i. Targeting Moiety

[0145] In one embodiment, the targeting moiety is an antibody, preferably an antibody specifically binding to a surface marker on a cell. Accordingly, in some embodiments, the chimeric molecule is an immunotoxin.

[0146] In another embodiment, the targeting moiety is an antibody fragment, preferably an antibody fragment specifically binding to a surface marker on a cell. A preferred antibody fragment is a single chain Fv. Herein the construction and characterization of cytotoxin-based immunotoxins wherein the cytotoxin is fused to a scFv are described. Other preferred antibody fragments to which a toxin or cytotoxic fragment can be fused include Fab, Fab', F(ab')2, Fv fragment, a helix-stabilized antibody, a diabody, a disulfide stabilized antibody, and a domain antibody.

[0147] The fusion of a cytotoxin to an antibody or antibody fragment can be either to the N-terminus or C-terminus of the antibody or antibody fragment. Such fusion typically is accomplished employing recombinant DNA technologies.

[0148] In another embodiment, the targeting moiety is a ligand specifically binding to a receptor on a cell surface. The ligand can be any ligand which binds to a cell surface marker. A preferred ligand is VEGF, Fas, TRAIL, a cytokine, a chemokine, a hormone. Other preferred ligands include, but are not limited to, TGFa, IL-2, IL- 15, IL-4, IL- 13, etc. ii. Target Cell Surface Markers

[0149] The targeting component of the chimeric molecule can be against a cell surface marker. The cell surface marker can be a protein or a carbohydrate. The cell surface antigen can be a tumor associated antigen. Preferably, the cell surface marker is exclusively expressed, preferentially expressed or expressed at clinically relevant higher levels on cancer cells or other aberrantly proliferating cells. Cell surface antigens that are targets for chimeric molecules are well known in the art, and summarized, e.g. , in Mufson, Front Biosci (2006) 1 1 :337-43; Frankel, Clin Cancer Res (2000) 6:326-334 and Kreitman, AAPS Journal (2006) 8(3):E532-E551.

[0150] Exemplary cell surface marker targets include cell surface receptors. Cell surface receptor that can be targeted using a toxin of the present invention include, but are not limited to, transferrin receptor, EGF receptor, CD19, CD22, CD25, CD21 , CD79, mesothelin and cadherin. Additional cell surface antigens subject to targeted immunotoxin therapy include without limitation MUC l , MAGE, PRAME, CEA, PSA, PSMA, GM-CSFR, CD56, HER2/neu, erbB-2, CD5, CD7. Other cell surface tumor associated antigens are known and find use as targets. [0151] The antigen targets can be found on numerous different types of cancer cells, including without limitation neuroblastoma, intestine carcinoma, rectum carcinoma, colon carcinoma, familiary adenomatous polyposis carcinoma, hereditary non-polyposis colorectal cancer, esophageal carcinoma, labial carcinoma, larynx carcinoma, hypopharynx carcinoma, tong carcinoma, salivary gland carcinoma, gastric carcinoma, adenocarcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, follicular thyroid carcinoma, anaplastic thyroid carcinoma, renal carcinoma, kidney parenchym carcinoma, ovarian carcinoma, cervix carcinoma, uterine corpus carcinoma, endometrial carcinoma, chorion carcinoma, pancreatic carcinoma, prostate carcinoma, testis carcinoma, breast carcinoma, urinary carcinoma, melanoma, brain tumors, glioblastoma, astrocytoma, meningioma, medulloblastoma, peripheral neuroectodermal tumors, Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, acute lymphatic leukemia (ALL), chronic lymphatic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), adult T-cell leukemia lymphoma, hepatocellular carcinoma, gall bladder carcinoma, bronchial carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma, choroids melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcome, liposarcoma, fibrosarcoma, Ewing sarcoma, and plasmocytoma. [0152] In some embodiments, the cell surface marker is mesothelin. Exemplary cancers whose growth, spread and/or progression can be reduced or inhibited by targeting mesothelin include ovarian cancer, mesothelioma, non-small cell lung cancer, lung adenocarcinoma, fallopian tube cancer, head and neck cancer, cervical cancer and pancreatic cancer. [0153] In some embodiments, the cell surface marker is CD22. Exemplary cancers whose growth, spread and/or progression can be reduced or inhibited by targeting CD22 include hairy cell leukemia, chronic lymphocytic leukemia (CLL), prolymphocyte leukemia (PLL), non- Hodgkin's lymphoma, Small Lymphocytic Lymphoma (SLL) and acute lymphatic leukemia (ALL). [0154] In some embodiments, the cell surface marker is CD25. Exemplary cancers whose growth, spread and/or progression can be reduced or inhibited by targeting CD25 include leukemias and lymphomas, including hairy cell leukemia, and Hodgkin's lymphoma.

[0155] In some embodiments, the cell surface marker is a carbohydrate, e.g., Lewis Y antigen. Exemplary cancers whose growth, spread and/or progression can be reduced or inhibited by targeting Lewis Y antigen include bladder cancer, breast cancer, ovarian cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancer and pancreatic cancer.

[0156] In some embodiments, the cell surface marker is CD33. Exemplary cancers whose growth, spread and/or progression can be reduced or inhibited by targeting CD33 include acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CML), and myeloproliferative disorders. iii. Cytotoxin Moiety

[0157] Cytotoxins for use in the present invention inhibit protein synthesis. A number of plant and bacterial toxins have been studied for their suitability as the toxin component of

immunotoxins. For example, the bacterial toxin known as Pseudomonas exotoxin A ("PE") has been studied for two decades as a toxin for use in chimeric molecules, e.g. , immunotoxins.

Typically, PE has been truncated or mutated to reduce its non-specific toxicity while retaining its toxicity to cells to which it is targeted by the antibody portion of the immunotoxin. Over the years, numerous mutated and truncated forms of PE have been developed and clinical trials employing some of them are ongoing. [0158] Bacterial protein toxins are well known in the art, and are discussed in such sources as Burns, D., et al, eds., BACTERIAL PROTEIN TOXINS, ASM Press, Herndon VA (2003), Aktories, K. and Just, I., eds., BACTERIAL PROTEIN TOXINS (HANDBOOK OF

EXPERIMENTAL PHARMACOLOGY), Springer- Verlag, Berlin, Germany (2000), and Alouf, J. and Popoff, M., eds., THE COMPREHENSIVE SOURCEBOOK OF BACTERIAL

PROTEIN TOXINS, Academic Press, Inc., San Diego, Calif. (3rd Ed., 2006).

[0159] In some embodiments, the cytotoxin moiety is an ADP-ribosyltransferase.

Pseudomonas exotoxin A ("PE"), diphtheria toxin ("DT") and cholix toxin ("CT"), cholera exotoxin ("CET") irreversibly ribosylate elongation factor 2 ("EF-2") in eukaryotic cells, causing the death of affected cells by inhibiting their ability to synthesize proteins. Since EF-2 is essential for protein synthesis in eukaryotic cells, inactivation of the EF-2 in a eukaryotic cell causes death of the cell. The sequences and structure of PE, DT, CT and CET are well known in the art. Mutated forms of DT suitable for use in immunotoxins are known in the art. See, e.g., U.S. Patent Nos. 5,208,021 and 5,352,447. DT does not share significant sequence identity or structural similarity with PE. Since most persons in the developed world have been immunized against diphtheria, DT-based immunotoxins can generally only be used in compartments of the body, such as the brain, that cannot be accessed by antibodies.

[0160] ADP-ribosylating cytotoxins and variants thereof that find use are described, for example, in co-pending application PCT US2009/046292 and U.S. Patent Publ. No.

2009/0142341 , the disclosures of both of which are hereby incorporated herein by reference in their entirety for all purposes.

[0161] In some embodiments, the toxin moiety is a ribosome inactivating agent, for example a shiga toxin, a ricin toxin or a pokeweed antiviral protein (PAP) toxin. Shiga toxins and ricin toxin act to inhibit protein synthesis by functioning as N-glycosidases, cleaving several nucleobases from ribosomal RNA. PAP depurinates 25S ribosomal RNA. Ribosomal inactivating proteins are reviewed, e.g. , in Stirpe and Battelli, Cell Mol Life Sci. (2006)

63(16): 1850-66.

[0162] Variants of cytotoxins useful in immunotoxins are reviewed, e.g., in Kreitman, The AAPS Journal (2006) 8(3):E532-551 and the references cited therein. 1. Pseudomonas Exotoxin A [0163] In preferred embodiments of the present invention, the toxin is a Pseudomonas exotoxin ("PE") or a variant thereof. The term "Pseudomonas exotoxin" as used herein refers to a PE that has been modified from the native sequence to reduce or to eliminate non-specific binding. Such modifications may include, but are not limited to, elimination of domain la, various amino acid deletions in domains lb, II and III, single amino acid substitutions and the addition of one or more sequences at the carboxyl terminus such as KDEL (SEQ ID NO:4) and REDL (SEQ ID NO:3). See Siegall, et al, J. Biol. Chem. 264:14256-14261 (1989). In a preferred embodiment, the cytotoxic fragment of PE retains at least 50%, preferably 75%, more preferably at least 90%, and most preferably 95% of the cytotoxicity of native PE when delivered to a cell bearing mesothelin. In a most preferred embodiment, the cytotoxic fragment, when delivered by an antibody or ligand, is more toxic than native PE.

[0164] Native Pseudomonas exotoxin A ("PE") is an extremely active monomeric protein (molecular weight 66 kD), secreted by Pseudomonas aeruginosa, which inhibits protein synthesis in eukaryotic cells. The native 613 amino acid sequence of PE is provided in U.S. Patent No. 5,602,095, incorporated herein by reference. The method of action is inactivation of the ADP-ribosylation and inactivation of elongation factor 2 (EF-2). The exotoxin contains three structural domains that act in concert to cause cytotoxicity. Domain la (amino acids 1 -252) mediates cell binding. Domain II (amino acids 253-364) is responsible for translocation into the cytosol and domain III (amino acids 400-613) mediates ADP ribosylation of elongation factor 2. The function of domain lb (amino acids 365-399) remains undefined, although a large part of it, amino acids 365-380, can be deleted without loss of cytotoxicity. See Siegall, et al., (1989), supra.

[0165] The term "PE" as used herein includes cytotoxic fragments of the native sequence, and conservatively modified variants of native PE and its cytotoxic fragments. Cytotoxic fragments of PE include those which are cytotoxic with or without subsequent proteolytic or other processing in the target cell {e.g. , as a protein or pre-protein). Cytotoxic fragments and variants of PE have been investigated for years as agents for clinical use; several of these fragments and variants are described below. For convenience, residues of PE which are deleted or mutated are typically referred to in the art by their position in the 613 amino acid sequence of native PE (SEQ ID NO: l ). As noted, the 613-amino acid sequence of native PE is well known in the art. [0166] In preferred embodiments, the PE has been modified to reduce or eliminate nonspecific cell binding. Frequently, this is achieved by deleting domain la. as taught in U.S. Patent 4,892,827, although it can also be achieved by, for example, mutating certain residues of domain la. U.S. Patent 5,512,658, for instance, discloses that a mutated PE in which Domain la is present but in which the basic residues of domain la at positions 57, 246, 247, and 249 are replaced with acidic residues (glutamic acid, or "E")) exhibits greatly diminished non-specific cytotoxicity. This mutant form of PE is sometimes referred to as "PE4E".

[0167] One derivative of PE in which Domain la is deleted has a molecular weight of 40 kDa and is correspondingly known as PE40. See, Pai, et al , Proc. Nat 'l Acad. Sci. USA 88:3358-62 (1991 ); and Kondo, et al. , J. Biol. Chem. 263:9470-9475 (1988). Another derivative is PE25, containing the 1 1 -residue fragment from domain II and all of domain III. In some embodiments, the derivative of PE contain only domain III.

[0168] In some embodiments, the cytotoxic fragment PE38 is employed. PE38 is a truncated PE pro-protein composed of PE amino acids 253-364 and 381 -613 which is activated to its cytotoxic form upon processing within a cell (see e.g., U.S. Patent No. 5,608,039, and Pastan et al., Biochim. Biophys. Acta 1333:C1 -C6 (1997)). In some embodiments, the lysine residues at positions 590 and 606 of PE in PE38 are mutated to glutamines, while the lysine at position 613 is mutated to arginine, to create a form known as "PE38QQR." See, e.g., Debinski and Pastan, Bioconj. Chem., 5: 40-46 (1994). This form of PE was originally developed in the course of increasing the homogeneity of immunotoxins formed by chemically coupling the PE molecules to the targeting antibodies.

[0169] In some embodiments, the cytotoxic fragment PE35 is employed. PE35 is a 35 kD carboxyl-terminal fragment of PE in which amino acid residues 1 -279 have deleted and the molecule commences with a methionine residue at position 280, followed by amino acids 281 - 364 and 381 -613 of native PE. PE35 and PE40 are disclosed, for example, in U.S. Patents 5,602,095 and 4,892,827.

[0170] Further, several means are known for increasing the cytotoxicity of PE by altering residues in domain III from the native sequence. Studies have determined that certain amino acid sequences and repeats of these sequences could be used in place of the native sequence of residues 609-613 of PE to increase the cytotoxicity of the resulting PE compared to PE made with the native sequence (the native sequence of residues 609-613 and specific mutations that increase cytotoxicity are discussed in more detail below in the section entitled Pseudomonas exotoxin A". More recently, it has been determined that a substitution of glycine, alanine, valine or other residues for the arginine present at position 490 of the native PE sequence would increase cytotoxicity, with substitution of the arginine by alanine being particularly

advantageous. See, e.g., U.S. Published Patent Application 2007/0189962; Bang et al., Clin Cancer Res, 1 1 : 1545-1550 (2005). While PEs of the invention using the native domain III sequence are expected to be useful by themselves, if desired the cytotoxicity of the PE can be augmented by using one or more of these substitutions or mutations. Any particular substitution or mutation can be tested to determine whether it retains adequate cytotoxicity for in vitro use and whether it has sufficiently low non-specific toxicity for in vivo use using assays known in the art, including those described in WO 2009/032954.

[0171] In some embodiments, the PE toxin is modified to remove epitopes recognized by

T cells and/or B cells. The presence of epitopes or subepitopes have been mapped in domain III.

Binding of antibodies which recognize those epitopes can be reduced or eliminated by substitutions of the residues normally present at certain positions. It has been demonstrated that the binding of these antibodies can be reduced by substituting an alanine, glycine, serine or glutamine for one or more amino acid residues selected from the group consisting of D403, R412, R427, E431 , R432, R458, D461 , R467, R505, R513, E522, R538, E548, R551 , R576, K590, and L597 in a PE (the positions are made with reference to SEQ ID NO: ] ; see, e.g. , WO 2007/016150, U.S. Published Patent Application 2009/0142341 and WO 2009/032954). In some embodiments, the PE toxin is PE-6X, wherein alanine, glycine or serine residues are substituted in place of amino acid residues R432, R467, R490, R513, E548 and 590, the residue positions corresponding to SEQ ID NO: l . In some embodiments, the PE toxin is PE-8X, wherein alanine, glycine or serine residues are substituted in place of amino acid residues D406, R432, R467, R490, R513, E548, K590 and Q592, the residue positions corresponding to SEQ ID 1M0: 1 . In PE-6X and PE-8X, all of domain I and part or all of domain II may also be removed, for example, as described above for PE35, PE38 and PE40.

[0172] Since the presence of these residues prior to their substitution maintains an epitope or subepitope in domain III, for ease of reference, the residues at these positions can be referred to as "maintaining" the immunogenicity of their respective epitopes or subepitopes, while substituting them with alanine or the like reduces the immunogenicity of PE domain III resulting from the native epitope or subepitope. While PEs of the invention using the native domain III sequence are expected to be useful by themselves, therefore, if desired substitutions of one of more of the residues identified above can be made to reduce further the immunogenicity of the PEs of the invention. Any particular substitution or mutation can be tested to determine whether it retains adequate cytotoxicity for in vitro or in vivo use using assays known in the art, including those set forth WO 2009/032954 and in PCT/US2009/046292.

[0173] In some embodiments, the PE toxin is modified to remove amino acid segment(s) that are targets of lysosomal proteases, i.e. , are lysosomal resistant ("LR"). Exemplary lysosomal resistant variants of PE .are described, e.g., in Weldon, et al., Blood (2009) 1 13:3792-3800 and in WO 2009/032954. For example, in some PE-LR cytotoxins, residues 1 -273 and 285-394 are removed, the positions corresponding to SEQ ID NO: 1 . In some embodiments, a cytotoxic, lysosomal resistant PE fragment selected from PE25LR, PE35LR, PE38LR or PE40LR is used. In some embodiments, the PE toxin is PE-LR/6X, wherein residues 1 -273 and 285-394 are removed and alanine, glycine or serine residues are substituted in place of amino acid residues R432, R467, R490, R513, E548 and K590, the residue positions corresponding to SEQ ID NO: l . In some embodiments, the PE toxin is PE-LR/8X, wherein residues 1 -273 and 285-394 are removed and alanine, glycine or serine residues are substituted in place of amino acid residues D406, R432, R467, R490, R513, E548, K590 and Q592, the residue positions corresponding to SEQ ID NO: l .

[0174] As noted above, some or all of domain l b may be deleted, and the remaining portions joined by a linker or directly by a peptide bond. Some of the amino portion of domain II may be deleted. And, the C-terminal end may contain the native sequence of residues 609-613 (REDLK; SEQ ID NO:5), or may contain a variation found to maintain the ability of the construct to translocate into the cytosol, such as REDL (SEQ ID NO:3) or KDEL (SEQ ID NO:4), and repeats of these sequences. See, e.g., U.S. Patents 5,854,044; 5,821 ,238; and 5,602,095 and WO 99/51643. While in preferred embodiments, the PE is PE4E, PE40, PE38, or PE38QQR, any form of PE in which non-specific cytotoxicity has been eliminated or reduced to levels in which significant toxicity to non-targeted cells does not occur can be used in the immunotoxins of the present invention so long as it remains capable of translocation and EF-2 ribosylation in a targeted cell. [0175] In some preferred embodiments, the toxicity of the PE is increased by mutating the arginine (R) at position 490 of the native sequence of PE. The R is mutated to an amino acid having an aliphatic side chain that does not comprise a hydroxyl. Thus, the R can be mutated to glycine (G), alanine (A), valine (V), leucine (L), or isoleucine (I). In preferred embodiments, the substituent is G, A, or I. Alanine is the most preferred. Surprisingly, the mutation of the arginine at position 490 to alanine doubles the toxicity of the PE molecule. The discovery of this method of increasing the toxicity of PE is disclosed in co-owned international application PCT/US2004/039617, which is incorporated herein by reference.

Conservatively Modified Variants of PE

[0176] Conservatively modified variants of PE or cytotoxic fragments thereof have at least 80% sequence similarity, preferably at least 85% sequence similarity, more preferably at least 90% sequence similarity, and most preferably at least 95% sequence similarity at the amino acid level, with the PE of interest, such as PE38 or PE40.

[0177] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acid sequences which encode identical or essentially identical amino acid sequences, or if the nucleic acid does not encode an amino acid sequence, to essentially identical nucleic acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0178] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Assaying for Cytotoxicity of PE

[0179] Pseudomonas exotoxins employed in the invention can be assayed for the desired level of cytotoxicity by assays well known to those of skill in the art. Exemplary toxicity assays are described in, e.g., WO 00/73346, Example 2. Thus, cytotoxic fragments of PE and

conservatively modified variants of such fragments can be readily assayed for cytotoxicity. A large number of candidate PE molecules can be assayed simultaneously for cytotoxicity by methods well known in the art. For example, subgroups of the candidate molecules can be assayed for cytotoxicity. Positively reacting subgroups of the candidate molecules can be continually subdivided and reassayed until the desired cytotoxic fragment(s) is identified. Such methods allow rapid screening of large numbers of cytotoxic fragments or conservative

2. Diphtheria Toxin

[0180] In some embodiments, the cytotoxin moiety is a diphtheria toxin. Diphtheria toxin ("DT") is an exotoxin secreted by Corynebacterium diphtheriae, the pathogen bacterium that causes diphtheria. "DT" refers to a protein secreted by toxigenic strains of Corynebacterum diphtheriae. It is initially synthesized as a 535 amino acid polypeptide which undergoes proteolysis to form the toxin, which is composed of two subunits, A and B, joined by a disulfide bond. The B subunit, found at the carboxyl end, is responsible for cell surface binding and translocation; the A subunit, which is present on the amino end, is the catalytic domain, and causes the ADP ribosylation of Elongation Factor 2 ("EF-2"), thereby inactivating EF-2. See generally, Uchida et al., Science 175 :901 -903 (1972); Uchida et al, J Biol Chem 248:3838-3844 (1973). Mutated forms of DT suitable for use in immunotoxins are known in the art. See, e.g., U.S. Patent Nos. 5,208,021 and 5,352,447. Once again, for convenience of reference, the term "DT" as used herein refers to the native toxin, but more commonly is used to refer to forms in which the B subunit has been deleted and in which modifications have been made in the A subunit to reduce non-specific binding and toxicity.

3. Cholix Toxin ("CT")

[0181] In some embodiments, the cytotoxin moiety is a cholix toxin. Jorgensen, R. et al , J Biol Chem 283(16): 10671 -10678 (2008) (hereafter, "Jorgensen") recently reported that some strains of Vibrio cholerae, the causative agent of cholera, contain a ADP-ribosyltransferase, which they termed cholix toxin (also referred to herein as "CT"). Like PE, CT ribosylates EF-2. Jorgensen stated that CT's primary structure shows a 32% sequence identity with PE, and has a potential furin protease cleavage site for cellular activation, like that of PE, and contains a C- terminal KDEL sequence (SEQ ID NO:4), similar to the C-terminal sequence of PE, that likely targets the toxin to the endoplasmic reticulum of the host cell (Jorgensen, at page 10673).

Jorgensen further reports that CT, like PE, is organized in three structural domains: domain la (residues 1 -264), a receptor binding domain, a short domain lb (residues 387-423), of unknown function, which with domain la comprise "a 13-stranded antiparallel β-jellyroll", domain II (residues 265-386), a translocation domain consisting of six a-helices, and domain III, a catalytic domain with an α/β topology (Jorgensen, at page 10675). In fact, Figure 3b of Jorgensen superpositions the structures of CT and PE, showing that the two structures are almost indistinguishable from one another.

[0182] Mature cholix toxin (CT) is a 70.7 kD, 634 residue protein. The sequence, with an eight residue leader sequence consisting of a 6-histidine tag flanked by a methionine on each side, is publicly available on-line in the Entrez Protein database under accession number 2Q5T A.

[0183] A preferred CT is a truncated version of CT in which the receptor binding domain, domain la, is deleted, to create a 40 kD version of CT corresponding to PE40 and referred to herein as "CT40." Given the similarity of CT and PE, it is expected that additional variants of CT, such as a CT38 or CT35 variant, can be made that correspond to variants of PE as described in the preceding section. For example, it is anticipated that some or all of CT domain lb can be deleted which, with the deletion of domain la, would create a CT variant akin to PE38.

Similarly, it is anticipated that the carboxyl terminus of CT, which ends with KDELK (SEQ ID No:6), can be varied by replacing it with one of the various C-terminal sequences mentioned above as maintaining the toxicity of PE. In preferred embodiments, if the C-terminal sequence of CT is replaced, the C-terminal sequence used as a replacement is one suitable for use in humans. In some preferred embodiments, the C-terminal sequence of CT (KDELK; SEQ ID No:6) is replaced with the terminal sequence of PE, REDLK (SEQ ID No:5).

[0184] Similarly, it is anticipated that the NAD domain of CT, which at least comprises amino acid residues GGEDETVIG (SEQ ID No:7) can be varied by replacing it with another NAD domain sequence. In preferred embodiments, if the NAD domain sequence of CT is replaced, the NAD domain sequence used as a replacement is one suitable for use in humans. In some preferred embodiments, the NAD domain sequence of CT (GGEDETVIG (SEQ ID No:7) is replaced with the NAD binding site of PE comprising the amino acid sequence GGRLETILG) (SEQ ID No:8).

[0185] Exemplary variants of cholix toxins and immunotoxins comprising a cholix toxin that find use in the present compositions and methods are described, e.g., in co-pending application PCT/US2009/046292.

4. Cholera Exotoxin ("CET")

[0186] In some embodiments, the cytotoxin moiety is a cholera exotoxin ("CET"). Mature cholera exotoxin is a 634 residue protein. As shown in Figure 9C of PCT/US2009/046292, the amino acid sequence of CET differs from that of cholix toxin in the following 14 amino acid positions: 90, (CT =H; CET = N), 213 (CT = M; CET = I), 245 (CT = V; CET = A), 266 (CT = G; CET = K), 270 (CT = S; CET = E), 295 (CT = T; CET = P), 342 (CT = D, CET = A), 345 (CT = R, CET = Q), 376 (CT = T, CET =1), 400 (CT =S; CET =P), 523, (CT = D; CET = E), 553 (CT = E; CET = R), 622 (CT = T; CET = A), and 629 (CT = R; CET =Q). [0187] In some embodiments, the cytotoxin is a truncated version of CET in which the receptor binding domain, domain la, is deleted, to create a 40 kD version of CET corresponding to PE40, referred to herein as "CET40." In one embodiment, the CET is a CET40. Given the similarity of CET and PE, it is expected that additional variants of CE such as a CET38 or CET35 variant, can be made that correspond to variants of PE as described in the preceding section. For example, it is anticipated that some or all of CET domain lb can be deleted which, with the deletion of domain la, would create a CET variant akin to PE38. Similarly, it is anticipated that the carboxyl terminus of CET, which ends with KDELK (SEQ ID No:6), can be varied by replacing it with one of the various C-terminal sequences mentioned above as maintaining the toxicity of PE. In preferred embodiments, if the C-terminal sequence of CET is replaced, the C-terminal sequence used as a replacement is one suitable for use in humans. In some preferred embodiments, the C-terminal sequence of CET (KDELK; SEQ ID No:6) is replaced with the terminal sequence of PE, REDLK (SEQ ID No:5).

[0188] Similarly, it is anticipated that the NAD domain of CET, which comprises at least amino acid residues GGEDETVIG (SEQ ID No:7) can be varied by replacing it with another NAD domain sequence. In preferred embodiments, if the NAD domain sequence of CET is replaced, the NAD domain sequence used as a replacement is one suitable for use in humans. In some preferred embodiments, the NAD domain sequence of CET (GGEDETVIG (SEQ ID No:7) is replaced with the NAD binding site of PE comprising the amino acid sequence GGRLETILG (SEQ ID No:8)). 5. Shiga Toxin

[0189] In some embodiments, the cytotoxin moiety is a shiga toxin or a shiga-like toxin. Shiga toxins are a family of related toxins with two major groups, Stxl and Stx2. The most common sources for Shiga toxin are the bacteria Shigella dysenteriae and the Shigatoxigenic group of Escherichia coli (STEC), which includes serotype 0157:H7 and other enterohemorrhagic E. coli. Shiga toxin has two subunits— designated A and B— with a stoichiometry of AB5. The

B subunit is a pentamer that binds to globotriaosylceramide (Gb3). Following this, the A subunit is internalised and cleaved into two parts. The A l component then binds to the ribosome, disrupting protein synthesis. Stx-2 has been found to be approximately 400 times more toxic (as quantified by LD50 in mice) than Stx- 1 . 6. Ricin

[0190] In some embodiments, the cytotoxin moiety is ricin toxin. Ricin is a protein toxin that is extracted from the castor bean (Ricinus communis). The tertiary structure of ricin is a globular, glycosylated heterodimer of approximately 60-65 kDA, comprised of Ricin A and Ricin B chains. Ricin toxin A chain (RTA) and ricin toxin B chain (RTB) are of similar molecular weight, approximately 32 kDA and 34 kDA respectively. Ricin A Chain is an

N-glycoside hydrolase composed of 267 amino acids. Ricin B Chain is a lectin composed of 262 amino acids that is able to bind terminal galactose residues on cell surfaces. RTA cleaves a glycosidic bond within the large rRNA of the 60S subunit of eukaryotic ribosomes. RTA specifically and irreversibly hydrolyses the N-glycosidic bond of the adenine residue at position 4324 (A4324) within the 28S rRNA, but leaves the phosphodiester backbone of the RNA intact. The ricin targets A4324 that is contained in a highly conserved sequence of 12 nucleotides universally found in eukaryotic ribosomes. The sequence, 5'-AGUACGAGAGGA-3' (SEQ ID No:9), termed the sarcin-ricin loop, is important in binding elongation factors during protein synthesis. The depurination event rapidly and completely inactivates the ribosome, resulting in toxicity from inhibited protein synthesis. A single RTA molecule in the cytosol is capable of depurinating approximately 1500 ribosomes per minute. 7. Pokeweed Antiviral Protein

[0191] In some embodiments, the cytotoxin moiety is a pokeweed antiviral protein. Pokeweed antiviral protein (PAP) is another ribosome-inactivating proteins (RIPs) that inactivate ribosomes by depurinating rRNA at a specific site. iv. Exemplary Antibodies and Immunotoxins

[0192] Numerous antibodies for use in an immunotoxin are known in the art and find use in the present compositions and methods. Exemplary antibodies against tumor antigens include without limitation antibodies against the transferrin receptor (e.g. , HB21 and variants thereof), antibodies against CD22 (e.g., RFB4 and variants thereof), antibodies against CD25 (e.g., anti- Tac and variants thereof), antibodies against mesothelin (e.g., SS I , SSP1 , MN, H 1 , HN2 and variants thereof) and antibodies against Lewis Y antigen (e.g., B3 and variants thereof).

[0193] Antibodies for inclusion in an immunotoxin and that find use in the present invention have been described, e.g. , in U.S. Patent Nos. 5,242,824 (anti -transferrin receptor); 5,846,535 (anti-CD25); 5,889, 157 (anti-Lewis Y); 5,981 ,726 (anti-Lewis Y); 5,990,296 (anti-Lewis Y); 7,081 ,518 (anti-mesothelin); 7,355,012 (anti-CD22 and anti-CD25); 7,368,1 10 (anti- mesothelin); 7,470,775 (anti-CD30); 7,521 ,054 (anti-CD25); 7,541 ,034 (anti-CD22); in U.S. Patent Publ. No. 2007/0189962 (anti-CD22), and reviewed in, e.g., Frankel, Clin Cancer Res (2000) 6:326-334 and Kreitman, AAPS Journal (2006) 8(3):E532-E551.

[0194] Numerous immunotoxins successfully used in anticancer and acute graft-versus-host disease are also known in the art, and find use in the present compositions and methods.

Exemplary immunotoxins can be found, for example, on the worldwide web at clinicaltrials.gov and include without limitation LMB-2 (Anti-Tac(Fv)-PE38), BL22 and HA22 (RFB4(dsFv)- PE38), SS I P (SS l (dsFv)-PE38), HB21 -PE40. Additional immunotoxins of use are described in the patents listed above and herein, and are reviewed in, e.g. , Frankel, Clin Cancer Res (2000) 6:326-334 and Kreitman, AAPS Journal (2006) 8(3):E532-E551 .

[0195] HA22 is a recently developed, improved form of BL22. In HA22, residues SSY in the CDR3 of the antibody variable region heavy chain ("V_H") were mutated to THW. Compared to its parental antibody, RFB4, HA22 has a 5-10-fold increase in cytotoxic activity on various CD22-positive cell lines and is up to 50 times more cytotoxic to cells from patients with CLL and HCL (Salvatore, G., et al., Clin Cancer Res, 8(4):995- 1002 (2002); see also, co-owned application PCT/US02/30316, International Publication WO 03/027135). Further improved versions of HA22 are described in U.S. Patent Publ. No. US-2007-0189962-A 1 , also published in International Publication WO 2005/052006A2.

[0196] SS 1P has been shown to specifically kill mesothelin expressing cell lines and to cause regressions of mesothelin expressing tumors in mice (Hassan, R. et al., Clin Cancer Res 8:3520-6 (2002); Onda, M. et al., Cancer Res 61 :5070-7 (2001 )). Based on these studies and appropriate safety data, 2 phase I trials with SS 1P are being conducted at the National Cancer Institute in patients with mesothelin expressing cancers (Chowdhury, P. S. et al., Proc Natl Acad Sci USA 95:669-74 (1998); Hassan, R. et al., Proc Am Soc Clin Oncol 21 :29a (2002)). In addition, other therapies targeting mesothelin are in preclinical development (Thomas, A.M. et al., J Exp Med 200:297-306 (2004)). Human anti-mesothelin antibodies H 1 and HN2 are described in co-pending, co-owned U.S. Provisional Appl. No. 61/162,778.

[0197] HA22-LR, HA22-LR/6X, HA22-LR/8X, SS 1 P-LR, SS 1P-LR/6X, SS 1 P-LR/8X are lysosomal resistant variants of the HA22 and SS 1 P immunotoxins where cleavage clusters for lysosomal proteases have been removed. Lysosomal resistant variants are described, e.g., in Weldon, et al, Blood, (2009) 1 13(16):3792-800; in WO 2009/032954, and in co-pending , co-owned U.S. Prov. Appl. No. 61/241620.

[0198] Exemplary immunotoxins comprising a cholix toxin and cholera exotoxin that also find use in the present compositions and methods are described, e.g., in co-pending application PCT/US2009/046292.

4. Pharmaceutical Compositions and Administration

[0199] In one aspect the present invention provides a pharmaceutical composition or a medicament comprising a JAK3 inhibitor and/or at least one therapeutic foreign protein of the present invention, preferably a targeted toxin, and optionally a pharmaceutically acceptable carrier. A pharmaceutical composition or medicament can be administered to a patient for the treatment of a condition, including, but not limited to, a malignant disease or cancer. b. Formulation

[0200] Pharmaceutical compositions or medicaments for use in the present invention can be formulated by standard techniques using one or more physiologically acceptable carriers or excipients. Suitable pharmaceutical carriers are described herein and in Remington: The Science and Practice of Pharmacy, 21^s Ed., University of the Sciences in Philadelphia, Lippencott Williams & Wilkins (2005). The JA 3 inhibiors or therapeutic foreign proteins of the present invention can be formulated for administration by any suitable route, including via inhalation, topically, nasally, orally, parenterally, or rectally. Thus, the administration of the pharmaceutical composition may be made by intradermal, subdermal, intravenous, intramuscular, intranasal, inhalationally, intracerebral, intratracheal, intraarterial, intraperitoneal, intravesical, intrapleural, intracoronary, subcutaneously or intratumoral injection, with a syringe or other devices.

Transdermal administration is also contemplated, as are inhalation or aerosol administration. Tablets and capsules can be administered orally, rectally or vaginally. [0201] The compositions for administration will commonly comprise a solution of the JAK3 inhibitor or therapeutic foreign protein, preferably a targeted toxin, dissolved in a

pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of the JAK3 inhibitor or therapeutic foreign protein in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.

[0202] The tpharmaceutical compositions of this invention are suited for parenteral administration, including intravenous administration or administration into a body cavity.

[0203] The JAK3 inhibitors or therapeutic foreign proteins, preferably targeted toxins, of the present invention can be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are preferably prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use. In addition, they may also contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, of the active ingredient.

[0204] Controlled release parenteral formulations of the pharmaceutical compositions of the present invention can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, Banga, A.J., THERAPEUTIC PEPTIDES AND PROTEINS: FORMULATION, PROCESSING, AND DELIVERY SYSTEMS, Technomic Publishing Company, Inc., Lancaster, PA, (1995) incorporated herein by reference. Particulate systems include

microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic protein as a central core. In microspheres the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μιη are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μηι so that only nanoparticles are administered intravenously. Microparticles are typically around 100 μπι in diameter and are administered subcutaneously or intramuscularly. See, e.g., reuter J., COLLOIDAL DRUG DELIVERY SYSTEMS, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, TREATISE ON CONTROLLED DRUG DELIVERY, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339 (1992), both of which are incorporated herein by reference.

[0205] Polymers can be used for ion-controlled release of pharmaceutical compositions of the present invention. Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer R., Accounts Chem. Res. , 26:537-542 (1993)). For example, the block copolymer, polaxamer 407 exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res., 9:425-434 (1992); and Pec et al., J. Parent. Sci. Tech., 44(2):58-65 (1990)). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm. , 1 12:215-224 ( 1994)). In yet another aspect, liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et al., LIPOSOME DRUG DELIVERY SYSTEMS, Technomic Publishing Co., Inc., Lancaster, PA

(1993)). Numerous additional systems for controlled delivery of therapeutic proteins are known. See, e.g., U.S. Pat. No. 5,055,303, 5, 1 88,837, 4,235,871 , 4,501 ,728, 4,837,028 4,957,735 and 5,019,369, 5,055,303; 5,5 14,670; 5,413,797; 5,268, 164; 5,004,697; 4,902,505; 5,506,206, 5,271 ,961 ; 5,254,342 and 5,534,496, each of which is incorporated herein by reference.

[0206] Suitable formulations for transdermal application include an effective amount of a composition of the present invention with a carrier. Preferred carriers include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. For example, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the composition optionally with carriers, optionally a rate controlling barrier to deliver the composition to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin. Matrix transdermal formulations may also be used.

[0207] Suitable formulations for topical application, e.g., to the skin and eyes, are preferably aqueous solutions, ointments, creams or gels well-known in the art. Such may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

[0208] For oral administration, a pharmaceutical composition or a medicament can take the form of, for example, a tablet or a capsule prepared by conventional means with a

pharmaceutically acceptable excipient. Preferred are tablets and gelatin capsules comprising the active ingredient, i.e., a composition of the present invention, together with (a) diluents or fillers, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose (e.g., ethyl cellulose,

microcrystalline cellulose), glycine, pectin, polyacrylates and/or calcium hydrogen phosphate, calcium sulfate, (b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt, metallic stearates, colloidal silicon dioxide, hydrogenated vegetable oil, corn starch, sodium benzoate, sodium acetate and/or polyethyleneglycol; for tablets also (c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium

carboxymethylcellulose, polyvinylpyrrolidone and/or hydroxypropyl methylcellulose; if desired (d) disintegrants, e.g., starches (e.g., potato starch or sodium starch), glycolate, agar, alginic acid or its sodium salt, or effervescent mixtures; (e) wetting agents, e.g., sodium lauryl sulphate, and/or (f) absorbents, colorants, flavors and sweeteners. [0209] Tablets may be either film coated or enteric coated according to methods known in the . art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives, for example, suspending agents, for example, sorbitol syrup, cellulose derivatives, or hydrogenated edible fats; emulsifying agents, for example, lecithin or acacia; non-aqueous vehicles, for example, almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils; and preservatives, for example, methyl or propyl-p- hydroxybenzoates or sorbic acid. The preparations can also contain buffer salts, flavoring, coloring, and/or sweetening agents as appropriate. If desired, preparations for oral

administration can be suitably formulated to give controlled release of the active composition.

[0210] For administration by inhalation the JAK3 inhibitor or therapeutic protein, preferably an antibody and/or targeted toxin may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, 1 , 1 , 1 ,2- tetrafluorethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the composition, preferably an antibody and/or targeted toxin and a suitable powder base, for example, lactose or starch.

[0211] The compositions can also be formulated in rectal compositions, for example, suppositories or retention enemas, for example, containing conventional suppository bases, for example, cocoa butter or other glycerides.

[0212] Furthermore, the compositions can be formulated as a depot preparation. Such long- acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the composition can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. [0213] The compositions can, if desired, be presented in a pack or dispenser device that can contain one or more unit dosage forms containing the active ingredient. The pack can, for example, comprise metal or plastic foil, for example, a blister pack. The pack or dispenser device can be accompanied by instructions for administration. c. Dosa2e

[0214] In one embodiment of the present invention, a pharmaceutical composition or medicament is administered to a patient at a therapeutically effective dose to prevent, treat, or control a disease or malignant condition, such as cancer and to inhibit the neutralizing antibody response to the therapeutic foreign protein. The pharmaceutical composition or medicament is administered to a patient in an amount sufficient to elicit an effective therapeutic or diagnostic response in the patient. An effective therapeutic or diagnostic response is a response that at least partially arrests or slows the symptoms or complications of the disease or malignant condition. An amount adequate to accomplish this is defined as "therapeutically effective dose."

[0215] The dosage of the JAK3 inhibitors and therapeutic foreign proteins, preferably targeted toxins, or compositions administered is dependent on the species of warm-blooded animal (mammal), the body weight, age, individual condition, surface area of the area to be treated and on the form of administration. The size of the dose also will be determined by the existence, nature, and extent of any adverse effects that accompany the administration of a particular compound in a particular subject. A unit dosage for administration to a mammal of about 50 to 70 kg may contain between about 5 and 500 mg of the active ingredient. Typically, a dosage of the compound of the present invention, is a dosage that is sufficient to achieve the desired effect. [0216] Optimal dosing schedules can be calculated from measurements of therapeutic foreign protein, preferably targeted toxin, accumulation in the body of a subject. In general, dosage is from 1 ng to 1 ,000 mg per kg of body weight and may be given once or more daily, weekly, monthly, or yearly. Persons of ordinary skill in the art can easily determine optimum dosages, dosing methodologies and repetition rates. One of skill in the art will be able to determine optimal dosing for administration of a chimeric protein, preferably a targeted toxin, to a human being following established protocols known in the art and the disclosure herein.

[0217] Optimum dosages, toxicity, and therapeutic efficacy of compositions may vary depending on the relative potency of individual compositions and can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio, LD₅o/ED₅₀. Compositions that exhibit large therapeutic indices are preferred. While compositions that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compositions to the site of affected tissue to minimize potential damage to normal cells and, thereby, reduce side effects. [0218] The data obtained from, for example, animal studies (e.g. rodents and monkeys) can be used to formulate a dosage range for use in humans. The dosage of compounds of the present invention lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration. For any composition for use in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography (HPLC). In general, the dose equivalent of a chimeric protein, preferably a targeted toxin is from about 1 ng/kg to 100 mg/kg for a typical subject.

[0219] A typical targeted toxin composition of the present invention for intravenous administration would be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to about 100 mg per patient per day may be used. Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, 21 ^st Ed., University of the Sciences in Philadelphia, Lippincott Williams & Wilkins (2005).

[0220] Exemplary doses of the compositions described herein, include milligram or microgram amounts of the composition per kilogram of subject or sample weight (e.g., about 1 microgram per-kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a composition depend upon the potency of the composition with respect to the desired effect to be achieved. When one or more of these compositions is to be administered to a mammal, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular mammal subject will depend upon a variety of factors including the activity of the specific composition employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated. [0221] In one embodiment of the present invention, a pharmaceutical composition or medicament comprising a chimeric protein, preferably a targeted toxin, of the present invention is administered, e.g., in a daily dose in the range from about 1 mg of compound per kg of subject weight (1 mg/kg) to about l g/kg. In another embodiment, the dose is a dose in the range of about 5 mg/kg to about 500 mg/kg. In yet another embodiment, the dose is about 10 mg/kg to about 250 mg/kg. In another embodiment, the dose is about 25 mg/kg to about 150 mg/kg. A preferred dose is about 10 mg/kg. The daily dose can be administered once per day or divided into subdoses and administered in multiple doses, e.g., twice, three times, or four times per day. However, as will be appreciated by a skilled artisan, compositions described herein may be administered in different amounts and at different times. The skilled artisan will also appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or malignant condition, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or, preferably, can include a series of treatments. [0222] Exemplary doses of JAK3 inhibitors {e.g., CP690,550) are in the range of about 5- 1000 mg, for example, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 50 mg, 100 mg, 500 mg, or 1000 mg once or twice daily. In formulations or devices for controlled delivery over an extended period of time, the JAK3 inhibitor can be administered at a dose of about 5-75 mg/kg/day, for example, about 5, 10, 15, 20, 25, 30, 50, or 75 mg/kg/day. [0223] Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease or malignant condition treated. d. Administration

[0224] The compositions of the present invention can be administered for therapeutic treatments. In therapeutic applications, compositions are administered to a patient suffering from a disease or malignant condition, such as cancer, in an amount sufficient to cure or at least partially arrest the disease and its complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. An effective amount of the compound is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. [0225] Determination of an effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. Generally, an efficacious or effective amount of an immunoconjugate is determined by first administering a low dose or small amount of the immunoconjugate, and then incrementally increasing the administered dose or dosages, adding a second or third medication as needed, until a desired effect of is observed in the treated subject with minimal or no toxic side effects.

[0226] Single or multiple administrations of the compositions are administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the proteins of this invention to effectively treat the patient. Preferably, the dosage is administered once but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy.

Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.

[0227] To achieve the desired therapeutic effect, compositions may be administered for multiple days at the therapeutically effective daily dose. Thus, therapeutically effective administration of compositions to treat a disease or malignant condition described herein in a subject may require periodic (e.g., daily) administration that continues for a period ranging from three days to two weeks or longer. Typically, compositions will be administered for at least three consecutive days, often for at least five consecutive days, more often for at least ten, and sometimes for 20, 30, 40 or more consecutive days. While consecutive daily doses are a preferred route to achieve a therapeutically effective dose, a therapeutically beneficial effect can be achieved even if the compounds or compositions are not administered daily, so long as the administration is repeated frequently enough to maintain a therapeutically effective concentration of the composition in the subject. For example, one can administer a composition every other day, every third day, or, if higher dose ranges are employed and tolerated by the subject, once a week. [0228] Among various uses of the targeted toxins of the present invention are included a variety of disease conditions caused by specific human cells that may be eliminated by the toxic action of the fusion protein. For example, the targeted cells might express a cell surface marker such as mesothelin, CD22 or CD25. 5. Therapeutic Regimes a. Patients Subject to Treatment

[0229] In some embodiments, a therapeutic foreign protein and JAK3 inhibitor are

co-administered to a patient who has been receiving the therapeutic foreign protein, as described above and herein. The patient may or may not have already developed or produced antibodies against the foreign protein. In cases where the patient has already developed or produced neutralizing antibodies, co-administration of the JAK3 inhibitor therapeutically reduces or inhibits production of the neutralizing antibody response. In cases where the patient has not mounted an antibody response against the foreign protein, co-administration of the JAK3 inhibitor prevents production of the neutralizing antibody response. [0230] In some embodiments, the therapeutic foreign protein and JAK3 inhibitor are co-administered to a patient who has not before received the therapeutic foreign protein, but who is scheduled to commence treatment. In cases where the patient has not before been

administered the foreign protein, and therefore not mounted an antibody response against the foreign protein, co-administration of the JAK3 inhibitor prevents production of the neutralizing antibody response. b. Co-Administration of Foreign Protein and JAK3 inhibitor

[0231] The foreign protein and the JAK3 inhibitor can be administered concurrently or sequentially. The foreign protein and the JAK3 inhibitor can be administered by the same or different route of administration. Formulation, dosing, scheduling, and routes of administration for the foreign protein and the JAK3 inhibitor are as described above and herein. In some embodiments, the therapeutic foreign protein and the JAK3 inhibitor are formulated and administered together as a mixture in pharmaceutically acceptable excipients.

[0232] In some embodiments, a patient has already been receiving a therapeutic foreign protein and produced neutralizing antibodies, rendering the therapeutic foreign protein less effective or inefficacious for its intended purpose. In this case, administration of the therapeutic foreign protein can be temporarily discontinued and the JAK3 inhibitor can be administered alone until the detectable levels of antibodies against the foreign protein are eliminated or reduced to sufficiently low levels to allow for the efficacy of the therapeutic foreign protein for its intended purpose. Administration of the therapeutic foreign protein can then be resumed in conjunction with administration.

[0233] Co-administering the foreign protein with the JAK3 inhibitor also allows for administering a reduced dose of the foreign protein. For example, the dosage of the foreign protein can be reduced by about 10%, 20%, 30%, 40%, 50%, or more, when co-administering the foreign protein with the JAK3 inhibitor in comparison to when the foreign protein is administered without the JAK3 inhibitor. c. Assaying for Production of Neutralizing Antibodies

[0234] The effectiveness of co-administration of the JAK3 inhibitor in reducing, inhibiting or preventing the production of neutralizing antibodies can be measured using any method known in the art. For example, the presence or levels in a biological of antibodies that specifically bind to the administered foreign protein can be measured using known techniques including without limitation ELISA, surface plasmon resonance, Western blot, flow cytometry,

immunohistochemistry, as appropriate. The biological sample can be any biological sample that would contain neutralizing antibodies. Usually, the biological sample is a fluid sample, for example, blood, serum, plasma, mucous, saliva, urine. In some embodiments, the biological sample is a solid tissue. Because the antibody response in a patient against the foreign protein will be polyclonal, detection of binding of antibodies in the biological sample to the foreign protein is a sufficient indication that the patient has mounted a neutralizing antibody response to the foreign protein.

[0235] In one embodiment, an immunoassay is used to determine the presence or levels of antibodies in one or more biological samples taken from the patient. For example, an immunoassay plate can be coated with the foreign protein or with an antibody bound to the foreign protein. The foreign protein is then exposed to the biological sample. Labeled secondary antibody, e.g. , that binds to the Fc portion of immunoglobulin, is then bound to any antibodies in the biological sample that are bound to the immobilized foreign protein. Detection of labeled secondary antibody indicates the presence of neutralizing antibodies in the biological sample that bind to the foreign protein. The label can be any detectable label using techniques known in the art, including without limitation a radioisotope, a fluorophore, a chemiluminescent label, an enzyme, etc. The levels of neutralizing antibodies that specifically bind to the foreign protein can also be quantified. Methods for detecting antibodies against therapeutic foreign proteins ( .ka., anti-drug antibodies) are described, e.g., in Smith, et al., Regul Toxicol

Pharmacol (2007) 49(3):230-7; Bourdage, et al., J Immunol Methods (2007) 327(1 -2): 10-17; Lofgren, et ah, J Immunol (2007) 178(1 1 ):7467-72; and Stubenrauch, et al., J P harm Biomed Anal (2010) PMID 20083366.

[0236] The presence or levels of antibodies against the foreign protein can be measured before and after administration of the foreign protein, and/or before and after administration of the JAK3 inhibitor. The presence or levels of antibodies against the foreign protein also can be measured over a predetermined period of time during co-administration of both the foreign protein and the JAK3 inhibitor. For example, biological samples may be taken at regular intervals, e.g., once daily, once weekly, once bi-weekly (i.e., every other week or twice monthly), or once a month, as needed or desired. [0237] The detectable presence or increased levels of antibodies that specifically bind to foreign protein after administration of the foreign protein indicate that the patient develops or produces neutralizing antibodies to the foreign protein.

[0238] In a patient who has been receiving foreign protein and who produces detectable levels of antibodies that specifically bind to the foreign protein, administration of the JAK3 inhibitor with or without foreign protein, will reduce or eliminate the production of antibodies against the therapeutic foreign protein. A baseline measurement of levels of antibodies that specifically bind to the foreign protein can be taken before the first administration of the JAK3 inhibitor, and then one or more measurements of levels of antibodies that specifically bind the foreign protein are taken after a regime of the JAK3 inhibitor administration has begun. Reduction or inhibition of the neutralizing antibody response is demonstrated when the levels of antibodies that specifically bind the foreign protein in the biological sample are reduced by at least 10%, for example, by at least about 25%, 50%, 75% or completely eliminated after administration of the JAK3 inhibitor has begun.

[0239] In a patient who has not yet produce detectable levels of antibodies that specifically bind to the foreign protein, co-administration of the JAK3 inhibitor with foreign protein will prevent the production of antibodies against the therapeutic foreign protein. A baseline measurement of levels of antibodies that specifically bind to the foreign protein can be taken before the first administration of the JAK3 inhibitor, and then one or more measurements of levels of antibodies that specifically bind the foreign protein are taken after a regime of the JAK3 inhibitor administration has begun. Prevention of the neutralizing antibody response is demonstrated when the levels of antibodies that specifically bind the foreign protein in the biological sample are undetectable or remain at sufficiently low levels such that the foreign protein is efficacious for its intended purpose after administration of the JAK3 inhibitor has begun.

EXAMPLES The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1 : CP-690.,550 reduces immunogenicity of SS I P

[0240] To examine the effects of CP-690,550 administered during the immunization, 4 week Alzet osmotic minipumps delivering compound or PEG at a flow rate of 0.26 μΐ/hr were implanted s.c. under isoflurane anesthesia 3 days prior to Immunization (i.p.). Mice received 5-20 mg/kg/day of CP-690550. Immunization was done using 1 week interval schedules shown in Figure 1. The tested groups were as listed in Table 1 :

Table 1

[0241] Results are shown in Figures 2-7.

Example 2: CP-690,550 reduces immunogenicity of HA22 [0242] CP-690,550 was administered immediately prior and during the immunization of Balb/c mice with the immunotoxing, HA22. CP-690,550 was given via 4-week Alzet osmotic minipumps delivering compound or vehicle (5% ethanol/distilled water) at a flow rate of 0.1 1 μΐ/hr). Minipumps were implanted s.c. under isoflurane anesthesia 5 days prior to immunization intraperiotoneally (i.p.) Mice received 20 mg/kg/day of CP-690,550. HA22 was administered using a 1 -week interval schedule.

Table 2

[0243] Results are shown in Figures 8-10.

Example 3 CP-690,550 reduces immunogenicity of keyhole limpet hemocyanin (KLH)

[0244] To examine the effect of CP-690,550 on the immunogenicity of KLH, Alzet osmotic minipumps delivering CP-650,550 or vehicle (PEG solution) were implanted s.c, as described above. Five mice of each group were immunized with KLH (5 ug/mouse) i.p. every week. At day 21 , blood was drawn from each mice and total Ig concentration was determined by ELISA. The KLH-specific antibody production of the non CP -690,550 treatment group (closed square) and CP-690,550 treatment group (diamonds) are shown as average values in Figure 1 1 . All isotypes of KLH-specific antibodies were observed in the CP-690,550 treated and non-treated groups. However, IgG l and IgG2a levels were lower in CP-690,550 treated mice (Figure 12).

[0245] Figure 13 shows the results of an experiment to measure total number of splenocytes in mice with or without CP-690550 treatment. CP-690,550 was implanted at Day (-3), and KLH (50 micro g) was injected s.c. at day 0, then sacrificed mice at day 10. (n=3). As shown in Figure 13, CP-690,550 treatment reduced the total number of splenocytes (about 3 fold less) It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS: 1 . A method of reducing, inhibiting or preventing a neutralizing antibody response to a therapeutic foreign protein in a patient in need thereof, comprising

co-administering to the patient the foreign protein and a JAK3 inhibitor, thereby reducing, inhibiting or preventing the neutralizing antibody response to the therapeutic foreign protein.

2. The method of claim 1 , wherein the JAK3 inhibitor is CP 690,550.

3. The method of claim 1 , wherein the foreign protein is protein not functionally expressed in the patient, a bacterial protein, a viral protein, a plant protein, or an antibody.

4. The method of claim 1 , wherein the foreign protein is Factor VIII.

5. The method of claim 1 , wherein the foreign protein is a chimeric molecule comprising a targeting moiety and a cytotoxin moiety.

6. The method of claim 5, wherein the chimeric molecule is an immunotoxin comprising an antibody against a cell surface antigen on a tumor cell and a cytotoxin moiety.

7. The method of claim 6, wherein the cell surface antigen is selected from the group consisting of CD 19, CD21 , CD22, CD25, CD30, CD33, CD79b, transferrin receptor, EGF receptor, mesothelin, cadherin and Lewis Y.

8. The method of claim 6, wherein the antibody is selected from the group consisting of B3, RFB4, SS I , SS I P, SS 1P-LR, MN, HN 1 , HN2 and HB21.

9. The method of claim 5, wherein the cytotoxin moiety is selected from Pseudomonas exotoxin A, diphtheria toxin, cholix toxin, cholera exotoxin, shiga toxin, ricin toxin and pokeweed antiviral protein (PAP).

10. The method of claim 6, wherein the immunotoxin is selected from the group consisting of LMB-2, LMB-7, LMB-9, BL22, HA22, HA22-LR, HA22-LR/6X, HA22- LR/8X, SS 1 P, SS 1P-LR, SS 1 P-LR/6X and SS 1P-LR/8X.

1 1. The method of claim 1 , wherein the foreign protein and the JA 3 inhibitor are administered concurrently.

12. The method of claim 1 , wherein the foreign protein and the JA 3 inhibitor are administered sequentially.

13. The method of claim 1 , wherein the JAK3 inhibitor is administered in an extended-release formulation.

14. The method of claim 1 , wherein the patient has already produced neutralizing antibodies to the foreign protein.

15. The method of claim 1 , wherein the patient has not produced neutralizing antibodies to the foreign protein.

16. The method of claim 1 , wherein the patient is human.

17. A composition comprising a mixture of a therapeutic protein that elicits neutralizing antibodies against the protein in a human and a JAK3 inhibitor.

18. The composition of claim 17, wherein the JAK3 inhibitor is CP 690,550

19. The composition of claim 17, wherein the therapeutic protein is a protein not functionally expressed in a patient, a bacterial protein, a viral protein, a plant protein, or an antibody.

20. The composition of claim 17, wherein the therapeutic protein is Factor VIII.

21. The composition of claim 17, wherein the therapeutic protein is a chimeric molecule comprising a targeting moiety and a cytotoxin moiety.

22. The composition of claim 21 , wherein the chimeric molecule is an immunotoxin comprising an antibody against a cell surface antigen on a tumor cell and a cytotoxin moiety.

23. The composition of claim 22, wherein the cell surface antigen is selected from the group consisting of CD 19, CD21 , CD22, CD25, CD30, CD33, CD79b, transferrin receptor, EGF receptor, mesothelin, cadherin and Lewis Y.

24. The composition of claim 21 , wherein the cytotoxin moiety is selected from Pseudomonas exotoxin A, diphtheria toxin, cholix toxin, cholera exotoxin, shiga toxin, ricin toxin and pokeweed antiviral protein (PAP).

25. The composition of claim 22, wherein the immunotoxin is selected from the group consisting of LMB-2, LMB-7, LMB-9, BL22, HA22, HA22-LR, HA22-LR/6X, HA22-LR/8X, SS1P, SS1P-LR, SS1P-LR/6X and SS 1 P-LR/8X.