Antibodies binding to IL-4 and / or IL-13 and uses thereof

Abstract

The present invention relates to antibodies that bind to IL-4 and / or IL-13 and their uses. In particular, the present invention relates to novel humanized anti-IL-4 and IL-13 antibodies and fragments thereof, as well as novel bispecific antibodies and fragments thereof that specifically bind to IL-4 and IL-13. The present invention also includes the use of the antibodies to treat or prevent IL-4 and / or IL-13 mediated diseases or disorders, including allergic asthma and dermatitis.

Description

[0001] The present application is a divisional application of an invention application filed on October 14, 2008, with Chinese application number 201710131539.4 and invention name “Antibodies binding to IL-4 and / or IL-13 and uses thereof”. Field of the Invention

[0002] The present invention relates to novel anti-IL-4 antibodies, anti-IL-13 antibodies and bispecific anti-IL-4 / anti-IL-13 antibodies and their use in the improvement, treatment and prevention of diseases or disorders in mammals, including humans, caused by improper IL-4 and / or IL-13 activity or metabolism. The antibodies of interest may block the binding and / or signal transduction of ligands such as IL-4 or IL-13 to receptors or receptor complexes such as IL-4Rα, IL-13Rα1 and IL-13Rα2. The present invention also discloses preventive, immunotherapeutic and diagnostic compositions containing the antibodies of interest and their use in methods for preventing or treating diseases in mammals, including humans, which are caused by improper metabolism and / or activity of lymphoid cells and non-lymphoid cells, including monocytes, fibroblasts and endothelial cells. These diseases include autoimmune deficiencies and diseases caused by or characterized by inflammation, such as allergic asthma and dermatitis. Background of the Invention

[0003] Interleukin-4 (IL-4) is a pleiotropic cytokine with a broad spectrum of biological effects on lymphoid B and T cells and many non-lymphoid cells, including monocytes, endothelial cells and fibroblasts. For example, IL-4 stimulates the proliferation of several cell lines that are dependent on IL-2 and IL-3, induces the expression of class II major histocompatibility complex on resting B cells, and enhances IgG4 and IgE secretion by human B cells. IL-4 is associated with Th2-type immune responses, is produced by Th2 cells, and promotes the differentiation of Th2 cells. IL-4 is implicated in a variety of disorders, such as allergies and asthma.

[0004] IL-13 is a recently discovered 112 amino acid cytokine secreted by activated T lymphocytes, B lymphocytes and activated mast cells (Minty, A. et al., Nature, 1993, 362, 248-250, and McKenzie, AN et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 3735-3739).

[0005] IL-13 is described as an IL-4-like cytokine because it shares many common biological properties with IL-4. It is expressed in B cells (Defrance, T. et al., J. Exp. Med., 1994, 179, 135-143, Punnonen, J. et al., Proc. Natl. Acad. Sci. (USA), 1993, 90, 3730-3734, Fior, R. et al., Eur. Cytokine Network, 1994, 5, 593-600), monocytes (Muzio, MRF et al., Blood, 1994, 83, 1738-1743, De Its activity is indeed similar to that of IL-4 on WaalMalefyt, R. et al., J. Immunol, 1993, 151, 6370-6381, Doyle, A. et al., Eur. J. Immunol. 1994, 24, 1441-1445, Montaner, LJ et al., J. Exp. Med., 1993, 178, 743-747, Sozzani, P. et al., J. Biol. Chem., 1995, 270, 5084-5088) and other non-hematopoietic cells (Herbert, JM et al., Febs Lett., 1993, 328, 268-270, and Derocq, JM et al., Febs Lett. 1994, 343, 32-36). On the other hand, in contrast to IL-4, it has no specific effect on resting or activated T cells (Zurawuki, G. et al., Immunol. Today, 1994, 15, 19-26).

[0006] AJMinty and the review article on IL-13 describe in detail the multiple biological activities of IL-13 on monocytes / macrophages, B lymphocytes, and some hematopoietic precursors. In addition, there are several data showing that this cytokine has pleiotropic effects on other cell types. These non-hematopoietic cells that are directly affected by IL-13 are endothelial cells, microglia, keratinocytes, and kidney and colon cancers.

[0007] One of the analysis stages of the signals emitted by intracellular biomolecules is the identification of their membrane receptors. Studies on the IL-13 receptor have shown that IL-13 and IL-4 have a common receptor, or at least some components of a common receptor complex, as well as common signal transduction elements (Zurawski SM et al., Embo Journal, 1993, 12, 2663-2670, Aversa, G. et al., J. Exp. Med., 1993, 178, 2213-2218, Vita, N. et al., Biol. Chem., 1995, 270, 3512-3517, Lefort, S. et al., Febs Lett., 1995, 366, 122-126). This receptor is present on the surface of many types of cells, and its number varies depending on the cell type considered. AJ Minty pointed out the comparable distribution of IL-13 and IL-4 receptors (Interleukin-13 for Cytokines in Health and Disease. Eds. DG Remick and JS Fri, Marcel Decker, NY 1996).

[0008] Cell surface receptors and receptor complexes bind to IL-4 and / or IL-13 with different affinities. The main components of receptors and receptor complexes that bind to IL-4 and / or IL-13 are IL-4Rα, IL-13Rα1, and IL-13Rα2. These chains are expressed on the cell surface as monomers or heterodimers of IL-4Rα / IL-13Rαl (type II IL-4R) or IL-4Rα / γc (type I IL-4R). IL-4Rα monomers and IL-4Rα / γc heterodimers bind to IL-4 but not to IL-13. IL-13Rα1 and IL-13Rα2 monomers bind to IL-13 but not to IL-4. The IL-4Rα / IL-13Rα1 heterodimer binds to both IL-4 and IL-13 (Murata et al., Int. J. Hematol., 1999, 69, 13-20).

[0009] Th2-type immune response promotes antibody production and humoral immunity, and is carefully designed to resist extracellular pathogens. Th2 cells are mediators (humoral immunity) that produce Ig and produce IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13 (Tanaka, et al., Cytokine Regulation of Humoral Immunity, 251-272, Snapper, editor, John Wiley and Sons, New York (1996)). The feature of Th2-type immune response is the production of certain types of cytokines (such as IL-4, IL-13) and specific types of antibodies (IgE, IgG4), which belong to typical allergic reactions and may cause tearful eyes and asthma symptoms, such as airway inflammation and contraction of airway muscle cells in the lungs.

[0010] According to their biological functions, IL-4 and IL-13 are both cytokines with important therapeutic significance, playing a key role in many diseases, including asthma (Curr Opin Allergy Clin Immunol 2005, Vo. 5, 161-166). IL-4 has been shown to be able to inhibit autoimmune diseases, and both IL-4 and IL-13 have been shown to have the potential to enhance anti-tumor immune responses. Since both cytokines are involved in the pathogenesis of allergic diseases, inhibitors of these cytokines should be able to provide therapeutic benefits.

[0011] Accordingly, there is a need for improved agents that can inhibit IL-4, IL-13, and for a single agent that can inhibit both IL-4 and IL-13. SUMMARY OF THE INVENTION

[0012] The present invention provides novel humanized monoclonal and bispecific antibodies and fragments and derivatives thereof that specifically bind to IL-4 and / or IL-13. Some anti-IL-4 and / or IL-13 mono- or bispecific antibodies and fragments thereof may be altered to prevent the formation of intrachain disulfide bonds, thereby forming molecules that are stable during preparation and in vivo use. The antibodies of the present invention neutralize IL-4 and / or IL-13 activity in the bioassays described herein.

[0013] The present invention includes the amino acid sequences of the variable heavy and light chains of the antibodies and their corresponding nucleic acid sequences.

[0014] Another embodiment of the present invention includes cell lines and vectors carrying the antibody sequences of the present invention.

[0015] Another embodiment of the invention is the use of the antibody in the preparation of a pharmaceutical composition for the treatment of diseases and disorders related to IL-4 and / or IL-13 function and metabolism. In particular, the invention is relevant to the treatment of cancer, autoimmune deficiencies, and diseases caused by or characterized by inflammation, such as allergic asthma and dermatitis.

[0016] This application also relates to the following:

[0017] 1. Humanized antibody that specifically binds to IL-4.

[0018] 2. The antibody of item 1, comprising a variable light chain domain consisting of SEQ ID NO: 3.

[0019] 3. The antibody of item 1, comprising a variable heavy chain domain selected from SEQ ID NO: 4 and SEQ ID NO: 5.

[0020] 4. The optional antibody of any one of items 1-3, wherein the antibody is an antibody fragment.

[0021] 5. The antibody of item 1, which comprises a constant region C H1 , C H2 and C H3 Structural domain.

[0022] 6. A polypeptide as described in item 5, wherein the IgG antibody is an IgG4 antibody.

[0023] 7. A nucleic acid encoding the polypeptide of item 1.

[0024] 8. A vector containing the nucleic acid of item 7.

[0025] 9. A cell containing the vector of item 8.

[0026] 10. A humanized antibody that specifically binds to IL-13.

[0027] 11. The antibody of claim 10, comprising a variable light chain domain consisting of SEQ ID NO: 1.

[0028] 12. The antibody of claim 11, comprising a variable heavy chain domain consisting of SEQ ID NO: 2.

[0029] 13. The optional antibody of any one of items 10-12, wherein said antibody is an antibody fragment.

[0030] 14. The antibody of any one of items 10-12, comprising a C constant region H1 , C H2 and C H3 Structural domain.

[0031] 15. A polypeptide as described in item 14, wherein the IgG antibody is an IgG4 antibody.

[0032] 16. A nucleic acid encoding the polypeptide of item 15.

[0033] 17. A vector containing the nucleic acid of item 16.

[0034] 18. A cell containing the vector of item 17.

[0035] 19. A bispecific antibody or bispecific antibody fragment that specifically binds to IL-13 and IL-4.

[0036] 20. The bispecific antibody or antibody fragment of claim 19, further comprising a variable light chain and a variable heavy chain.

[0037] 21. The bispecific antibody or antibody fragment of claim 20, wherein the variable light chain comprises the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:3.

[0038] 22. The bispecific antibody or antibody fragment of claim 20, wherein the variable heavy chain comprises the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:5.

[0039] 23. The bispecific antibody or antibody fragment of claim 20, wherein the variable heavy chain comprises the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:4.

[0040] 24. The bispecific antibody or antibody fragment of claim 20, wherein the variable light chain comprises the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:3, and the variable heavy chain comprises the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:4.

[0041] 25. The bispecific antibody or antibody fragment of item 24, wherein the SEQ ID NO: 1 and SEQ ID NO: 3 are connected together by a peptide connector, and the SEQ ID NO: 2 and SEQ ID NO: 4 are connected together by a peptide connector.

[0042] 26. The bispecific antibody or antibody fragment of item 25, wherein the peptide connector consists of SEQ ID NO:6.

[0043] 27. A bispecific antibody that specifically binds IL-13 and IL-13, wherein the variable light chain comprises SEQ ID NO: 1 and SEQ ID NO: 3, the variable heavy chain comprises SEQ ID NO: 2 and SEQ ID NO: 4, and further comprises a constant region domain.

[0044] 28. The bispecific antibody of item 27, wherein the constant region domain is composed of CH1, CH2, CH3 and CL.

[0045] 29. A pharmaceutical composition comprising the humanized antibody of item 1 or item 8 and a pharmaceutically acceptable carrier.

[0046] 30. A pharmaceutical composition comprising the optional bispecific antibody or antibody fragment of items 19-28 and a pharmaceutically acceptable carrier.

[0047] 31. The humanized antibody of item 1 or item 10, which is further conjugated to an effector molecule.

[0048] 32. The humanized antibody of claim 31, wherein the effector molecule is selected from the group consisting of: heterologous polypeptides, drugs, radionucleotides and toxins.

[0049] 33. The optional bispecific antibody or antibody fragment of any one of items 19-28, further conjugated to an effector molecule.

[0050] 34. The bispecific antibody or fragment of claim 33, wherein the effector molecule is selected from the group consisting of: heterologous polypeptides, drugs, radionucleotides and toxins.

[0051] 35. A method for treating an allergic disease in a mammal, comprising the step of administering to said mammal a therapeutically effective amount of the optional bispecific antibody or antibody fragment according to items 19-28.

[0052] 36. A method for treating an allergic disease in a mammal, comprising the step of administering to said mammal a therapeutically effective amount of the humanized antibody according to item 1 or 10.

[0053] 37. A method for treating cancer, comprising the step of administering to said mammal a therapeutically effective amount of the optional bispecific antibody or antibody fragment according to items 19-28.

[0054] 38. A method for treating cancer, comprising the step of administering a therapeutically effective amount of the humanized antibody according to item 1 or 10 to said mammal.

[0055] 39. A method for treating asthma in a mammal, comprising the step of administering to said mammal a therapeutically effective amount of the optional bispecific antibody or antibody fragment according to items 19-28.

[0056] 40. A method for treating asthma in a mammal, comprising the step of administering to said mammal a therapeutically effective amount of the humanized antibody according to item 1 or 10.

[0057] 41. A method for treating a disease associated with abnormal production of IL-4 and / or IL-13 in a mammal, comprising the step of administering to the mammal a therapeutically effective amount of the optional bispecific antibody or antibody fragment according to items 19-28.

[0058] 42. A method for treating a disease associated with abnormal production of IL-4 and / or IL-13 in a mammal, comprising the step of administering a therapeutically effective amount of the humanized antibody according to item 1 or 10 to the mammal.

[0059] 43. A method of inhibiting a TH-2 mediated response in a mammal, comprising the step of administering to said mammal a therapeutically effective amount of an optional bispecific antibody or antibody fragment according to items 19-28.

[0060] 44. A method for inhibiting TH-2 mediated responses in a mammal, comprising the step of administering to said mammal a therapeutically effective amount of the humanized antibody according to item 1 or 10.

[0061] 45. A nucleic acid encoding the bispecific antibody of item 27.

[0062] 46. ​​A vector containing the nucleic acid of item 45.

[0063] 47. A cell containing a vector as described in item 46.

[0064] Other features and advantages are described herein and will be set forth in the following detailed description and accompanying drawings. Sequence Listing <110> Sanofi-Aventis US Inc Rao, Ercole V. Mikol (Vincent) D. Li (Danxi) Jochen Kruip Davison, Matthew <120> Antibodies binding to IL-4 and / or IL-13 and uses thereof <130> US2008 / 020 <160> 7 <170> PatentIn Version 3.5 <210> 1 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Humanized mouse / mouse VL3 region <400> 1 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr 20 25 30 Gly Gln Ser Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp 65 70 75 80 Pro Val Gln Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Ala 85 90 95 Glu Asp Ser Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 2 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Humanized mouse / mouse VH2 region <400> 2 Glu Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Gly Gly 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Ser 20 25 30 Ser Ile Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Gly Asp Gly Arg Ile Asp Tyr Ala Asp Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Ser Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Glu Met Thr Ser Leu Arg Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Asp Gly Tyr Phe Pro Tyr Ala Met Asp Phe Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 3 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Humanized mouse / mouse VL1 region <400> 3 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly 1 5 10 15 Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Tyr Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 4 <211> 124 <212> PRT <213> Synthetic Sequence <220> <223> Humanized Mouse / Mouse VH1 Region <400> 4 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Pro Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe 50 55 60 Gln Gly Arg Ala Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Leu Lys Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val 100 105 110 Trp Gly Ala Gly Thr Leu Val Thr Val Ser Ser Ala 115 120 <210> 5 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Humanized mouse / mouse VH2 region <400> 5 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Ala Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe 50 55 60 Gln Gly Arg Ala Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Leu Lys Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val 100 105 110 Trp Gly Ala Gly Thr Leu Val Thr Val Ser Ser Ala 115 120 <210> 6 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Connector <400> 6 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 7 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 7 ggaggcggag ggtccggagg cggaggatcc 30 BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 This is a schematic diagram of a bispecific anti-IL-4 / IL-13 antibody molecule containing four polypeptide chains. The two lighter chains consist of N-VL hB-B13 -Connector-VL h8D4-8 -CL-C (CL, light chain constant region), the two heavier chains consist of N-VH hB-B13 -Linker-VH h8D4-8The linker sequence (G4S)2 is GGGGSGGGGS (SEQ ID NO: 6).

[0066] Figure 2 The amino acid sequences of the variable domains of the humanized B-B13 anti-IL-13 antibody (SEQ ID NOs: 1 and 2) and the amino acid sequences of the variable domains of the humanized 8D4-8 anti-IL-4 antibody (SEQ ID NOs: 3, 4, and 5) are shown. Underlined amino acid changes are made. Bold indicates CDRs.

[0067] Detailed description of the invention

[0068] The present invention is not limited to the specific methodology, protocol, cell line, vector or reagent described herein, because they can be changed without departing from the spirit and scope of the present invention. Moreover, the terms used herein are for the purpose of illustrating specific embodiments only and are not intended to limit the scope of the present invention. Unless otherwise defined, all technical and scientific terms and any acronyms used herein have the same meaning as those commonly understood by those of ordinary skill in the art of the present invention. Any similar or equivalent methods and materials described herein can be used in practicing the present invention, and only exemplary methods, equipment and materials are described herein.

[0069] All patents and publications mentioned herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing reported proteins, enzymes, vectors, host cells and methodologies that might be used with and in the present invention. However, nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosures by virtue of prior invention.

[0070] Prior to teaching the preparation and use of IL-4 and / or IL-13 related methods and products of interest, the following non-limiting definitions of certain terms and phrases are provided to guide the skilled artisan.

[0071] "Interleukin-4" (IL-4) refers to a naturally occurring or endogenous mammalian IL-4 protein or a protein having the same amino acid sequence as the naturally occurring or endogenous corresponding mammalian IL-4 protein (e.g., a recombinant protein, a synthetic protein (i.e., a protein prepared by synthetic organic chemistry)). Accordingly, as defined herein, the term includes mature IL-4 protein, polymorphic or allelic variants, other isoforms of IL-4, and modified or unmodified forms (e.g., lipidated or glycosylated) of the above. Naturally occurring or endogenous IL-4 includes wild-type proteins such as mature IL-4, polymorphic or allelic variants, and other isoforms and mutant forms that occur naturally in mammals (e.g., humans, non-human primates). These proteins can be recovered or isolated from sources such as naturally occurring IL-4. These proteins and proteins having the same amino acid sequence as the naturally occurring or endogenous corresponding IL-4 are named after the corresponding mammal. For example, when the corresponding mammal is a human, the protein is called human IL-4. Several mutant IL-4 proteins are known in the art, such as disclosed in WO 03 / 038041.

[0072] "Interleukin-13" (IL-13) refers to a naturally occurring or endogenous mammalian IL-13 protein or a protein having the same amino acid sequence as the corresponding naturally occurring or endogenous mammalian IL-13 protein (e.g., a recombinant protein, a synthetic protein (i.e., a protein prepared by synthetic organic chemistry)). Accordingly, as defined herein, the term includes mature IL-13 protein, polymorphic or allelic variants, other isoforms of IL-13 (e.g., produced by alternative splicing or other cellular processes), and modified or unmodified forms of the foregoing (e.g., lipidated or glycosylated). Naturally occurring or endogenous IL-13 includes wild-type protein such as mature IL-13, polymorphic or allelic variants, and other isoforms and mutant forms that occur naturally in mammals (e.g., humans, non-human primates). For example, the IL-13 used herein includes human IL-13 variants in which the Arg at position 110 of mature human IL-13 is replaced by Gin (position 110 of mature IL-13 corresponds to position 130 of the precursor protein), and Gin is associated with asthma (specific and non-specific asthma) and other IL-13 variants. (Heinzmann et al., Hum Mol Genet 9: 549-559 (2000)). These proteins can be recovered or separated from sources such as naturally occurring IL-13. These proteins and proteins with the same amino acid sequence as the corresponding naturally occurring or endogenous IL-13 are named after the corresponding mammals. For example, when the corresponding mammal is a human, the protein is called human IL-13. Several mutant IL-13 proteins are known in the art, such as those disclosed in WO 03 / 035847.

[0073] In the case of antibody chain polypeptide sequences, the phrase "substantially identical" is understood to mean antibody chains that exhibit at least 70%, 80%, 90%, 95% or more sequence identity to a reference polypeptide sequence. In the case of nucleic acid sequences, the term is understood to mean nucleotide sequences that exhibit at least greater than 85%, 90%, 95% or 97% or more sequence identity to a reference nucleic acid sequence.

[0074] The term "identity" or "homology" may refer to the percentage of nucleotide bases or amino acid residues in a candidate sequence that are identical to the residues in the corresponding sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage identity over the entire sequence and not considering any conservative substitutions as part of the sequence identity. Neither N-terminal or C-terminal extensions or insertions should be construed as reducing the identity or homology. Methods and computer programs for comparison are readily available and well known in the art. Sequence identity can be measured using sequence analysis software.

[0075] Phrases and terms such as "functional fragments, variants, derivatives or analogs" of antibodies or antigens and their various forms refer to compounds or molecules that have the same biological activity as the full-length antibody or antigen of interest. For example, a functional fragment or analog of an anti-IL-4 antibody is a fragment or analog that can bind to an IL-4 molecule, or a fragment or analog that can prevent or substantially reduce the ability of a ligand or an agonist or antagonist antibody to bind to IL-4.

[0076] A "substitution" variant is one in which at least one amino acid residue in the native sequence has been removed and a different amino acid has been inserted at the same position. The substitution may be single, where only one amino acid is substituted in the molecule, or multiple, where two or more amino acids are substituted in the same molecule. Multiple substitutions may be located at consecutive sites. Likewise, an amino acid may be substituted by multiple residues, where such a variant includes both substitutions and insertions. An "insertion" variant is one in which one or more amino acids are inserted adjacent to an amino acid at a specific position in a native sequence. Adjacent to an amino acid means linked to the α-carboxyl or α-amino functional group of the amino acid. A "deletion" variant is one in which one or more amino acids in the native amino acid sequence have been removed. Typically, a deletion variant has one or two amino acids deleted in a specific region of the molecule.

[0077] The term "antibody" is used in the broadest sense, specifically covering monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), antibody fragments or synthetic polypeptides carrying one or more CDRs or derived from CDR sequences, as long as these polypeptides exhibit the desired biological activity. Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins with the same structural characteristics. In general, antibodies are considered to be Igs with a defined or recognized specificity. Therefore, although antibodies exhibit binding specificity to specific targets, immunoglobulins include antibodies and other antibody-like molecules that lack target specificity. The antibodies described in the present invention may be of any type (such as IgG, IgE, IgM, IgD, IgA, etc.), or subclass (such as IgG1, IgG2, IgG 2a , IgG3, IgG4, IgA1, IgA2, etc.) ("type" and "species", as well as "subtype" and "subclass" are used interchangeably herein). Natural or wild-type (i.e., obtained from a population member that has not been artificially manipulated) antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, which are composed of two identical light chains (L) and two identical heavy chains (H). Each heavy chain has a variable domain (V H ), followed by multiple constant domains. Each light chain has a variable domain (V L ), and the other end has a constant domain. The so-called "unmanipulated" means that it has not been treated to contain or express foreign antigen-binding molecules. Wild-type can refer to the most common allele or species found in a population or to antibodies obtained from unmanipulated animals, compared to alleles or polymorphisms, or variants or derivatives obtained by some form of manipulation, such as mutagenesis, using recombinant methods, etc., to change the amino acids of the antigen-binding molecule.

[0078] As used herein, "anti-IL-4 antibody" means an antibody that can specifically bind to IL-4 as defined herein or a polypeptide (derivative) derived from these antibodies, including but not limited to molecules that inhibit or substantially reduce the binding of IL-4 to its receptor or inhibit IL-4 activity.

[0079] As used herein, "anti-IL-13 antibody" means an antibody or polypeptide (derivative) derived from such antibody that can specifically bind to IL-13 as defined herein, including but not limited to molecules that inhibit or substantially reduce the binding of IL-13 to its receptor or inhibit the activity of IL-13.

[0080] With respect to the variable domains of antibodies, the term "variable" refers to certain parts of related molecules that have extensive sequence differences between antibodies and are used for specific recognition and binding of a particular antibody to its specific target. However, the variability is not evenly distributed throughout the variable domain of an antibody. The variability is concentrated in three segments called complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) or hypervariable regions, which are located in the variable domains of both the light and heavy chains. The more conserved portions of the variable domains are called framework (FR) regions or framework sequences. Each variable domain of a native heavy and light chain includes four FR regions, which primarily adopt a β-sheet configuration, connected by three CDRs, which form loops that connect the β-sheet structure and in some cases form part of the β-sheet structure. The CDRs of each chain are usually linked adjacently by the FR regions and, with the help of the CDRs from the other chain, contribute to the formation of the antibody target binding site (epitope or determinant) (see Kabat et al. Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD (1987)). As used herein, the numbering of immunoglobulin amino acid residues is based on the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated. A CDR may have the ability to specifically bind to a cognate epitope.

[0081] The term "hinge" or "hinge region" as used herein refers to the flexible polypeptide comprising amino acids between the first and second constant domains of an antibody.

[0082] The term "antibody fragment" refers to a complete or full-length chain or portion of an antibody, generally the target binding region or variable region. Examples of antibody fragments include, but are not limited to, F ab 、F ab' 、F (ab')2 and F v Fragment. A "functional fragment" or "analog of an anti-IL-4 and / or IL-13 antibody" is a fragment or analog that can prevent or substantially reduce the ability of the receptor to bind to a ligand or initiate signal transduction. As used herein, a functional fragment is generally synonymous with an "antibody fragment" and, with respect to an antibody, can refer to a fragment that can prevent or substantially reduce the ability of the receptor to bind to a ligand or initiate signal transduction, such as a F v 、F ab 、F (ab')2 Etc. v The fragment is a dimer formed by the variable domain of a heavy chain and the variable domain of a light chain in a non-covalent manner (V H -V LIn this configuration, the three CDRs of each variable domain interact to determine the V H -V L The target binding site on the surface of the dimer is the same as that of the intact antibody. The six CDRs together confer target binding specificity to the intact antibody. However, even a single variable domain (or an F-terminal domain comprising only three target-specific CDRs) v half of the target), and still has the ability to recognize and bind to the target.

[0083] "Single-chain F v "、"sF v " or "scAb" antibody fragments include the V H and V L domains, wherein these domains are located on a single polypeptide chain. v The polypeptide also includes a segment located at V H and V L The polypeptide linker between the regions, which is usually a flexible molecule, allows the sFv to form the desired structure suitable for target binding.

[0084] The term "diabody" refers to antibody fragments with two antigen-binding sites, which may include the light chain variable domain (V L ) is connected to the heavy chain variable domain (V H ). By using a linker that is too short so that the two variable domains on the same chain cannot pair, the diabody domains are forced to pair with the binding domains of another chain, generating two antigen binding sites.

[0085] F ab The fragment contains the variable and constant domains of the light chain and the variable and first constant domains of the heavy chain (C H1 ). ab' Fragment and F ab The difference between the fragments is that the former is in C H1 Several residues are added to the carboxyl terminus of the domain to include one or more cysteines from the antibody hinge region. (ab')2 The disulfide bond at the hinge cysteine ​​of the pepsin digestion product can generate F ab' Fragments. Additional enzymatic and chemical treatments of antibodies can generate other functional fragments of interest.

[0086] The term "linear Fab" refers to the tetravalent antibody described by Miller et al. (Miller et al. (2003), J Immunol. 170: 4854-4861). "Linear Fab" is composed of a series of identical CH1-VH domains, paired with the same light chain at each CH1-VH position. These molecules were developed to increase the titer of the antibody and enhance its functional affinity through the avidity effect, however, they are monospecific.

[0087] The term "bispecific antibodies (BsAbs)" refers to molecules that combine the antigen-binding sites of two antibodies in the same molecule. Thus, bispecific antibodies are able to bind to two different antigens simultaneously. In addition to their use for diagnostic purposes, bispecific antibodies pave the way for the development of new therapeutic approaches by diverting powerful effector systems to the diseased area or increasing the neutralizing or stimulatory activity of antibodies.

[0088] Initial attempts to couple the binding specificities of two intact antibodies directed against different target antigens were made using chemically fused heteroconjugate molecules (Staerz et al. (1985), Nature 314:628-631).

[0089] Bispecific antibodies have been prepared from hybrid hybridomas using the heterohybridoma technique and have been shown in vitro to have properties similar to those observed for heteroconjugates (Milstein & Cuello (1983) Nature 305:537-540).

[0090] Despite promising results with heteroconjugates or bispecific antibodies prepared from cell fusion as described above, several factors make their large-scale therapeutic use impractical. These factors include the rapid clearance of large heteroconjugates from the body, the labor-intensive techniques required to produce either class of molecules, the extensive purification required to separate the heteroconjugates from the monoconjugates or monospecific antibodies, and the generally low yields.

[0091] Genetic engineering is increasingly being used to design, alter, and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions.

[0092] A variety of recombinant techniques have been developed to efficiently produce bispecific antibodies, either as antibody fragments (Carter et al. (1995), J. Hematotherapy 4:463-470; Pluckthun et al. (1997) Immunotechology 3:83-105; Todorovska et al. (2001) J. Immunol. Methods 248:47-66) or as full-length IgG formats (Carter (2001) J. Immunol. Methods 248:7-15).

[0093] Combining two different scFvs produces a bispecific antibody format with minimal molecular weight called sc-BsAbs or Ta-scFvs (Mack et al. (1995), Proc. Acad. Sci. USA. 92: 7021-7025; Mallender et al. (1994) J. Biol. Chem. 269: 199-206). In genetic engineering, bispecific antibodies are constructed by fusing two scFvs via a dimerization functional group such as a leucine zipper (Kostelny et al. (1992) J. Immunol. 148: 1547-53; de Kruif et al. (1996) J. Biol. Chem. 271: 7630-4).

[0094] As mentioned above, diabodies are small bivalent bispecific antibody fragments. These fragments contain VH linked to VL on the same peptide chain by using a linker that is too short (less than 12 amino acids) to allow pairing between the two domains on the same chain. These domains are forced to intermolecularly pair with complementary domains on another chain, thereby generating two antigen binding sites. These dimeric antibody fragments, or "diabodies", are bivalent and bispecific. (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA. 90: 6444-6448). The size of diabodies is similar to that of Fab fragments. Polypeptide chains of VH and VL domains connected by a linker of 3 to 12 amino acids mainly form dimers (diabodies), while those connected by a linker of 0 to 2 amino acid residues are favorable for the formation of trimers (triabodies) and tetramers (tetrabodies). In addition to the length of the linker, the exact mode of oligomerization appears to depend on the composition and orientation of the V-domains (Hudson et al. (1999), J Immunol Methods 231: 177-189). The final structure of diabody molecules is poorly predictable.

[0095] Although constructs based on sc-BsAbs and diabodies show interesting clinical potential, it has been shown that such non-covalently bound molecules are not sufficiently stable under physiological conditions. The overall stability of the scFv fragment depends on the stability of the VL and VH domains themselves and the stability of the domain interface. It has been shown that insufficient stability of the VH-VL interface of the scFv fragment is the main cause of irreversible scFv inactivation, because the transient opening of the interface allowed by the peptide linker exposes hydrophobic patches that favor aggregation, thus causing instability and low yield ( and Plückthun (2001), J. Mol. Biol. 305:989-1010).

[0096] US5,989,830 discloses an alternative method for preparing bispecific bivalent antigen binding proteins from VH and VL domains. Such a two-headed antibody fragment is obtained by expressing a bicistronic vector encoding two polypeptide chains, one of which has two VHs connected in series by a peptide connector (VH1-connector-VH2), and the other polypeptide chain consists of complementary VL domains connected in series by a peptide connector (VL1-connector-VL2). US5,989,830 describes that each connector should contain at least 10 amino acid residues.

[0097] US2005 / 0003403 A1 describes a multivalent protein complex (PPC) of increased valency. The multivalent protein complex comprises two polypeptide chains, which are generally arranged laterally to each other. Each polypeptide chain generally comprises 3 or 4 "v-regions" comprising amino acid sequences that are capable of forming an antigen binding site when matched with corresponding v-regions on the opposing polypeptide chain. Up to about 6 "v-regions" may be used on each polypeptide chain. The v-regions on each polypeptide chain are linearly connected to each other and may be connected by discrete linker regions. When arranged in the form of a PPC, the v-regions on each polypeptide chain form a single antigen binding site. The complex may contain one or several binding specificities.

[0098] However, the use of such molecules showed aggregation, instability and poor expression yield (Wu et al. (2001) Prot. Eng. 14: 1025-1033). These are typical stability problems that may occur when expressing single-chain based antibodies. and Plückthun (2001), J. Mol. Biol. 305:989-1010).

[0099] It is therefore an object of the present invention to provide bispecific multivalent antibodies with which the formation of aggregates can be avoided. Furthermore, it should be stable so that it can be used in therapeutic applications.

[0100] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0101] The monoclonal antibodies herein specifically include "chimeric" antibodies, in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence derived from a particular species or belonging to a particular antibody class or subclass (type or subtype), wherein the rest of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, as well as antibody fragments thereof, as long as these antibody fragments exhibit the desired biological activity of binding to IL-4 and / or IL-13 or affecting the activity or metabolism of IL-4 and / or IL-13 (US Patent No. 4,816,567; and Morrison et al., Proc Natl Acad Sci USA 81:6851 (1984)). Thus, CDRs from antibodies of the same class can be transplanted to FRs of antibodies of different classes or subclasses.

[0102] Monoclonal antibodies are highly specific antibodies, directed against a single target site, epitope or determinant. Moreover, unlike conventional (polyclonal) antibody preparations that usually include different antibodies directed against different determinants (epitopes) of an antigen, each monoclonal antibody is directed against a single determinant on the target. In addition to its specificity, the synthesis of monoclonal antibodies in host cells is advantageous, free from contamination by other immunoglobulins, and provides the relevant genes and mRNA encoding its antibody chains for cloning. The modifier "monoclonal" indicates the nature of antibodies derived from a basic homologous antibody group, which should not be construed as requiring any particular method to produce the antibody. For example, the monoclonal antibodies used in the present invention can be separated from phage antibody libraries using well-known techniques or purified from polyclonal preparations. The parental monoclonal antibodies used in the present invention can be prepared by the hybridoma method described in Nature 256:495 (1975) by Kohler et al., or by well-known recombinant methods in the art.

[0103] The term "multivalent antibody" as used herein refers to an antibody that contains two or more antigen binding sites and is thus capable of binding to two or more antigens of the same or different structures. The term "bivalent" means that the antibody contains two antigen binding sites. The term "tetravalent" means that the antibody contains four antigen binding sites.

[0104] The term "antigen binding site" as used in the present invention refers to a portion of an antibody that includes a region that specifically binds to part or all of an antigen and is complementary to part or all of the antigen. When the antigen is large, the antibody may only bind to a specific portion of the antigen, which is called an epitope. The antigen binding domain can be provided by one or more antibody variable domains. Preferably, the antigen binding domain is formed by the combination of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0105] The term "antigen" as used herein refers to a molecule or a portion of a molecule that can be bound by the antibodies of the present invention. An antigen may have one or more than one epitope. Examples of antigens that can be recognized by the antibodies of the present invention include, but are not limited to, serum proteins such as cytokines, e.g., IL-4, IL5, IL9, and IL-13, bioactive peptides, cell surface molecules such as receptors, transporters, ion channels, viral and bacterial proteins.

[0106] The term "monospecific" as used in the present invention means that the multivalent antibody of the present invention can only recognize one antigen and all antigen binding sites are identical.

[0107] The term "bispecific" as used in the present invention means that the multivalent antibody of the present invention can recognize two different epitopes on the same or two different antigens.

[0108] The term "multispecific" as used in the present invention means that the multivalent antibody of the present invention can recognize multiple different epitopes on the same or multiple different antigens.

[0109] The term "connector" as used in the present invention refers to a peptide that is adjusted to connect the variable domains of the antibody structure of the present invention. The peptide connector may contain any amino acids, preferably glycine (G) and serine (S). The connectors may be the same or different between or within the heavy chain polypeptide and the light chain polypeptide. In addition, the length of the connector may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. The preferred peptide connector unit of the heavy chain domain and the light chain domain is GGGGS. The number of connector units of the heavy chain and the light chain may be the same (symmetrical order) or different from each other (asymmetric order).

[0110] The peptide linker is preferably long enough to provide a sufficient degree of flexibility to prevent the antibody portions from interfering with each other's activities, for example through steric hindrance, thereby allowing the protein to fold properly and, if necessary, allow the antibody molecule to interact with two or more receptors on the same cell that may be widely separated; however, it is preferably short enough to allow the antibody portions to remain stable in the cell.

[0111] Thus, the length, composition and / or conformation of the peptide linker can be readily selected by one skilled in the art to optimize the desired properties of the multivalent antibody.

[0112] "Humanized" forms of non-human (eg, murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, F v 、F ab 、F ab' 、F (ab')2 or other target binding subsequences), which contain sequences derived from non-human immunoglobulins compared to human antibodies. In general, the humanized antibody essentially includes one, typically two, variable domains in total, in which all or substantially all CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or substantially all FR regions are FR regions of human immunoglobulin template sequences. The humanized antibody may also include at least a portion of an immunoglobulin constant region (F c), usually the constant region of the selected human immunoglobulin template. In general, the goal is to have an antibody molecule with the lowest immunogenicity in the human body. Therefore, it is possible that one or more amino acids in one or more CDRs can also be replaced with amino acids with lower immunogenicity in the human host, but the specific binding function of the one or more CDRs to IL-4 and / or IL-13 is not substantially minimized. Alternatively, the FR may be non-human, but the amino acids with the highest immunogenicity are replaced with amino acids with lower immunogenicity. However, the CDR transplantation discussed above is not the only way to obtain humanized antibodies. For example, modifying only the CDR region may not be sufficient, because it is not uncommon for the framework region residues to participate in determining the three-dimensional structure of the CDR loop and the overall affinity of the antibody to its ligand. Therefore, any method can be implemented to modify the non-human parent antibody molecule into an antibody molecule with lower immunogenicity to humans, and global sequence identity with human antibodies is not always required. Thus, humanization can also be achieved, for example, by replacing only a few residues, in particular residues that are exposed on the antibody molecule and not buried in the interior of the molecule and therefore not easily accessible to the host's immune system. This approach is taught herein in terms of replacing "mobile" or "flexible" residues on an antibody molecule, with the goal of reducing or mitigating the immunogenicity of the resulting molecule, without destroying the specificity of the antibody for its epitope or determinant. See, e.g., Studnicka et al., Prot Eng 7(6)805-814, 1994; Mol Imm 44:1986-1988, 2007; Sims et al., J Immunol 151:2296 (1993); Chothia et al., J Mol Biol 196:901 (1987); Carter et al., Proc Natl Acad Sci USA 89:4285 (1992); Presta et al., J Immunol 151:2623 (1993), WO 2006 / 042333 and U.S. Pat. No. 5,869,619.

[0113] The humanization method of interest is based on the influence of the molecular flexibility of antibodies during and at the time of immune recognition. Protein flexibility is related to the molecular motion of protein molecules. Protein flexibility is the ability of the entire protein, a partial protein or a single amino acid residue to adopt a set of conformations, wherein the conformations are significantly different from each other. Information about protein flexibility can be obtained by performing protein X-ray crystallography experiments (see, for example, Kundu et al., 2002, Biophys J 83: 723–732.), nuclear magnetic resonance experiments (see, for example, Freedberg et al., J Am Chem Soc 1998, 120 (31): 7916-7923) or performing molecular dynamics (MD) simulations. Performing MD simulations of proteins on a computer allows the determination of the motion of all protein atoms over a period of time by calculating the physical interactions between atoms. The output of the MD simulation is the trajectory of the protein under study during the simulation time period. The trajectory is a collection of protein conformations, also referred to as snapshots, which are periodically sampled, for example, every 1 picosecond (ps) during the simulation. It is by analyzing a collection of snapshots that one can quantify the flexibility of the amino acid residues of a protein. Thus, for a polypeptide containing a flexible residue, a flexible residue is a residue that adopts a collection of different conformations. MD methods are well known in the art, see, for example, Brooks et al., Proteins: A Theoretical Perspective of Dynamics, Structure and Thermodynamics (Wiley, New York, 1988). Several software programs can perform MD simulations, such as Amber (see Case et al. (2005) J Comp Chem 26: 1668-1688), Charmm (see Brooks et al. (1983) J Comp Chem 4: 187-217; and MacKerell et al. (1998) The Encyclopedia of Computational Chemistry vol. 1: 271-177, Schleyer et al. eds. Chichester: John Wiley & Sons) or Impact (see Rizzo et al., J Am Chem Soc; 2000; 122(51): 12898-12900).

[0114] Most protein complexes enjoy a relatively large and flat embedded surface, and the flexibility of binding partner proteins has been shown to provide a source of their elasticity, allowing them to conformationally adapt to each other (Structure (2000) 8, R137-R142). Thus, instances of "induced fit" have been shown to play a major role in protein-protein interfaces. In addition, a steadily growing body of data suggests that proteins actually bind ligands of a variety of shapes, sizes, and compositions (Protein Science (2002) 11: 184-187), and conformational diversity appears to be a key component of the ability to recognize different partner proteins (Science (2003) 299, 1362-1367). Flexible residues are involved in protein-protein partner protein binding (Structure (2006) 14, 683-693).

[0115] Flexible residues can adopt many conformations, which provide a set of interaction regions that are likely to be recognized by memory B cells and trigger an immunogenic response. Therefore, antibodies can be humanized by modifying many residues from the framework regions so that the set of conformations and the set of recognition regions displayed by the modified antibodies are as similar as possible to those adopted by human antibodies.

[0116] This can be achieved by modifying a limited number of residues in the following manner: (1) constructing a homology model of the parent mAb and performing MD simulations; (2) analyzing the flexible residues and identifying the most flexible residues of the non-human antibody molecule, as well as identifying residues or motifs that are likely to be sources of heterogeneity or degradation reactions; (3) identifying human antibodies that exhibit the most similar set of recognition regions to the parent antibody; (4) determining the flexible residues to be mutated, and residues or motifs that are likely to be sources of heterogeneity and degradation are also mutated; (5) checking for the presence of known T cell or B cell epitopes. Flexible residues can be found using the MD calculations taught herein by using an implicit solvent model that describes the reasons for the interaction of the aqueous solvent with the protein atoms during the simulation. Once a set of flexible residues is identified within the variable light and heavy chains, a set of human heavy and light chain variable region frameworks that are highly similar to the antibody of interest are identified. For example, a BLAST search can be performed on a set of flexible residues in a database of antibody human germline sequences. It can also be performed by comparing the dynamics of the parent mAb with the dynamics of a germline canonical structure library. CDR residues and neighboring residues were excluded from the search to ensure that high affinity for the antigen was retained.

[0117] Flexible residues are then replaced. When several human residues show similar homology, the selection is also driven by the residue properties that may affect the solution behavior of the humanized antibody. For example, polar residues are preferred on exposed flexible loops, rather than hydrophobic residues. Residues that are potential sources of instability and heterogeneity are also mutated, even if they are found on CDRs. These residues include exposed methionine, because sulfoxide formation may be due to proteolytic cleavage of oxygen, acid-sensitive bonds such as Asp-Pro dipeptide bonds (Drug Dev Res (2004) 61: 137-154); deamidation sites (J Chromatog (2006) 837: 35-43) and N-glycosylation sites such as Asn-X-Ser / Thr sites at exposed asparagine residues and subsequent small amino acids such as Gly, Ser, Ala, His, Asn or Cys. Typically, exposed methionine is replaced by Leu, exposed asparagine is replaced by glutamine or aspartic acid, or the following residue is replaced. For glycosylation sites (Asn-X-Ser / Thr), either Asn or Ser / Thr residues can be replaced.

[0118] The formed composite sequence is checked for known B cell or linear T cell epitopes. For example, a search can be performed using the publicly available IEDB. If a known epitope is found in the composite sequence, another set of human sequences is retrieved and replaced.

[0119] Unlike the resurfacing method of US Patent No. 5,639,641, this method explores both B cell-mediated and T cell-mediated immunogenic responses. This method also avoids the problem of loss of activity that is sometimes observed using CDR transplantation (US Patent No. 5,530,101). In addition, stability and solubility issues are also considered during the design and selection process, thereby producing antibodies optimized for low immunogenicity, high antigen affinity and improved biophysical properties.

[0120] For example, US Patent No. 5,639,641 discloses strategies and methods for surface remodeling antibodies and other methods for reducing the immunogenicity of antibodies in different hosts. Briefly, in a preferred method, (1) a position alignment of an antibody heavy chain and light chain variable region library is generated to generate positions exposed on the surface of the heavy chain and light chain variable region frameworks, wherein at least about 98% of the aligned positions of all variable regions are identical; (2) a set of heavy chain and light chain variable region framework surface exposed amino acid residues are defined for use in non-human, such as rodent antibodies (or antibody fragments thereof); (3) a set of heavy chain and light chain variable region framework surface exposed amino acid residues that are most consistent with the rodent surface exposed amino acid residues are identified; and (4) any atom of any residue in the rodent antibody CDR is excluded from the residue; In addition to the amino acid residues within, the exposed amino acids on the surface of the heavy chain and light chain variable region frameworks determined in step (2) are replaced with the exposed amino acid residues on the surface of the heavy chain and light chain variable region frameworks identified in step (3) to generate a humanized, for example, rodent antibody that retains binding specificity.

[0121] Antibodies can be humanized using a number of other techniques including CDR grafting (EPO 0 239 400; WO 91 / 09967 and US Patent Nos. 5,530,101 and 5,585,089), veneering or resurfacing (EPO 0 592 106; EPO 0 519596; Padlan, 1991, Molec Imm 28(4 / 5):489-498; Studnicka et al., 1994, Prot Eng 7(6):805-814 and Roguska et al., 1994, PNAS 91:969-973) and chain shuffling (US Patent No. 5,565,332). Human antibodies can be prepared by many methods known in the art, including but not limited to phage display methods (see U.S. Patent Nos. 4,444,887, 4,716,111, 5,545,806 and 5,814,318 and WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735 and WO 91 / 10741), using transgenic animals such as rodents, using chimeric cells, etc.

[0122] "Antibody homologue" or "homologue" refers to any molecule that specifically binds to IL-4 and / or IL-13 as taught herein. Thus, antibody homologues include natural or recombinant antibodies, whether modified or not, that retain the desired biological property (e.g., binding to IL-4 or IL-13), such as F ab or F v Molecules, single-chain antibodies, and polypeptides carrying one or more CDR regions, etc. The amino acid sequence of the homologues need not be identical to the naturally occurring antibody amino acid sequence, but may be altered or modified to carry substituted amino acids, inserted amino acids, deleted amino acids, amino acids other than the 20 amino acids normally present in the protein, etc., to obtain polypeptides with enhanced or other advantageous properties.

[0123] An antibody with a homologous sequence is an antibody whose amino acid sequence has sequence homology with the amino acid sequence of the IL-4, IL-13 or bispecific IL-4 / IL-13 antibody of the present invention. Preferably, the homology is located in the amino acid sequence of the variable region of the antibody of the present invention. "Sequence homology" as applied to the amino acid sequences herein is defined as having at least about 90%, 91%, 92%, 93%, 94% or more sequence homology with other amino acid sequences, more preferably at least about 95%, 96%, 97%, 98% or 99% sequence homology, as determined, for example, by the FASTA search method according to Pearson & Lipman, Proc Natl Acad Sci USA 85, 2444-2448 (1988).

[0124] Chimeric antibodies are antibodies with different antibody portions that are derived from different sources, such as different antibodies, different kinds of antibodies, different animal species, such as antibodies having variable regions derived from mouse monoclonal antibodies paired with human immunoglobulin constant regions, etc. Thus, a humanized antibody is a chimeric antibody. Methods for producing chimeric antibodies are known in the art, see, for example, Morrison, 1985, Science 229: 1202; Oi et al., 1986, BioTechniques 4: 214; Gillies et al., 1989, J Immunol Methods 125: 191-202 and US Patent Nos. 5,807,715, 4,816,567 and 4,816,397.

[0125] Artificial antibodies include scFv fragments, chimeric antibodies, diabodies, triabodies, tetrabodies and mru (see review by Winter & Milstein, 1991, Nature 349:293-299; and Hudson, 1999, Curr Opin Imm 11:548-557), each of which has antigen binding or epitope binding ability. v Fragment (scF v ) in which the antibody V H and V L The domains are connected by flexible peptides. Generally, the linker is a peptide of about 15 amino acids. If the linker is much smaller, for example 5 amino acids, a diabody is formed. The smallest binding unit of an antibody is a CDR, usually a heavy chain CDR2 with sufficient specific recognition and binding ability. This fragment is called a molecular recognition unit or MRU. Several such MRus can be connected together by a short linker peptide to form an artificial binding protein with a higher affinity than a single MRU.

[0126] Functional equivalents of the target antibody are also included within the scope of the present invention. The term "functional equivalent" includes antibodies with homologous sequences, antibody homologs, chimeric antibodies, artificial antibodies and modified antibodies, for example, wherein each functional equivalent is defined according to its ability to bind to IL-4 and / or IL-13, inhibit IL-4 and / or IL-13 signaling or function, or inhibit IL-4 and / or IL-13 binding to its receptor. The skilled person will understand that a group of molecules named "antibody fragments" overlaps with a group named "functional equivalents". Methods for preparing functional equivalents that retain IL-4 and / or IL-13 binding ability are known to those skilled in the art and are disclosed, for example, in WO 93 / 21319, EPO Ser. No. 239,400, WO 89 / 09622, EPO Ser. No. 338,745 and EPO Ser. No. 332,424.

[0127] Functional equivalents of the present application also include modified antibodies, such as antibodies modified by covalent attachment of any type of molecule to the antibody. For example, modified antibodies include antibodies modified by known protection / blocking groups such as glycosylation, acetylation, pegylation, deamidation, phosphorylation, amidation, derivatization, proteolytic cleavage, attachment to cell ligands, attachment to toxins or cytotoxic moieties or other proteins. Covalent attachment does not require such antibodies, which are immune to generating anti-idiotypic reactions. The modification can be achieved by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis, etc. In addition, the modified antibody may contain one or more atypical amino acids.

[0128] A number of techniques are available to those skilled in the art to achieve optimization of binding affinity. Generally, these techniques include replacing multiple amino acid residues at the target site, followed by screening and analyzing the binding affinity of the mutant polypeptide to the associated antigen or epitope.

[0129] Once the antibody is identified and isolated, it can generally be used to generate variant antibodies or mutants or mutant proteins, wherein, for example, one or more amino acid residues in one or more hypervariable regions of the antibody are changed. Alternatively or additionally, one or more changes (e.g., substitutions) of framework region residues may be introduced into the antibody, wherein these changes result in an increase in the binding affinity of the antibody mutant to IL-4 and / or IL-13. Examples of modified framework region residues include residues that directly bind to the antigen non-covalently (Amit et al., Science 233: 747-753 (1986)); residues that affect CDR conformation or interact with it (Chothia et al., J Mol Biol 196: 901-917 (1987)); and / or residues that participate in V L -V HThe invention relates to the modification of one or more of the framework region residues that are present at the interface of the antibody (EP 239 400). In certain embodiments, modification of one or more of these framework region residues results in an increase in the binding affinity of the antibody to the cognate antigen. For example, in this embodiment of the invention, about 1 to about 5 framework region residues may be changed. Sometimes, this is sufficient to produce an antibody mutant suitable for preclinical testing, even if no residue in the hypervariable region is changed. Normally, however, an antibody mutant may include one or more hypervariable region changes. The constant region may also be changed to obtain desired or more desired effector properties.

[0130] The hypervariable region residues that are altered may be changed randomly, especially when the initial binding affinity of the parent antibody allows the randomly generated antibody mutants to be readily screened for altered binding in the assays taught herein.

[0131] One method for obtaining antibody mutants, such as CDR mutants, is "alanine scanning mutagenesis" (Cunningham & Wells, Science 244: 1081-1085 (1989) and Cunningham & Wells, Proc Nat Acad Sci USA 84: 6434-6437 (1991)). One or more hypervariable region residues are replaced by alanine or polyalanine residues. These hypervariable region residues that show functional sensitivity to the substitution are then refined by introducing further or other mutations at or at the substitution site. Therefore, although the site for introducing amino acid sequence changes is determined in advance, the nature of the mutation itself does not need to be determined in advance. Depending on the desired properties of the scanned residues, similar substitutions can be attempted using other amino acids.

[0132] A more systematic method for identifying amino acid residues to be modified includes identifying hypervariable region residues that are involved in binding to IL-4 and / or IL-13 and hypervariable region residues that are rarely or not involved in binding to IL-4 and / or IL-13. An alanine scan of non-binding hypervariable region residues is performed, wherein each ala mutant is tested for enhanced binding to IL-4 and / or IL-13. In another embodiment, these residues that are significantly involved in IL-4 and / or IL-13 binding are selected for modification. Modification may include deletion of residues or insertion of one or more residues adjacent to the target residue. However, normally modification includes substitution of residues with another amino acid. Conservative substitutions may be the first substitution. If the substitution results in a change in biological activity (e.g., binding affinity), another conservative substitution may be performed to determine whether more substantial changes are obtained.

[0133] By selecting amino acids whose properties differ more substantially from those normally located at a particular site, more substantial modifications can be made to the range and biological properties exhibited by the antibody. Thus, the substitution can be made while maintaining: (a) the structure of the polypeptide backbone in the area of ​​the substitution, such as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the size of the side chain.

[0134] For example, naturally occurring amino acids can be divided into the following groups based on common side chain properties:

[0135] (1) Hydrophobic: methionine (M or met), alanine (A or ala), valine (V or val), leucine (L or leu) and isoleucine (I or ile);

[0136] (2) Neutral, hydrophilic: cysteine ​​(C or cys), serine (S or ser), threonine (T or thr), asparagine (N or asn) and glutamine (Q or gln);

[0137] (3) Acidic: aspartic acid (D or asp) and glutamic acid (E or glu);

[0138] (4) Basic: histidine (H or his), lysine (K or lys) and arginine (R or arg);

[0139] (5) residues that affect chain orientation: glycine (G or gly) and proline (P or pro), and

[0140] (6) Aromatic: tryptophan (W or trp), tyrosine (Y or tyr), phenylalanine (F or phe).

[0141] Non-conservative substitutions require exchanging an amino acid with an amino acid from another group. Conservative substitutions require exchanging one amino acid for another within the group.

[0142] Preferred amino acid substitutions include those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity, and (4) confer or modify other physiological-chemical or functional properties of these analogs. Analogs may include various mutant proteins of sequences other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made to the naturally occurring sequence (preferably in the portion of the polypeptide outside the region forming intermolecular contacts). Except for changes in the size or conformation of the R group or side chain, conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., amino acid replacements should not tend to interrupt helices present in the parent sequence, or disrupt other types of secondary structure that characterize the parent sequence (Proteins, Structures and Molecular Principles, Creighton, ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (Branden & Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354:105 (1991).

[0143] Typically, antibody mutants with improved biological properties have an amino acid sequence identity or similarity of at least 75% to the amino acid sequence of the heavy or light chain variable domain of the parent anti-human IL-4 and / or IL-13 antibody, at least 80%, at least 85%, at least 90%, and usually at least 95%. With respect to the parent antibody sequence, identity or similarity is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., identical residues) or similar (i.e., amino acid residues from the same group based on the same side chain properties, see above) to the parent antibody residues after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage sequence identity.

[0144] Alternatively, the heavy and light chain FR and CDR regions or the F region of the anti-IL-4, anti-IL-13 or bispecific IL-4 / IL-13 antibody may be used to identify the antibody. cSystematic mutation of the region can generate antibody mutants. Another method for generating antibody mutants includes affinity maturation using phage display (Hawkins et al., J Mol Biol 254: 889-896 (1992) and Lowman et al., Biochemistry 30 (45): 10832-10838 (1991)). It is known that phage coat protein fusions (Smith, Science 228:1315 (1985); Scott & Smith, Science 249:386 (1990); Cwirla et al., Proc Natl Acad Sci USA 8:309 (1990); Devlin et al., Science 249:404 (1990); Wells & Lowman, Curr Opin Struct Biol 2:597 (1992) and US 5,223,409) can be used to link the phenotype of the displayed protein or peptide to the genotype of the phage particle encoding them. ab Domains have also been displayed on bacteriophage (McCafferty et al., Nature 348:552 (1990); Barbas et al., Proc Natl Acad Sci USA 88:7978 (1991); and Garrard et al., Biotechnol 9:1373 (1991)).

[0145] Monovalent phage display consists of displaying a panel of protein variants as fusions to the phage coat protein on the phage particle (Bass et al., Proteins 8:309 (1990). Affinity maturation or improvement of the balanced binding affinity of multiple proteins has been achieved previously by sequential application of mutagenesis, monovalent phage display, and functional analysis (Lowman & Wells, J Mol Biol 234:564578 (1993); U.S. Patent No. 5,534,617), such as by focusing on the CDR regions of antibodies (Barbas et al., Proc Natl Acad Sci USA 91:3809 (1994); and Yang et al., J Mol Biol 254:392 (1995)).

[0146] Many (e.g., 10 6 The invention relates to a library of protein variants having differences in specific positions of the sequence of a protein, wherein each phage particle contains DNA encoding a specific protein variant. After multiple rounds of affinity purification using an immobilized antigen, a single phage clone is isolated and the amino acid sequence of the displayed protein is deduced from the DNA.

[0147] After the antibody mutants are generated, the biological activity of the molecule relative to the parent antibody can be determined according to the teachings herein. As noted above, this involves determining the binding affinity and / or other biological activities or physical properties of the antibody. In a preferred embodiment of the present invention, a group of antibody mutants are prepared and screened for their binding affinity to the antigen. Optionally, one or more additional biological activity assays may be performed on one or more antibody mutants selected from the screen to determine that the antibody mutants have new or improved properties. In a preferred embodiment, the antibody mutants retain the ability to bind to IL-4 and / or IL-13 with a similar or better / higher binding affinity than the parent antibody.

[0148] The antibody mutant thus selected may be further modified, usually depending on the intended use of the antibody. Such modifications may include further changes in the amino acid sequence, fusion with a heterologous polypeptide, and / or covalent modification. For example, serine may generally be used to replace cysteine ​​residues that are not involved in maintaining the proper conformation of the antibody mutant to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​may be added to the antibody to improve stability (particularly when the antibody is an antibody fragment such as an F v fragment).

[0149] Another type of antibody mutant has a changed glycosylation pattern. This can be achieved by deleting one or more carbohydrate moieties found on the antibody and / or adding one or more glycosylation sites not present on the antibody. The glycosylation of the antibody is generally N-connected to Asn or O-connected to Ser or Thr. The tripeptide sequence, asparagine-X-serine and asparagine-X-threonine are common recognition sequences for the carbohydrate moiety to be enzymatically connected to the asparagine side chain, where X is any amino acid except proline. For example, N-acetylgalactosamine, galactose, fucose or xylose are connected to hydroxyamino acids, most of which are serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. Adding or replacing one or more serine or threonine residues to the sequence of the original antibody can increase the probability of O-linked glycosylation.

[0150] Ideally, the effector function of the antibodies of the present invention can be modified to enhance the utility of the antibodies. c The homodimeric antibody thus generated may have improved internalization ability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC), see Caron et al., J Exp Med 176:1191-1195 (1992) and Shopes, Immunol 148:2918-2922 (1993). Alternatively, a polypeptide having two F cSuch antibodies may have enhanced complement lysis and ADCC capabilities, see Stevenson et al., Anti-Cancer Drug Design 3:219 230 (1989).

[0151] Covalent modification of antibodies is encompassed within the scope of the present invention. This can be achieved by chemical synthesis or by enzymatic or chemical cleavage of antibodies (if feasible). Other types of covalent modifications of antibodies can be introduced into antibody molecules by reacting the targeted amino acid residues of the antibody with an organic derivatizing agent, wherein the organic derivatizing agent can react with selected side chains or with N-terminal or C-terminal residues.

[0152] Cysteinyl residues can react with α-haloacetates (and corresponding amines) such as chloroacetic acid or chloroacetamide to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues can also be derivatized, for example, by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidazolyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridine disulfide, methyl 2-pyridine disulfide, p-chloromercuribenzoate, 2-chloromercura-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0153] Histidyl residues can be derivatized by reaction with diethyl pyrocarbonate at pH 5.5-7.0. p-Bromophenacyl bromide can also be used, the reaction preferably being carried out in 0.1 M sodium cacodylate at pH 6.0.

[0154] Lysinyl and α-terminal residues can be reacted with succinic acid or other carboxylic acid anhydrides to reverse the charge of the residue. Other suitable reagents for derivatization to obtain residues containing α-amino groups include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea and 2,4-pentanedione. Amino acids can be catalyzed by transaminases with glyoxylate.

[0155] Arginyl residues can be modified by reaction with one or more conventional reagents such as phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and ninhydrin. Derivatization of arginine residues generally requires alkaline reaction conditions. Moreover, these reagents react with the ε-amino group of lysine as well as arginine.

[0156] Specific modification of tyrosyl residues can be performed using aromatic diazo compounds or tetranitromethane. For example, N-acetylimidazole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. 125 I or 131I can iodinate tyrosyl residues to prepare labeled proteins for use in radioimmunoassays.

[0157] The carboxyl side chain groups (asparagyl or glutamyl) can be modified by reaction with carbodiimide (R=C=C-R'), wherein R and R' can be different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. In addition, aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0158] Glutaminyl and asparaginyl residues are often deamidated under neutral or alkaline conditions to yield the corresponding glutamyl and asparagine residues, respectively. The deamidated forms of these residues fall within the scope of the present invention.

[0159] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine and histidine side chains (Creighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine and amidation of any C-terminal carboxyl group.

[0160] Another type of covalent modification involves chemical or enzymatic coupling of glycosides to the antibody. These methods do not require production of antibodies capable of N-linked or O-linked glycosylation in host cells. Depending on the coupling method used, the sugar can be attached to: (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups, such as the sulfhydryl groups of cysteine; (d) free hydroxyl groups, such as the hydroxyl groups of serine, threonine or hydroxyproline; (e) aromatic residues such as phenylalanine, tyrosine or tryptophan; or (f) the amide group of glutamine. WO 87 / 05330 and Aplin & Wriston, CRC Crit Rev Biochem, pp. 259-306 (1981) describe such methods.

[0161] Any carbohydrate moiety on the antibody can be removed chemically or enzymatically. For example, chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid or a comparable compound, resulting in the cleavage of most or all sugars other than the linked sugar (N-acetylglucosamine or N-acetylgalactosamine) while maintaining the integrity of the antibody. For example, Hakimuddin et al., Arch Biochem Biophys 259:52 (1987) and Edge et al., Anal Biochem 118:131 (1981) describe chemical deglycosylation. Enzymatic cleavage of carbohydrate moieties on the antibody can be achieved by any of the many endoglycosidases and exoglycosidases described in, for example, Thotakura et al., Meth Enzymol 138:350 (1987).

[0162] Another type of covalent modification of the antibody includes linking the antibody to one of a number of nonproteinaceous polymers such as polyethylene glycol, polypropylene glycol, or polyoxylalkylenes in the manner described in US Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337.

[0163] Another preferred technique for obtaining mutants or mutant proteins is affinity maturation by phage display (Hawkins et al., J Mol Biol 254:889-896 (1992) and Lowman et al., Biochemistry 30 (45): 10832-10838 (1991)). Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody mutants thus obtained are displayed on phage particles as fusions to proteins on the phage particles in a monovalent form. Phages expressing a variety of mutants can be cycled through multiple rounds of binding selection, followed by isolation and sequencing of mutants that exhibit high affinity.

[0164] Methods for selecting new binding polypeptides can use libraries of structurally related polypeptides. Libraries of structurally related polypeptides fused to, for example, bacteriophage coat proteins are generated by mutagenesis and displayed on the surface of particles. These particles are then contacted with the target molecule, and particles with the highest affinity for the target are separated from particles with low affinity. High-affinity binders are then amplified by infecting a suitable bacterial host, and the competitive binding step is repeated. The process is repeated until a polypeptide with the desired affinity is obtained.

[0165] Alternatively, polyvalent phage (McCafferty et al. (1990) Nature 348:552-554 and Clackson et al. (1991) Nature 352:624-628) can be used to express random point mutations (e.g., generated using an error-prone DNA polymerase) to generate a library of phage antibody fragments, which can then be screened for affinity to IL-4 and / or IL-13 (Hawkins et al. (1992) J Mol Biol 254:889-896).

[0166] Preferably, during affinity maturation, the replicable expression vector is under tight control of transcriptional regulatory elements, and the culture conditions are adjusted so that the amount or number of particles displaying more than one copy of the fusion protein is less than about 1%. Also preferably, the amount of particles displaying more than one copy of the fusion protein is less than 10% of the amount of particles displaying a single copy of the fusion protein. Preferably, the amount is less than 20%.

[0167] Functional equivalents can be created by interchanging framework regions or different CDRs from different antibody chains within a composite FR from multiple antibodies. Thus, for example, for a given set of CDRs, by substituting different heavy chain, e.g., IgG 1-4 ,IgM,IgA 1-2 Different classes of antibodies may generate different IL-4 and / or IL-13 antibody types and isotypes. Similarly, artificial antibodies within the scope of the present invention may be generated by embedding a given set of CDRs into an entirely synthetic framework region.

[0168] Antibody fragments and functional equivalents of the present invention include molecules whose specific binding to IL-4 and / or IL-13 can be detected. The detectable degree of binding includes all values ​​within the range of at least 10-100% of the binding capacity of the target antibody, preferably at least 50%, 60% or 70%, more preferably at least 75%, 80%, 85%, 90%, 95% or 99%. Equivalents with greater than 100% affinity of the target antibody are also included.

[0169] Generally, CDRs are important for epitope recognition and antibody binding. However, residues comprising CDRs can be changed without interfering with the ability of the antibody to recognize and bind to the associated epitope. For example, changes that do not affect epitope recognition but increase the binding affinity of the antibody to the epitope can be made. Based on the understanding of the main antibody sequence, several studies have investigated the effects of introducing one or more amino acid changes to multiple sites of the antibody sequence on antibody properties, such as binding and expression levels (Yang et al., 1995, J Mol Biol 254: 392-403; Rader et al., 1998, Proc Natl Acad Sci USA 95: 8910-8915; and Vaughan et al., 1998, Nature Biotechnology 16, 535-539).

[0170] Therefore, equivalents of the desired antibody can be generated by changing the sequence of the heavy and light chain genes in CDR1, CDR2 or CDR3 or the framework regions using methods such as oligonucleotide-mediated site-directed mutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling or mutant strains of E. coli (Vaughan et al., 1998, Nat Biotech 16:535-539; and Adey et al., 1996, Chap. 16, pp. 277-291, in Phage Display of Peptides and Proteins, eds. Kay et al., Academic Press). Methods of altering the nucleic acid sequence of primary antibodies can result in improved antibody affinity (Gram et al., 1992, Proc Natl Acad Sci USA 89:3576-3580; Boder et al., 2000, Proc Natl Acad Sci USA 97:10701-10705; Davies & Riechmann, 1996, Immunotech 2:169-179; Thompson et al., 1996, J Mol Biol 256:77-88; Short et al., 2002, J Biol Chem 277:16365-16370; and Furukawa et al., 2001, J Biol Chem 276:27622-27628).

[0171] Repeated rounds of "polypeptide selection" can be used to select for higher and higher affinity binding, for example, by selecting multiple amino acid changes selected over multiple rounds of selection. Additional rounds of selection can be performed on other regions or amino acids of the ligand after the first round of selection, where the first round of selection involves the first region of amino acid selection of the ligand or antibody polypeptide. Multiple rounds of selection are repeated until the desired affinity properties are achieved.

[0172] Improved antibodies also include antibodies with improved characteristics that are prepared by standard techniques of animal immunization, hybridoma formation, and selection of antibodies with specific characteristics.

[0173] "Antagonist" refers to a molecule that is capable of inhibiting one or more biological activities of a target molecule (e.g., signal transduction by IL-4 and / or IL-13). Antagonists can interfere with receptor binding to ligand and vice versa by inactivating or killing cells activated by the ligand and / or interfering with signal transduction following receptor or ligand activation (e.g., tyrosine kinase activation) or ligand binding to the receptor. The antagonist can completely block the receptor-ligand interaction, or substantially reduce such interaction.

[0174] "Agonist" refers to a compound including proteins, polypeptides, peptides, antibodies, antibody fragments, conjugates, macromolecules, small molecules that activate one or more biological activities of IL-4 and / or IL-13. Agonists can interact with the binding of receptors to ligands by acting as mitogens for cells activated by the ligands and / or by interfering with cell inactivation or signal transduction inhibition after the ligands bind to the receptors, and vice versa. For the purposes of the present invention, all such interference positions of agonists are considered equivalent.

[0175] The terms "cell", "cell line" and "cell culture" include their progeny. It is also understood that all progeny may not be exactly the same, for example in terms of DNA content, due to intentional or unintentional mutations. Variant progeny having the same desired function or biological property, wherein the desired function or biological property is screened for the original cell, are included.

[0176] The term "vector" means a nucleic acid construct, a vector, which contains nucleic acid, a transgenic, an exogenous gene or a target gene, which is effectively connected to a suitable regulatory sequence to express the transgenic in a suitable host. Such regulatory sequences include, for example, promoters that affect transcription, optional operator sequences that control transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences that regulate transcription and translation termination. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host's genome, or in some cases may be integrated into the host cell's genome. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are common forms of vector use. However, the present invention is intended to include such other forms of vectors that function as equivalent vectors, which are or are known in the art, such as viruses, synthetic molecules carrying nucleic acids, liposomes, etc.

[0177] For therapeutic purposes, "mammal" refers to any animal that can be classified as a mammal, including humans, domestic and farm animals, non-human primates, and zoo, sport, or pet animals, such as dogs, horses, cats, cows, etc.

[0178] The antibody of interest may be screened or used in assays described herein or known in the art. Such assays generally require a detectable, such as labeled, reagent. As used herein, the term "label" refers to a detectable compound or composition that can be conjugated directly or indirectly to a molecule or protein, such as an antibody. The label may itself be detectable (e.g., a radioisotope label, a particle, or a fluorescent label), or, in the case of an enzyme label, may catalyze a chemical alteration of a substrate compound or composition that can be detected.

[0179] As used herein, "solid phase" means a non-aqueous matrix to which an entity or molecule, such as an antibody of the invention, can adhere. Examples of solid phases included herein include solid phases formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamide, polystyrene, polyvinyl alcohol, and polysiloxane. In some embodiments, depending on the context, the solid phase may include the wells of an assay plate; in other cases, it may be used for a purification column (e.g., an affinity chromatography column). Thus, the solid phase may be paper, beads, plastic, a chip, etc., may be prepared from many materials, such as nitrocellulose, agarose, polystyrene, polypropylene, silicon, etc., and may also take many conformations.

[0180] Genes or cDNAs encoding IL-4 and IL-13 are known in the art and can be cloned into plasmids or other expression vectors and expressed in any of a number of expression systems according to methods well known to those skilled in the art and, for example, see below.

[0181] The nucleotide sequence encoding the amino acid sequence mutant can be prepared by many methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of a mutant or non-mutant form of the previously prepared target molecule (see, e.g., Kunkel, Proc Natl Acad Sci USA 82:488 (1985)).

[0182] The recombinant expression of the antibody of the present invention or its fragment, derivative or analog (e.g., the heavy chain or light chain of the antibody of the present invention, the single-chain antibody of the present invention or the antibody mutant protein of the present invention) includes constructing an expression vector containing a polynucleotide encoding the antibody or antibody fragment described herein. Once the polynucleotide encoding the antibody molecule is obtained, a vector for producing the antibody can be produced by recombinant DNA techniques known in the art. An expression vector containing an antibody coding sequence and appropriate transcription and translation control signals is constructed. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0183] The expression vector is transferred into the host cell by conventional techniques, and the transfected cells are subsequently cultured by conventional techniques to produce the antibody or fragment of the present invention. In one aspect of the present invention, the vectors encoding the heavy chain and the light chain can be co-expressed in the host cell to express the entire immunoglobulin molecule described in detail herein.

[0184] Many host / expression vector systems can be used to express the antibody molecules of the present invention. These expression systems not only represent vehicles that can produce and subsequently purify the target coding sequence, but also represent cells that can in situ express the antibody molecules of the present invention when transformed or transfected with suitable nucleotide coding sequences. Bacterial cells such as Escherichia coli and eukaryotic cells are commonly used for the expression of recombinant antibody molecules, especially for the expression of full-length recombinant antibody molecules. For example, mammalian cells such as CHO cells are combined with vectors such as vectors carrying the major intermediate immediate early gene promoter element from human cytomegalovirus to be an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); and Cockett et al., Bio / Technology 8:2 (1990)). As is known in the art, plants and plant cell cultures, insect cells, etc. can also be used to produce target proteins.

[0185] In addition, host cells are selected that can modulate the expression of the inserted sequence or modify and process the gene product in a desired specific manner. These modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product may be important for protein function. Different host cells have properties and specific mechanisms for post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems can be selected to ensure the correct modification and processing of the expressed antibody of interest. Therefore, eukaryotic host cells that possess cellular machinery for appropriate processing of the primary transcript, glycosylation and phosphorylation of the gene product can be used. These mammalian host cells include, but are not limited to, CHO, COS, 293, 3T3, or myeloma cells.

[0186] Stable expression is preferred for long-term high-yield production of recombinant proteins. For example, cell lines that stably express antibody molecules can be transformed. Rather than using expression vectors containing viral replication origins, it is better to use DNA and selective markers regulated by suitable expression regulatory elements (such as promoters, enhancers, sequences, transcription terminators, polyadenylic acid sites, etc.) to transform host cells. After introducing foreign DNA, the transformed cells can be grown on enriched medium for 1 to 2 days and then transferred to selective medium. The selective markers of recombinant plasmids confer resistance to selection, so that cells stably integrate plasmids into chromosomes and expand into cell lines. These transformed cell lines can not only be used for antibody preparation, but also can be used for screening and evaluating compounds that interact directly or indirectly with antibody molecules.

[0187] A number of selection systems can be used, including but not limited to herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al., Proc Natl Acad Sci USA 48:202 (1992)), glutamate synthetase selection in the presence of methionine sulfoximine (Adv Drug Del Rev 58, 671, 2006 and see the website or literature of Lonza Group Ltd.), and the adenine phosphoribosyltransferase gene in tk, hgprt or aprt cells (Lowy et al., Cell 22:817 (1980)). Likewise, antimetabolite resistance can be used as the basis for selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Proc Natl Acad Sci USA 77:357 (1980); O'Hare et al., Proc Natl Acad Sci USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan et al., Proc Natl Acad Sci USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 (Wu et al., Biotherapy 3:87 (1991)); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clones, as described in, for example, Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press (1990); Dracopoli et al., eds., Current Protocols in Human Genetics, John Wiley & Sons (1994); and Colberre-Garapin et al., J Mol Biol 150:1 (1981).

[0188] The expression level of antibody molecules can be increased by vector amplification (see, for example, Bebbington et al., DNA Cloning, Vol. 3. Academic Press (1987)). When the marker in the vector system expressing the antibody is amplifiable, the increase in the inhibitor level in the culture will increase the copy number of the marker gene. Since the amplified region is associated with the antibody gene, the production of the antibody will also increase (Crouse et al., Mol Cell Biol 3: 257 (1983)).

[0189] Two or more expression vectors of the present invention can be used to co-transfect host cells, for example, the first vector encodes a polypeptide derived from a heavy chain, and the second vector encodes a polypeptide derived from a light chain. The two vectors may contain the same selection markers, which can make the expression of heavy and light chain polypeptides the same. Alternatively, a single vector that encodes and can express heavy and light chain polypeptides can be used. In this case, the light chain should be placed before the heavy chain to avoid producing too many non-toxic heavy chains (Proudfoot, Nature 322: 52 (1986); and Kohler, Proc Natl Acad Sci USA 77: 2197 (1980)). The coding sequences of heavy and light chains may include cDNA or genomic DNA.

[0190] Once the antibody molecules of the present invention are produced by animals, chemical synthesis or recombinant expression, the antibody molecules can be purified by any method known in the art for purifying immunoglobulin molecules, such as chromatography (e.g., ion exchange, affinity chromatography, etc., especially affinity chromatography for IL-4 and / or IL-13 after protein A and size exclusion chromatography), centrifugation, differential solubility or by any other standard technique for protein purification. In addition, the antibodies or antibody fragments of the present invention can be fused to the heterologous polypeptide sequences described herein or other polypeptide sequences known in the art to facilitate purification.

[0191] The antibodies of the present invention can be generated by any suitable method known in the art. The antibodies of the present invention may include polyclonal antibodies, although monoclonal antibodies are preferred due to antibody modification to optimize their use in the human body and to optimize the use of the antibodies themselves, because of their convenient production and manipulation of specific proteins. Methods for preparing polyclonal antibodies are known to those skilled in the art (Harlow et al., Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd edition (1988)).

[0192] The antibodies of the present invention preferably include monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma technology, such as described by Kohler et al., Nature 256:495 (1975); U.S. Patent No. 4,376,110; Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988) and Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas, Elsevier (1981), recombinant DNA methods such as methods for preparing and using transfectomas, or other methods known to the skilled person. Other examples of methods that can be used to produce monoclonal antibodies include, but are not limited to, human B cell hybridoma technology (Kosbor et al., Immunology Today 4: 72 (1983); and Cole et al., Proc Natl Acad Sci USA 80: 2026 (1983)) and EBV-hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss (1985)). These antibodies can be antibodies of any immunoglobulin class including IgG, IgM, IgE, IgA and IgD and any subclass thereof. Hybridomas that produce mAbs of the present invention can be cultured in vitro or in vivo.

[0193] In the hybridoma model, a host such as a mouse, a humanized mouse, a transgenic mouse carrying human immune system genes, a hamster, a rabbit, a rat, a camel or any other suitable host animal is immunized to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to IL-4 or IL-13. Alternatively, fence cells are immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103 (1986)).

[0194] In general, when preparing hybridomas that produce antibodies, peripheral blood lymphocytes ("PBL") can be used when cells of human origin are needed, or spleen cells or lymph node cells can be used when non-human mammalian sources are needed. Immortal cell lines are usually transformed mammalian cells, especially myeloma cells of rodent, bovine or human origin. Myeloma cell lines of rats or mice are generally used. Hybridoma cells can be cultured on a suitable culture medium, which preferably contains one or more substances that can inhibit the growth or survival of unfused immortal cells. For example, if the parental cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium used for hybridomas will generally contain hypoxanthine, aminopterin and thymidine ("HAT culture medium"), which are substances that prevent the growth of cells lacking HGPRT.

[0195] Preferred immortalized cell lines are cell lines that efficiently fuse and support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif, and SP2 / 0, FO or X63-Ag8-653 cells available from the American Type Culture Collection, Manassas, VA.

[0196] Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J Immunol 133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc, pp. 51-63 (1987)). The mouse myeloma cell line NSO (European Collection of Cell Cultures, Salisbury, Wilshire, UK) can also be used.

[0197] Another option is to use electrofusion rather than chemical fusion to form hybridomas. Instead of using fusion, B cells can be immortalized using, for example, Epstein Barr virus or other transforming genes, see, for example, Zurawaki et al., Monoclonal Antibodies, ed., Kennett et al., Plenum Press, pp. 19-33. (1980). Transgenic mice expressing immunoglobulins and severe combined immunodeficient (SCID) mice transplanted with human B lymphocytes can also be used.

[0198] The production of monoclonal antibodies against IL-4 and / or IL-13 in the culture medium in which the hybridoma cells are grown is measured. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be measured by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA), fluorescent cell measurement analysis (FACS) or enzyme-linked immunosorbent assay (ELISA). These techniques are known in the art and are within the skill of the technician. The binding affinity of the monoclonal antibody to IL-4 and / or IL-13 can be measured by, for example, Scatchard analysis (Munson et al., Anal Biochem 107: 220 (1980)).

[0199] After identifying hybridoma cells that produce antibodies of the desired specificity, affinity, and / or activity, these clones can be subcloned by limiting dilution techniques and cultured according to standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103 (1986). Suitable culture media include, for example, Dulbecco's modified Eagle's medium (D-MEM) or RPMI-1640 medium. In addition, hybridoma cells can be grown in animals as ascites tumors.

[0200] The monoclonal antibodies secreted by the subclones are suitably separated or isolated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as protein A-Sepharose, protein G-Sepharose, hydroxylapatite chromatography, gel exclusion chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0201] There are many methods for producing monoclonal antibodies in the art, so the present invention is not limited to their preparation only in hybridomas. For example, monoclonal antibodies can be prepared by recombinant DNA methods such as the methods described in US Patent No. 4,816,567. In this case, the term "monoclonal antibody" refers to an antibody derived from a single eukaryotic, phage or prokaryotic clone.

[0202] DNA encoding the monoclonal antibodies of the present invention can be easily isolated and sequenced using conventional methods (e.g., using oligonucleotide probes that specifically bind to genes encoding mouse antibody heavy and light chains or such chains from humans, humanized or other sources) (Innis et al., PCR Protocols. A Guide to Methods and Applications, Academic (1990) and Sanger et al., Proc Natl Acad Sci 74: 5463 (1977)). Hybridoma cells serve as a source of such DNA. Once isolated, the DNA can be placed in an expression vector, which is then transfected into a host cell that does not otherwise produce immunoglobulins, such as an E. coli cell, a NSO cell, a COS cell, a Chinese hamster ovary (CHO) cell or a myeloma cell, to obtain the synthesis of the monoclonal antibody in the recombinant host cell. The DNA may also be modified, for example, by replacing the homologous mouse sequences with coding sequences for human heavy and light chain constant domains (U.S. Pat. No. 4,816,567; and Morrison et al., Proc Natl Acad Sci USA 81:6851 (1984)) or by covalently attaching all or part of the coding sequences for non-immunoglobulin polypeptides to the immunoglobulin coding sequences. These non-immunoglobulin polypeptides may be substituted for the constant domains of an antibody of the invention, or may be substituted for the variable domains of one IL-4 or IL-13 binding site of an antibody of the invention to form a chimeric bivalent antibody.

[0203] These antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chains and modified heavy chains. The heavy chains are generally in the F c Any position of the region may be truncated to prevent heavy chain cross-linking. Alternatively, the relevant cysteine ​​residue may be substituted with another amino acid residue or deleted to prevent cross-linking.

[0204] Antibody fragments that recognize specific epitopes can be generated by known techniques. Traditionally, these fragments are obtained by proteolytic digestion of intact antibodies (see, for example, Morimoto et al., J Biochem Biophys Methods 24:107 (1992); and Brennan et al., Science 229:81 (1985)). For example, enzymes such as papain (producing F ab fragment) or pepsin (to produce F (ab')2 The F fragment of the present invention can be produced by proteolytic cleavage of the immunoglobulin molecule ab and F (ab')2 Fragment. (ab')2 The fragment contains the variable region, the light chain constant region and the heavy chain C H1 However, these fragments can be directly produced by recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, F (ab')2 -SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163 (1992). According to another method, F (ab’)2 Fragments can be isolated directly from recombinant host cell cultures. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain F v Fragment (F v )(WO93 / 16185).

[0205] For certain uses, including use of antibodies in humans and in vitro detection assays, chimeric, humanized or human antibodies may be preferred. Methods for producing chimeric antibodies are known in the art, for example, see Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J Immunol Methods 125:191 (1989); and U.S. Patent Nos. 5,807,715; 4,816,567 and 4,816,397.

[0206] Humanized antibodies are derived from antibody molecules that bind IL-4 and / or IL-13 and are produced in non-human species, in which one or more CDRs from these antibodies are inserted into the FR regions of human immunoglobulin molecules. Antibodies can be humanized using a variety of techniques known in the art, such as CDR grafting (EPO 239,400; WO 91 / 09967; and U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EPO 592,106; EPO 519,596; Padlan, Molecular Immunology 28:489 (1991); Studnicka et al., Protein Engineering 7:805 (1994); and Roguska et al., Proc Natl Acad Sci USA 91:969 (1994)), and chain shuffling (U.S. Patent No. 5,565,332).

[0207] Humanized antibodies have one or more amino acid residues from non-human sources. These non-human amino acid residues are usually referred to as "imported" residues, which are generally taken from the "imported" variable domains. According to the methods of Winter and colleagues (Jones et al., Nature 321: 522 (1986); Riechmann et al., Nature 332: 323 (1988) and Verhoeyen et al., Science 239: 1534 (1988)), humanization can be basically performed by replacing the corresponding sequence of a human antibody with a portion of a non-human CDR or CDRs sequence. Therefore, these "humanized" antibodies are chimeric antibodies (US Patent No. 4,816,567), in which sequences substantially smaller than the complete human variable domains have been replaced by corresponding sequences from non-human species. In practice, humanized antibodies are generally human antibodies, in which some CDRs residues and possibly some FR residues are replaced by similar sites of rodent antibodies. The heavy chain constant region and hinge region may be from any class or subclass to achieve the desired effect, such as a specific effector function.

[0208] Typically, framework residues in the human framework region can be substituted with corresponding residues from a CDR donor antibody, thereby altering and potentially improving antigen binding. Framework substitutions can be identified by methods known in the art, such as by modeling the interactions of CDRs with framework residues to identify important framework residues for antigen binding, and by sequence comparison to identify unusual framework residues at specific positions, for example, see U.S. Patent No. 5,585,089 and Riechmann et al., Nature 332:323 (1988).

[0209] More preferably, humanized antibodies retain high affinity to IL-4 and / or IL-13, and retain or obtain other favorable biological properties. Therefore, humanized antibodies are prepared by analyzing the parental sequence and a variety of conceptual humanized products using a three-dimensional model of the parental sequence and the humanized sequence. The three-dimensional immunoglobulin model is generally available and well known to those skilled in the art. A computer program that illustrates and displays the possible three-dimensional conformational structure of the selected candidate immunoglobulin sequence can be obtained. Checking this display allows analysis of the possible role of certain residues in the functioning of the candidate immunoglobulin sequence, i.e., analyzing the residues that affect the ability of the candidate immunoglobulin to bind IL-4 and / or IL-13. In this way, FR residues can be selected and merged from the receiving and importing sequences so that the desired antibody characteristics are maximized, such as increased affinity to the target antigen, although it is the CDR residues that directly and most substantially affect IL-4 and / or IL-13 binding. The CDR region may also be modified to contain one or more amino acids different from those obtained from the parent antibody from which the CDR was obtained to provide enhanced or different desired properties, such as greater affinity or greater avidity binding.

[0210] Certain portions of the antibody constant region can be manipulated and altered to provide antibody homologues, derivatives, fragments, etc., having properties different from or superior to those observed in the parent antibody. Thus, for example, many IgG4 antibodies form intrachain disulfide bonds near the hinge region. The intrachain bonds can destabilize the parent bivalent molecule to form a monovalent molecule containing a heavy chain and a light chain attached thereto. Such molecules can reassociate, but randomly.

[0211] It has been observed that modifying the amino acids in the hinge region of the IgG4 molecule can reduce the probability of intrachain bond formation, thereby stabilizing the IgG4 molecule, which will minimize the probability of bispecific molecule formation. If the therapeutic antibody is an IgG4 molecule, this modification is advantageous because the enhanced stability can minimize the probability of dissociation of the molecule during production and preparation and in vivo. Monovalent antibodies may not have the same effectiveness as bivalent parent molecules. For example, when a bivalent IgG4 is administered to a patient, the percentage of bivalent IgG4 decays to about 30% within two weeks. The amino acid substitution at position 228 enhances IgG4 stability. The serine at position 228 can be replaced by another amino acid, such as one of the remaining 19 amino acids. In particular, this change can be made to recombinant antibodies, wherein the nucleic acid coding sequence can be mutated to obtain an amino acid substitution at position 228. For example, S can be replaced by proline.

[0212] Another group of amino acids suitable for modification include those in the hinge region, which affect the binding of the heavy chain-containing molecule to the F cThe binding between the receptors and the internalization of the bound antibody. Such amino acids include residues from about 233 to about 237 (Glu-Leu-Leu-Gly-Gly) (SEQ ID NO: 49) in the IgG1 molecule; residues from about 252 to about 256 (Met-Ile-Ser-Arg-Thr) (SEQ ID NO: 50) and residues from about 318 (Glu) to about 331 (Pro), such as Lys 320 , Lys 322 and Pro 329 .

[0213] Fully human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be prepared by many methods known in the art, including the above-mentioned phage display methods using antibody libraries derived from human immunoglobulin sequences, see US Patent Nos. 4,444,887 and 4,716,111; and WO98 / 46645, WO 98 / 50433, WO 98 / 24893, WO98 / 16654, WO 96 / 34096, WO96 / 33735 and WO 91 / 10741. Human monoclonal antibodies can also be prepared using the techniques of Cole et al. and Boerder et al. (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss (1985); and Boerner et al., J Immunol 147:86 (1991)).

[0214] Transgenic mice can also be used to produce human antibodies. These mice cannot express functional endogenous immunoglobulins, but they also express certain human immunoglobulin genes. For example, human heavy chain and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells by random means or homologous recombination. Alternatively, in addition to human heavy chain and light chain genes, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells. By introducing human immunoglobulin loci through homologous recombination, mouse heavy chain and light chain immunoglobulin genes can be rendered functional separately or simultaneously. Specifically, homozygous deletions in the JH region prevent endogenous antibody production. Modified embryonic stem cells are expanded and microinjected into blastocysts to form chimeric mice. The chimeric mice are subsequently bred to produce homozygous offspring that express human antibodies (see, e.g., Jakobovitis et al., Proc Natl Acad Sci USA 90:2551 (1993); Jakobovitis et al., Nature 362:255 (1993); Bruggermann et al., Yearin Immunol 7:33 (1993); and Duchosal et al., Nature 355:258 (1992)).

[0215] Transgenic mice are immunized in the normal manner with IL-4 or IL-13 cytokines, such as all or part of IL-4 or IL-13. Monoclonal antibodies against IL-4 and IL-13 can be obtained from immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Therefore, it is possible to generate therapeutic IgG, IgA, IgM and IgE antibodies using this technology. For an overview, see Lonberg et al., Int Rev Immunol 13:65-93 (1995). For a discussion of the production of human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., WO 98 / 24893; WO 92 / 01047; WO 96 / 34096; and WO 96 / 33735; EPO No. 0 598 877; and U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598. In addition, see, e.g., Amgen (Fremont, CA), Genpharm (San Diego), and Jose, CA) and Medarex, Inc. (Princeton, NJ) are engaged in providing anti-IL-4 and / or IL-13 human antibodies using similar technologies as described above.

[0216] Similarly, mice with human peripheral blood leukocytes, spleen cells or bone marrow (e.g., tri-hybridoma technology of XTL Biopharmaceuticals, Israel) can be immunized to prepare human mAbs. A technique known as "guided selection" can be used to generate fully human antibodies that recognize selected epitopes. In this method, selected non-human monoclonal antibodies such as mouse antibodies are used to guide the selection of fully human antibodies that recognize the same epitope (Jespers et al., Bio / technology 12:899 (1988)).

[0217] When using recombinant technology, antibody variants can be produced in cells, periplasmic space, or directly secreted into culture medium. If antibody variants are produced in cells, the first step is to remove micro-fragments, which are either host cells or cleaved fragments, such as by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163 (1992) describes a method for separating antibodies secreted into the periplasmic space of E. coli. In brief, cell paste is contacted with sodium acetate (pH 3.5) and EDTA. Cell debris can be removed by centrifugation. When antibody variants are secreted into culture medium, the supernatant from these expression systems is generally first concentrated using commercially available protein concentration filters such as ultrafiltration components of Amicon or Millipore Pellicon. Protease inhibitors such as PMSF can be added to inhibit proteolysis, and antibiotics can also be added to prevent the growth of foreign contaminants.

[0218] Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. Whether protein A or protein G is suitable as an affinity ligand depends on the affinity of any immunoglobulin F on the antibody variant. c The type and isotype of the domain. Protein A can be used to purify antibodies based on human IgG1, IgG2 or IgG4 heavy chains (Lindmark et al., J Immunol Meth 62:1 (1983)). Protein G can be used for mouse isotypes and human IgG3 (Guss et al., EMBO J 5:1567 (1986)). The matrix to which the affinity ligand is bound is most commonly agarose, but other matrices can also be used. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene can allow faster flow rates and shorter processing times than achieved with agarose. When the antibody variant includes C H3 When the antibody or variant is to be recovered, Bakerbond ABX™ resin (JT Baker; Phillipsburg, NJ) can be used for purification. Other protein purification techniques such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin agarose, chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation can also be used, depending on the antibody or variant to be recovered.

[0219] Following any preliminary purification steps, the mixture containing the antibody or variant of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5-4.5, preferably at a low salt concentration (e.g., about 0-0.25 M salt).

[0220] The antibody of the present invention may be a bispecific antibody. The bispecific antibody may be a monoclonal antibody, preferably a human or humanized antibody having at least two different antigen binding specificities. In a preferred embodiment, the bispecific antibody, fragment thereof, etc. has binding specificity for IL-4 and IL-13.

[0221] The method of producing bispecific antibodies is well known. Traditionally, the recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain / light chain pairs, wherein the two heavy chains have different specificities (Milstein et al., Nature 305:537 (1983)). Due to the random distribution of immunoglobulin heavy chains and light chains, hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually carried out by an affinity chromatography step. Similar methods are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J10:3655 (1991). For example, Kufer et al., Trends Biotech 22:238-244, 2004 provides other methods for producing bispecific antibodies.

[0222] The variable domain of an antibody with the desired binding specificity can be fused to the constant domain sequence of an immunoglobulin. Preferably, yu includes at least part of the hinge region, C H2 and C H3 It can have a first heavy chain constant region (C H1 ), which contains the site required for light chain binding present in at least one fusion. DNAs encoding the immunoglobulin heavy chain fusion region and the immunoglobulin light chain (if desired) are inserted into separate expression vectors and co-transformed into a suitable host organism. For more details on the generation of bispecific antibodies, see, for example, Suresh et al., Meth Enzym 121:210 (1986).

[0223] The present invention also contemplates heteroconjugate antibodies. Heteroconjugate antibodies consist of two covalently linked antibodies. It has been suggested, for example, to target immune system cells to unwanted cells with such antibodies (US Patent No. 4,676,980). It is contemplated that the antibodies can be prepared in vitro using known methods of synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioester bonds. Examples of reagents suitable for this purpose include iminothiolate) and methyl-4-mercaptobutyrimidate, as well as those disclosed in US Patent No. 4,676,980.

[0224] In addition, single domain antibodies to IL-4 and / or IL- 13 may be generated. Some examples of this technology are described in WO9425591 for antibodies derived from camelid heavy chain Ig, and in US20030130496 for the isolation of single domain fully human antibodies from phage libraries.

[0225] Alternatively, certain techniques described for the production of single-chain antibodies (U.S. Patent No. 4,946,778; Bird, Science 242:423 (1988); Huston et al., Proc Natl Acad Sci USA 85:5879 (1988); and Ward, et al., Nature 334:544 (1989)) can also be adapted to produce single-chain antibodies. Single-chain antibodies are produced by combining F v The heavy and light chain fragments of the domain are connected by amino acid bridges to form a single polypeptide chain. Functional F in Escherichia coli v Fragment assembly techniques are also available (Skerra et al., Science 242:1038 (1988)).

[0226] The present invention includes antibodies fused or chemically conjugated (including covalent and non-covalent conjugation) to polypeptides in a recombinant manner. The fused or conjugated antibodies of the present invention can be used to simplify purification, see, for example, WO93 / 21232; EP 439,095; Naramura et al., Immunol Lett 39:91 (1994); US Patent No. 5,474,981; Gillies et al., Proc Natl Acad Sci USA 89:1428 (1992); and Fell et al., J Immunol 146:2446 (1991). The marker amino acid sequence can be a hexahistidine peptide, such as the tag provided by the pQE vector (QIAGEN, Inc., Chatsworth, CA), in addition, many of which can also be obtained through commercial channels, Gentz ​​et al., Proc Natl Acad Sci USA 86:821 (1989). Other peptide tags that can be used for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the "flag" tag.

[0227] It is also possible to create single peptide chain binding molecules, in which the heavy chain and light chain F v The single-chain antibody ("scF v ") and their construction methods are described in, for example, US Patent No. 4,946,778. Alternatively, F abCan be constructed and expressed in a similar manner. All fully and partially human antibodies may be less immunogenic than fully murine monoclonal antibodies, and fragments and single-chain antibodies may also be less immunogenic.

[0228] Antibodies or antibody fragments can be isolated from antibody phage libraries produced using the techniques described in McCafferty et al., Nature 348:552 (1990). Clarkson et al., Nature 352:624 (1991) and Marks et al., J Mol Biol 222:581 (1991) describe the use of phage libraries to separate murine and human antibodies, respectively. Subsequent publications describe the production of high-affinity (in the range of nM) human antibodies by chain shuffling (Marks et al., Bio / Technology 10:779 (1992)), as well as strategies for constructing very large phage libraries by combined infection and in vivo recombination (Waterhouse et al., Nucl Acids Res 21:2265 (1993)). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0229] Candidate anti-IL-4 and / or IL-13 antibodies are tested using enzyme-linked immunosorbent assay (ELISA), FACS, Western immunoblotting or other immunochemical techniques known in the art.

[0230] To determine whether a particular antibody homologue binds to human IL-4 and / or IL-13, any conventional binding assay may be used. Useful IL-4 and IL-13 binding assays include FACS analysis, ELISA assays, surface plasmon resonance (Biacore), radioimmunoassays, and the like, which detect the binding of antibodies to human IL-4 and / or IL-13 and the resulting function. The full-length and soluble forms of human IL-4 and IL-13 taught herein are useful in such assays. The binding of an antibody or homologue to IL-4 and / or IL-13 or a soluble fragment thereof can be conveniently detected by using a second antibody specific for an immunoglobulin of the species from which the antibody or homologue is derived.

[0231] To determine whether a particular antibody or homologue significantly blocks the binding of IL-4 and / or IL-13, any suitable competition assay may be used. Useful assays include, for example, ELISA assays, FACS assays, radioimmunoassays, etc., which quantify the competitive ability of an antibody or homologue to bind to IL-4 and / or IL-13. Preferably, the ability of a ligand to block the binding of labeled human IL-4 and / or IL-13 to an immobilized antibody or homologue is measured.

[0232] The antibodies of the invention may be described or specified in terms of an epitope or portion of IL-4 and / or IL-13 that the antibody recognizes or specifically binds to. The epitope or polypeptide portion may be specified as described herein, for example, by the position of the N-terminus and C-terminus, by the size of adjacent amino acid residues, conformational epitope, etc.

[0233] The antibodies of the invention may also be described or specified in terms of cross-reactivity. Antibodies that bind to IL-4 and / or IL-13 polypeptides that are at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identical (calculated using methods known in the art and described herein) to IL-4 and / or IL-13 are also included in the invention.

[0234] The antibodies of the present invention may also be described or specified in terms of their binding affinity to IL-4 and / or IL-13. The anti-IL-4 and / or IL-13 antibodies may have a binding affinity of less than about 10 -7 M, less than about 10 -6 M, or less than about 10 -5 M of K D Binding. A higher binding affinity of the antibody of interest may be beneficial, for example, having a binding affinity of about 10 -8 to about 10 -15 M, about 10 -8 to about 10 -12 M, about 10 -9 to about 10 -11 M, or about 10 -8 to about 10 -10 M is the equilibrium dissociation constant or K D The present invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the present invention as determined by any method known in the art to determine competitive binding, such as the immunoassay described herein. In some preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.

[0235] The present invention also includes conjugates containing an antibody of interest. The conjugates comprise two main components, the antibody of interest and a second component which may be a cell binding agent, a cytotoxic agent, or the like.

[0236] The term "cell binding agent" as used herein refers to an active agent that can specifically recognize and bind to a molecule on the cell surface. Therefore, the cell binding agent can be a CD antigen, a pathogen antigen such as a viral antigen, a differentiation antigen, a cancer antigen, a cell-specific antigen, a tissue-specific antigen, an Ig or Ig-like molecule, etc.

[0237] The cell binding agent can be any type currently known or newly known, including peptides, non-peptides, carbohydrates, nucleic acids, ligands, receptors, etc., or combinations thereof. The cell binding agent can be any compound that can bind to cells, whether in a specific or non-specific manner. In general, the cell binding agent can be an antibody (especially a monoclonal antibody), a lymphokine, a hormone, a growth factor, a vitamin, a nutrient transport molecule (such as transferrin), or any other cell binding molecule or substance.

[0238] Other examples of cell-binding agents that can be used include: polyclonal antibodies; monoclonal antibodies; and antibody fragments, such as F ab 、F ab' 、F (ab')2 and F v (Parham, J. Immunol. 131:2895-2902 (1983); Spring et al., J. Immunol. 113:470-478 (1974); and Nisonoff et al., Arch. Biochem. Biophys. 89:230-244 (1960)).

[0239] The second component may also be a cytotoxic agent. As used herein, the term "cytotoxic agent" refers to a substance that can reduce or block the function or growth of cells, and / or cause cell destruction. Thus, the cytotoxic agent may be a paclitaxel, maytansinoids, such as DM1 or DM4, CC-1065 or CC-1065 analogs, ricin, mitomycin C, etc. In certain embodiments, like any binding agent of the conjugate of the present invention, the cytotoxic agent is covalently linked to the antibody of interest directly or through a cleavable or non-cleavable linker.

[0240] Examples of suitable maytansines include maytansinol and maytansinol analogs. Maytansine inhibits microtubule formation and is highly toxic to mammalian cells.

[0241] Examples of suitable maytansinol analogs include those containing modified aromatic rings and those maytansinol analogs modified at other positions. Such suitable maytansines are disclosed in the following US Patents: 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.

[0242] Examples of suitable maytansinol analogs containing a modified aromatic ring include: (1) C-19-dechloro (U.S. Patent No. 4,256,746) (prepared, for example, by LAH reduction of ansamytocin P2); (2) C-20-hydroxy (or C-20-demethyl) + / - C-19-dechloro (U.S. Patent Nos. 4,361,650 and 4,307,016) (prepared, for example, by demethylation using Stretomyces or Actinomyces or by dechlorination using lithium aluminum hydride (LAH)); and (3) C-20-demethoxy, C-20-acyloxy (-OCOR), + / -dechloro (U.S. Patent No. 4,294,757) (prepared by acylation with an acyl chloride).

[0243] Examples of suitable maytansinol analogs modified at other positions include: (1) C-9-SH (U.S. Patent No. 4,424,219) (prepared by reaction of maytansinol with H2S or P2S5); (2) C-14 alkoxymethyl (demethoxy / CH2OR) (U.S. Patent No. 4,331,598); (3) C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Patent No. 4,450,254) (prepared from Nocardia (prepared by Streptomyces); (4) C-15-hydroxy / acyloxy (U.S. Patent No. 4,364,866) (prepared by transformation of maytansinol by Streptomyces); (5) C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated from Trewianudiflora); (6) C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared by demethylation of maytansinol by Streptomyces); and (7) 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by titanium trichloride / LAH reduction of maytansinol).

[0244] Cytotoxic conjugates can be prepared in vitro. In order to link a cytotoxic agent, drug or prodrug to an antibody, a linker is generally used. Suitable linkers are well known in the art and include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. For example, a conjugate can be constructed by a disulfide exchange reaction or by forming a thioether bond between the target antibody and the drug or prodrug.

[0245] As discussed above, the present invention provides isolated nucleic acid sequences encoding the antibodies or functional fragments thereof or variant variants disclosed herein, vector constructs containing nucleotide sequences encoding the IL-4 and / or IL-13 binding portions of the antibodies or functional fragments thereof of the present invention, host cells containing such vectors, and recombinant techniques for producing polypeptides.

[0246] The vector generally comprises components known in the art, and generally includes, but is not limited to, one or more of the following: a signal sequence, an origin of replication, one or more markers or selection genes, sequences that promote and / or enhance translation, enhancer elements, and the like. Thus, the expression vector includes a nucleotide sequence that is effectively linked to a suitable transcriptional or translational regulatory nucleotide sequence, such as those derived from mammalian, microbial, viral, or insect genes. Other examples of regulatory sequences include operators, mRNA ribosome binding sites, and / or other suitable sequences that control transcription and translation (e.g., initiation and termination thereof). When a regulatory sequence is functionally associated with a nucleotide sequence of an appropriate polypeptide, the nucleotide sequence is "effectively linked". Thus, if a promoter nucleotide sequence controls transcription of the nucleotide sequence, then the promoter nucleotide sequence is effectively linked to, for example, an antibody heavy chain sequence.

[0247] In addition, sequences encoding appropriate signal peptides that are not naturally associated with antibody heavy and / or light chain sequences can be incorporated into expression vectors. For example, a nucleotide sequence of a signal peptide (secretion leader) can be fused to a polypeptide sequence in frame so that the antibody is secreted into the periplasmic space or culture medium. A signal peptide that works in a desired host cell enhances extracellular secretion of an appropriate antibody or portion thereof. The signal peptide can be cleaved from the polypeptide when the cell secretes the antibody. Examples of such secretion signals are well known and include, for example, those described in US Patent Nos. 5,698,435, 5,698,417, and 6,204,023.

[0248] The vector can be a plasmid, a single-stranded or double-stranded viral vector, a single-stranded or double-stranded RNA or DNA phage vector, a phagemid, a cosmid, or any other vector of the desired transgene. Such vectors can be introduced into cells as polynucleotides using well-known techniques for introducing DNA and RNA into cells. In the case of phage and viral vectors, well-known infection and transduction techniques can also be used to introduce the vector into cells as a packaged or encapsulated virus. Viral vectors can be replicable or replication-defective. In the latter case, viral propagation generally only occurs in complementing host cells, and multiple vectors containing multiple viral components necessary for particle production are required. Cell-free translation systems can also be used to produce proteins using RNA derived from existing DNA constructs (see, for example, WO86 / 05807 and WO 89 / 01036, and US Patent No. 5,122,464).

[0249] The antibodies of the present invention can be expressed from any suitable host cell. Examples of host cells that can be used in the present invention include prokaryotes, yeasts or higher eukaryotic cells, and include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli, Bacillus subtilis, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, Serratia and Shigella, as well as Bacilli, Pseudomonas and Streptomyces) transformed with recombinant yeast expression vectors containing antibody coding sequences, plasmid DNA or cosmid DNA; yeast (e.g., Saccharomyces, Pichia, Actinomycetes) transformed with recombinant yeast expression vectors containing antibody coding sequences. cetes), Kluyveromyces, Schizosaccharomyces, Candida, Trichoderma, Neurospora, and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium and Aspergillus); insect cell systems infected with recombinant viral expression vectors (such as baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant viral expression vectors (such as cauliflower mosaic virus, CaMV; or tobacco mosaic virus, TMV), or transformed with recombinant plasmid expression vectors (such as Ti plasmid) containing antibody coding sequences; or mammalian cell systems (such as COS, CHO, BHK, 293 or 3T3 cells) containing recombinant expression constructs. The construct contains a promoter derived from the genome of a mammalian cell (eg, the metallothionein promoter) or a promoter derived from a mammalian virus (eg, the adenovirus late promoter; or the vaccinia virus 7.5K promoter).

[0250] Expression vectors for prokaryotic host cells generally contain one or more phenotypic selectable marker genes. Phenotypic selectable marker genes are, for example, genes encoding proteins that confer antibiotic resistance or provide autotrophic requirements. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids such as pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), pGEM1 (Promega Biotec, Madison, WI), pET (Novagen, Madison, WI) and pRSET (Invitrogen, Carlsbad, CA) series vectors (Studier, J Mol Biol 219: 37 (1991); and Schoepfer, Gene 124: 83 (1993)). Promoter sequences commonly used in recombinant prokaryotic host cell expression vectors include T7 (Rosenberg et al., Gene 56:125 (1987)), β-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615 (1978); and Goeddel et al., Nature 281:544 (1979)), tryptophan (trp) promoter system (Goeddel et al., Nucl Acids Res 8:4057 (1980)), and tac promoter (Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory (1990)).

[0251] Yeast vectors often contain a replication origin sequence, such as from the 2μ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, a polyadenylation sequence, a transcription termination sequence, and a selectable marker gene. Suitable promoter sequences for yeast vectors include promoters for metallothioneins, 3-phosphoglycerate kinase (Hitzeman et al., J Biol Chem 255:2073 (1980)) or other glycolytic enzymes (Holland et al., Biochem 17:4900 (1978)) such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase, among others. Other suitable vectors and promoters for yeast expression are further described in Fleer et al., Gene 107:285 (1991). Other yeast and yeast transformation protocols suitable promoters and vectors are well known in the art. Yeast transformation protocols are also well known. Hinnen et al., Proc Natl Acad Sci 75:1929 (1978) described such a protocol, which selects for Trp in a selective medium. + Transformant.

[0252] Any eukaryotic cell culture is feasible, whether it is derived from a vertebrate or invertebrate culture. Examples of invertebrate cells include plant and insect cells (Luckow et al., Bio / Technology 6:47 (1988); Miller et al., Genetic Engineering, Setlow et al., eds., vol.8, pp.277-9, Plenum Publishing (1986); and Maeda et al., Nature 315:592 (1985)). For example, the baculovirus system can be used to produce heterologous proteins. In the insect system, Autographa californicanuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. Antibody coding sequences can be cloned under the control of the AcNPV promoter (e.g., the polyhedrin promoter). Other identified hosts include Aedes, Drosophila melanogaster, and Bombyx mori. A variety of transfecting virus strains are publicly available, such as the L-1 variant of AcNPV and the Bm-5 strain of Bombyx mori NPV. In addition, plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts, as is well known in the art.

[0253] Vertebrate cells, propagation of vertebrate cells in culture (tissue culture) can be a routine procedure, although fastidious cell lines do exist which require, for example, specialized culture media with unique factors, feeder cells, etc., see Tissue Culture, Kruse et al., eds., Academic Press (1973). Examples of useful mammalian host cell lines are monkey kidney; human embryonic kidney line; baby hamster kidney cells; Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc Natl Acad Sci USA 77:4216 (1980)); mouse Sertoli cells; human cervical carcinoma cells (e.g., HeLa); canine kidney cells; human lung cells; human liver cells; mouse breast tumor; and NS0 cells.

[0254] Host cells are transformed with vectors to produce antibodies and cultured in conventional nutrient media containing growth factors, vitamins, minerals, etc., and inducers suitable for the cells and vectors used. Commonly used promoter sequences and enhancer sequences are derived from polyomavirus, adenovirus 2, simian virus 40 (SV40) and human cytomegalovirus (CMV). The DNA sequence derived from the SV40 viral genome can be used to provide other genetic elements (such as SV40 starting point, early and late promoters, enhancers, splicing and polyadenylation sites) to express structural gene sequences in mammalian host cells. Viral early and late promoters are particularly useful because both are easily obtained as fragments from the viral genome, and the fragments may also contain viral origins of replication. Exemplary expression vectors for mammalian host cells are available from commercial sources.

[0255] Commercially available culture media such as Ham's F10, minimal essential medium (MEM), RPMI-1640, and Dulbecco's modified Eagle medium (DMEM) are suitable for culturing host cells. In addition, Ham et al., Meth Enzymol 58:44 (1979) and Barnes et al., Anal Biochem 102:255 (1980), and any culture media described in US Patent Nos. 4,767,704; 4,657,866; 4,560,655; 5,122,469; 5,712,163; or 6,048,728 can also be used as a culture medium for host cells. Any of these media may be supplemented, as necessary, with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., chlorides, such as sodium, calcium, or magnesium chloride; and phosphates), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics, trace elements (defined as inorganic compounds, typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may be included at appropriate concentrations as a matter of design choice. Culture conditions, such as temperature, pH, etc., are those known in the art to be appropriate for the cells and to allow for desired expression of the transgene.

[0256] The polynucleotide of interest can be obtained and the nucleotide sequence of the polynucleotide determined by any method known in the art. For example, if the nucleotide sequence of an antibody is known, a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described by Kutmeier et al., Bio / Techniques 17:242 (1994)), and then the linked oligonucleotides amplified, e.g., by PCR.

[0257] Alternatively, polynucleotides encoding antibodies can be generated from nucleic acids of cells expressing the antibodies. If a clone containing nucleic acids encoding a particular antibody is not available, but the sequence of the antibody molecule is known, nucleic acids encoding immunoglobulins may be obtained from appropriate sources such as libraries, which may be nucleic acids specific to cells producing antibodies, such as hybridoma cells selected to express the antibodies of the present invention. Appropriate primers can be configured for PCR amplification. Then, the amplified nucleic acids generated by PCR can be cloned into replicable cloning vectors using any method well known in the art.

[0258] Once the nucleotide sequence and the corresponding antibody amino acid sequence are determined, methods known in the art for manipulating nucleotide sequences, such as DNA recombinant technology, site-directed mutagenesis, PCR, etc. (see, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory (1990); and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons (1998), can be used to manipulate the nucleotide sequence of the antibody to obtain the equivalent of the purpose described herein, to generate antibodies containing different amino acid sequences, for example, to cause amino acid substitutions, deletions and / or insertions.

[0259] The amino acid sequence of the heavy chain and / or light chain variable domain can be examined by well-known methods to identify CDR sequences, for example, by comparison with known amino acid sequences of other heavy chain and light chain variable regions to determine regions of high sequence variability. Using conventional DNA recombinant techniques, one or more CDRs can be inserted into the framework region, for example, into the human framework region to humanize a non-human antibody, as described above. The polynucleotide of interest generated by the combination of the framework region and one or more CDRs can encode an antibody that specifically binds to IL-4 and / or IL-13 or at least encodes its ED domain. For example, this method can be used to generate amino acid substitutions or deletions of one or more variable region cysteine ​​residues that participate in intrachain disulfide bonds to produce antibody molecules that lack one or more intrachain disulfide bonds.

[0260] The antibodies or antibody fragments of the present invention can be used to detect IL-4 and / or IL-13 in biological samples in vitro or in vivo, and thus cells expressing IL-4 and / or IL-13 can be detected. In one embodiment, the anti-IL-4 and / or IL-13 antibodies of the present invention are used to determine the presence and level of IL-4 and / or IL-13 in a tissue or in cells from the tissue. For example, in an immunoassay using the antibodies or antibody fragments of the present invention, the level of IL-4 and / or IL-13 in the tissue or biopsy sample can be determined. The tissue or its biopsy sample can be frozen or fixed. The same method or other methods can be used to determine other properties of IL-4 and / or IL-13, such as its level, cellular localization, mRNA level, its mutation, etc.

[0261] The above methods can be used, for example, to diagnose cancer in a subject known to be or suspected of having cancer, wherein the IL-4 and / or IL-13 levels measured in the patient are compared to a normal reference subject or standard. The assays of the invention can also be used to diagnose arthritis or other autoimmune diseases characterized by infiltration and enrichment of B cells and development of differentiated lymphoid tissue.

[0262] The present invention also provides monoclonal antibodies, humanized antibodies and epitope binding fragments thereof further labeled for research or diagnostic applications. In certain embodiments, the label is a radiolabel, a fluorophore, a chromophore, an imaging agent, or a metal ion.

[0263] Also provided is a diagnostic method, wherein the labeled antibody or epitope-binding fragment thereof is administered to a subject suspected of having cancer, arthritis, autoimmune disease or other IL-4 and / or IL-13 mediated disease, and the distribution of the marker in the subject is measured or monitored.

[0264] The antibodies and fragments thereof of the present invention can be used as affinity purification agents. In this process, the antibodies are fixed on a solid phase, such as dextran or agarose resin or filter paper, using methods known in the art. The fixed antibodies are contacted with a sample containing IL-4 and / or IL-13 or cells to be purified that carry the IL-4 and / or IL-13, and then the support is washed with a suitable solvent, which will remove almost all substances in the sample except IL-4 and / or IL-13 or cells to be purified, and IL-4 and / or IL-13 or cells to be purified are bound to the fixed antibodies of the present invention. Finally, the support is washed with another suitable solvent (such as glycine buffer, pH 5.0), which will release IL-4 and / or IL-13 or cells from the antibodies.

[0265] For diagnostic applications, the antibody of interest will usually be labeled with a detectable moiety. A variety of labels are available, which can generally be divided into the following categories: (a) radioactive isotopes, such as36 S. 14 C. 125 I. 3 H and 131 I (the antibody may be labeled with a radioactive isotope, for example, using the technique described in Current Protocols in Immunology, vol. 12, Coligen et al., eds., Wiley-Interscience, New York (1991), and the radioactivity may be measured by scintillation counting); (b) fluorescent labels, such as rare earth chelates (europium chelates), fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lissamine, phycoerythrin, and Texas Red; fluorescent labels may be conjugated to antibodies using techniques such as those disclosed in Current Protocols in Immunology (supra), wherein the fluorescence may be quantified by a fluorometer; and (c) a variety of enzyme substrate labels are available (US Pat. No. 4,275,149 provides a review), enzymes generally catalyze chemical changes in chromogenic substrates, which may be measured using a variety of techniques, for example, enzymes may catalyze changes in the color of the substrate, which may be measured spectrophotometrically; or, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying changes in fluorescence are well known, for example, using a luminometer, or the label may provide energy to a fluorescent acceptor. Examples of enzyme labels include luciferase (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al., Meth Enz, ed., Langone & Van Vunakis, Academic Press, New York, 73 (1981).

[0266] When these labels are used, suitable substrates are available, for example: (i) for horseradish peroxidase, hydrogen peroxidase can be used as a substrate, where the hydrogen peroxidase oxidizes a dye precursor (e.g., o-phenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB)); (ii) for alkaline phosphatase (AP), p-nitrophenyl phosphate can be used as a chromogenic substrate; and (iii) for β-D-galactosidase (β-D-Gal), a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) can be used, or a fluorescent substrate such as 4-methylumbelliferyl ketoacyl-β-D-galactosidase.

[0267] Other enzyme-substrate combinations are available to those skilled in the art. For a general review, see US Patent Nos. 4,275,149 and 4,318,980.

[0268] Sometimes, the label is conjugated to the antibody indirectly. For example, the antibody can be conjugated to biotin, and any of the above reporters can be conjugated to avidin, and vice versa. Biotin selectively binds to avidin, and therefore, the label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label, the antibody is conjugated to a small hapten (such as digoxigenin), and one of the above different types of labels or reporters is conjugated to an anti-digoxigenin antibody. Thus, indirect conjugation of the label to the antibody or mutant protein can be achieved using a second antibody.

[0269] In another embodiment of the invention, the antibody need not be labeled, and its presence can be detected using a labeled antibody that binds to the antibody (another form of a secondary antibody).

[0270] The antibodies of the present invention can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques (CRC Press, Inc. 1987).

[0271] Competitive binding assays depend on the ability of labeled standards and test samples to compete for binding to a limited amount of antibody. The amount of antigen in the test sample is inversely proportional to the amount of standard bound to the antibody. To facilitate determination of the amount of bound standard, the antibody is typically insoluble before or after competition. As a result, standards and test samples bound to the antibody can be conveniently separated from unbound standards and test samples.

[0272] Sandwich assays involve the use of two antibodies, each with the ability to bind to a different immunogenic portion (determinant or epitope) of the target to be detected. In a sandwich assay, the test sample to be analyzed is bound to a first antibody that is directly or indirectly immobilized on a solid support, and then a second antibody that is directly or indirectly labeled is bound to the already bound test sample, thereby forming an insoluble three-part complex, see, e.g., U.S. Patent No. 4,376,110. The second antibody itself may be labeled with a detectable moiety (direct sandwich assay), or may be measured with an anti-immunoglobulin antibody or other suitable member of a binding pair (e.g., antibody / antigen, receptor / ligand, enzyme / substrate) labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.

[0273] The present invention also includes reagent kits, for example, which include antibodies, fragments thereof, homologues thereof, derivatives thereof, etc., such as conjugates that are labeled or cytotoxic, and instructions for use of the antibodies, conjugates that kill specific types of cells, etc. The instructions may include instructions for using the antibodies, conjugates, etc. in vitro, in vivo, or ex vivo. The antibodies may be in liquid form or solid, usually lyophilized. The kit may contain other suitable reagents, such as buffers, reconstitution solutions, and other necessary components for the intended use. It is contemplated that the reagents are packaged in predetermined amounts and combined with instructions for their use, such as for therapeutic use or for performing diagnostic assays. When the antibody is labeled, such as with an enzyme, the kit may include a substrate and a cofactor required by the enzyme (e.g., a substrate precursor that provides a detectable chromophore or fluorophore). In addition, other additives, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), etc. may also be included. The relative amounts of the various reagents can be varied to provide concentrates of the reagent solutions, which provides user flexibility, space savings, reagent savings, etc. These reagents may also be provided in the form of a dry powder, usually lyophilized, including excipients which, when dissolved, provide a solution of the reagent having the appropriate concentration.

[0274] The antibodies of the present invention can be used to treat mammals. In one embodiment, for example, for the purpose of obtaining preclinical data, the target antibody or equivalent is administered to a non-human mammal. Exemplary non-human mammals to be treated include non-human primates, dogs, cats, rodents, and other mammals in which preclinical studies have been conducted. Such mammals can be established animal models for diseases that require antibody treatment, or can be used to study the toxicity of the target antibody. In each such embodiment, a dose escalation study can be performed in a mammal.

[0275] The antibodies, with or without a second component (e.g., a therapeutic moiety conjugated thereto), whether administered alone or in combination with a cytotoxic factor, can be used as therapeutic agents. The present invention relates to antibody-based therapies wherein the antibodies of the invention are administered to an animal, mammal or human to treat a disease, disorder or condition mediated by IL-4 and / or IL-13.

[0276] As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. It refers to preventing, curing, reversing, attenuating, ameliorating, minimizing, inhibiting or stopping the deleterious effects of a disease state, disease process, disease-causing agent (such as bacteria or virus) or other abnormal condition.

[0277] Thus, the present invention also encompasses multivalent antibodies, including bispecific anti-IL-4 / IL-13 antibodies, having effector molecules, atoms or other substances with diagnostic or therapeutic functions attached thereto. For example, the antibody may have a radioactive diagnostic label or a radioactive cytotoxic atom or metal or cytotoxic substance such as a ricin chain attached thereto for in vivo diagnosis or treatment of cancer.

[0278] In addition, the antibodies of the present invention can also be used in immunoassays, purification methods and other methods using immunoglobulins or fragments thereof. Such uses are well known in the art.

[0279] Accordingly, the present invention also provides a composition comprising the anti-IL-13 and / or anti-IL-4 antibodies or fragments thereof of the present invention, wherein the antibodies are conveniently combined with a pharmaceutically acceptable carrier, diluent or excipient, which is a conventional practice in the art.

[0280] The term "pharmaceutical composition" used in the present invention refers to a variety of preparations. The preparation containing a therapeutically effective amount of a multivalent antibody is in the form of a sterile liquid solution, liquid suspension or lyophilized form, optionally containing a stabilizer or excipient.

[0281] As used herein, the term "disorder" refers to any condition that can benefit from treatment with the antibodies of the invention. This includes chronic and acute disorders or diseases, including those pathological conditions that predispose mammals, especially humans, to the disorder. Non-limiting examples of disorders to be treated herein include cancer, inflammation, autoimmune disease, infection, cardiovascular disease, respiratory disease, neurological disease, and metabolic disease.

[0282] The antibodies of the present invention can be used to treat, inhibit or prevent diseases, such as allergic diseases, Th2-mediated diseases, IL-13-mediated diseases, IL-4-mediated diseases and / or IL-4 / IL-13-mediated diseases. Examples of such diseases include Hodgkin's disease, asthma, allergic asthma, atopic dermatitis, atopic allergy, ulcerative colitis, scleroderma, allergic rhinitis, COPD3 idiopathic pulmonary fibrosis, chronic transplant rejection, bleomycin-induced pulmonary fibrosis, radiation-induced pulmonary fibrosis, pulmonary granuloma, progressive systemic sclerosis, schistosomiasis, liver fibrosis, renal cancer, Burkitt's lymphoma, Hodgkin's disease, non-Hodgkin's disease, Sezary syndrome, asthma, toxic arthritis, dermatitis herpetiformis, chronic idiopathic urticaria, ulcerative colitis, scleroderma, hypertrophic scars, Whipple's disease, benign prostatic hyperplasia, IL-4 receptor-mediated Pulmonary disorders, conditions in which IL-4 receptor-mediated epithelial barrier disruption plays a role, digestive disorders in which IL-4 receptor plays a role, allergic reactions to drugs, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Grave's disease, preeclampsia, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, cystic fibrosis, allergic bronchopulmonary fungal disease, chronic obstructive pulmonary disease, bleomycin-induced lung disease and fibrosis, pulmonary alveolar proteinosis, adult respiratory distress syndrome, sarcoidosis, hyper-IgE syndrome, idiopathic hypereosinophil syndrome, autoimmune blistering disease, pemphigus vulgaris, bullous pemphigoid, myasthenia gravis, chronic fatigue syndrome, and kidney disease.

[0283] The term "allergic disease" refers to a pathological condition in which a patient is hypersensitive to and develops an immune response to a normally non-immunogenic substance. Allergic diseases are generally characterized by IgE activation of mast cells resulting in an inflammatory response (e.g., local, systemic) that can result in benign symptoms such as a runny nose, or life-threatening anaphylactic shock and death. Examples of allergic diseases include, but are not limited to, allergic rhinitis (e.g., hay fever), asthma (e.g., allergic asthma), allergic dermatitis (e.g., eczema), contact dermatitis, food allergies, and urticaria (hives).

[0284] As used herein, "Th2-mediated disease" refers to a disease whose pathology is (wholly or in part) caused by CD4 +The immune response regulated by Th2T lymphoid cells (Th2 immune response) is caused by the characteristic production of IL-4, IL-5, IL-9 and IL-13. Th2 immune response is associated with the production of certain cytokines (such as IL-4, IL-13) and certain types of antibodies (such as IgE) as well as humoral immunity. Th2-mediated diseases are characterized by the presence of elevated levels of Th2 cytokines (e.g., IL-4, IL-13) and / or certain classes of antibodies (e.g., IgE), and include, for example, allergic diseases (e.g., allergic rhinitis, atopic dermatitis, asthma (e.g., idiopathic asthma), allergic airway disease (AAD), anaphylactic shock, conjunctivitis), autoimmune disorders associated with elevated levels of IL-4 and / or IL-13 (e.g., rheumatoid arthritis, host vs graft disease, renal disease (e.g., nephrotic syndrome, lupus nephritis), and infections associated with elevated levels of IL-4 and / or IL-13 (e.g., viral, parasitic, fungal (e.g., Candida albicans (C. albicans)) infection). Some cancers are associated with elevated levels of IL-4 and / or IL-13, or with IL-4-induced and / or IL-13-induced cancer cell proliferation (e.g., B-cell lymphoma, T-cell lymphoma, multiple myeloma, head and neck cancer, breast cancer, and ovarian cancer). These cancers can be treated, inhibited, or prevented using the ligaud of the present invention.

[0285] As used herein, the term "cancer" refers to or describes a physiological condition in mammals, especially humans, that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.

[0286] As used herein, the term "autoimmune disease" refers to a non-malignant disease or disorder caused by and directed against an individual's own tissues. Examples of autoimmune diseases or disorders include, but are not limited to, inflammatory responses such as inflammatory skin diseases, including psoriasis and dermatitis; allergic conditions such as eczema and asthma; other conditions involving T cell infiltration and chronic inflammatory responses; atherosclerosis, diabetes (such as type I diabetes or insulin-dependent diabetes); multiple sclerosis and inflammatory disorders of the central nervous system.

[0287] The antibodies of the present invention can be used as a composition for separate administration, or can be used in combination with other active agents. The antibodies can be used in combination therapy with existing IL-13 therapies (e.g., existing IL-13 active agents such as anti-IL-13Rαl, IL-4 / 13Trap, anti-IL-13) plus anti-IL-4 antibodies, and existing IL-4 active agents (such as anti-IL-4R, IL-4 mutant proteins, IL-4 / 13Trap) plus anti-IL-13 antibodies and IL-4 antibodies (such as WO05 / 0076990 (CAT), WO03 / 092610 (Regeneron), WO00 / 64944 (Genetic Inst.) and WO2005 / 062967 (Tanox)).

[0288] The antibodies of the present invention can be administered and / or formulated with one or more additional treatments or active agents. When a ligand is administered with an additional therapeutic agent, the ligand can be administered before, simultaneously with, or after the administration of the additional therapeutic agent. In general, the ligand and additional active agent are typically administered in a manner that provides overlapping therapeutic effects. Additional active agents that can be administered or formulated with the ligands of the present invention include, for example, a variety of immunotherapeutic drugs, such as cyclosporine, methotrexate, doxorubicin or cisplatin, antibiotics, antifungal agents, antiviral agents, and immunotoxins. For example, when the antagonist is administered to prevent, inhibit or treat pulmonary inflammation or respiratory diseases (such as asthma), the antagonist can be administered in combination with: phosphodiesterase inhibitors (such as phosphodiesterase 4 inhibitors), bronchodilators (such as beta 2 agonists, anticholinergics, theophylline), short-acting beta agonists (such as albuterol, salbuiamol, bambuterol, fenoterol [sigmal] l, isoetherine, isoproterenol, leva [iota] buterol, austinerol, pirbuterol, terbutaline and tornlate), long-acting beta agonists (such as formoterol and salmeterol), short-acting anticholinergics (such as ipratropium bromide), and oxitropium), long-acting anticholinergics (such as tiotropium), theophylline (such as short-acting, long-acting), inhaled steroids (such as beclomethasone, beclomethasone, budesonide, flunisolide, fluticasone propionate, and triamcinolone), oral steroids (such as methylprednisolone, prednisolone, prednisolone, and prednisone), short-acting beta-agonists combined with anticholinergics (such as albuterol / salbutamol / ipratropium and fenoterol / ipratropium), long-acting beta-agonists combined with inhaled steroids (such as salmeterol / fluticasone and formoterol / budesonide), and mucolytics (such as erdosteine, acetylcysteine, bromhexine, carbocyslcine, guiiafencsin, and iodinated glycerol).

[0289] Other suitable co-therapeutic agents that can be co-administered with the antibodies of the invention to prevent, inhibit or treat asthma (e.g., allergic asthma) include corticosteroids (e.g., beclomethasone, budesonide, fluticasone), cromoglobin, nedocromil, beta agonists (e.g., albuterol, terbutaline, bambuterol, fenoterol, theaproterol, tolubuterol, salmeterol, fomtero), zafirlukast, salmeterol, prednisone, prednisolone, theophylline, zileutron, montelukast, and leukotriene modifiers. The ligands of the invention can be co-administered with a variety of co-therapeutic agents suitable for treating diseases (e.g., Th-2 mediated diseases, YL-A mediated diseases, IL-13 mediated diseases, IL-4 mediated diseases, and cancer), including cytokines, analgesics / antipyretics, antiemetics, and chemotherapeutic drugs.

[0290] As is well known in the art or described herein, the antibodies of the present invention can be provided in the form of pharmaceutically acceptable compositions. The terms "physiologically acceptable", "pharmacologically acceptable" and the like mean those that have been approved by a regulatory agency of a federal or state government, or that have been listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias for use in animals, especially humans.

[0291] Anti-IL-4, anti-IL-13 and bispecific anti-IL-4 / IL-13 antibodies can be administered to mammals, especially humans, in any acceptable manner. Methods of administration include, but are not limited to, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, epidural, inhalation and oral routes; if immunosuppressive therapy is desired, intralesional administration. Parenteral infusions include intramuscular, intradermal, intravenous, intraarterial or intraperitoneal administration. The antibody or composition can be administered by any convenient route, such as by infusion or rapid injection, absorption through epithelial or mucosal linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be necessary to introduce the therapeutic antibody or composition of the present invention into the central nervous system by any appropriate route, including intraventricular and intrathecal injections; intraventricular injections are by means of an intraventricular catheter, such as a catheter connected to a reservoir such as an Ommaya reservoir. Furthermore, the antibody is suitably administered as a pulse infusion, particularly with declining doses of the antibody.Preferably, the administration is by injection, preferably intravenous or subcutaneous, depending in part on whether the administration is brief or chronic.

[0292] A variety of other delivery systems are well known and can be used to administer the antibodies of the invention, including, for example, encapsulation in liposomes, microparticles, microcapsules (see Langer, Science 249:1527 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer; Lopez-Berestein et al., eds., p.353-365 (1989); and Lopez-Berestein, ibid., p.317-327) and recombinant cells capable of expressing the compound; receptor-mediated endocytosis (see Wu et al., J Biol Chem 262:4429 (1987)); construction of the nucleic acid as part of a retroviral or other vector, etc.

[0293] The active ingredient can also be encapsulated in microcapsules prepared, for example, by coascervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or microemulsions, respectively. These techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, A. Osal, ed. (1980).

[0294] Pulmonary administration may also be employed, for example using an inhaler or nebulizer, and formulation with an aerosolizing agent. Antibodies may also be administered to the lungs of a patient in the form of a dry powder composition, see, for example, US Patent No. 6,514,496.

[0295] In one embodiment, it may be desirable to administer the therapeutic antibody or composition of the invention locally to the area in need of treatment; this may be accomplished by, for example, but not limited to, local infusion, topical application, injection, via a catheter, as a suppository, or implant; the implant being a porous, non-porous, or gel-like material, including a film such as a sialastic film or fiber. Preferably, when administering the antibody of the invention, care should be taken to use a material that does not absorb or adsorb to proteins.

[0296] In another embodiment, the antibody can be delivered via a controlled release system. In one embodiment, a pump can be used (see Langer, Science 249: 1527 (1990); Sefton, CRC Crit Ref Biomed Eng 14: 201 (1987); Buchwald et al., Surgery 88: 507 (1980); and Saudek et al., N Engl J Med 321: 574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer et al., eds., CRC Press (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen et al., eds., Wiley (1984); Ranger et al., J Macromol Sci Rev Macromol Chem 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann Neurol 25:351 (1989); and Howard et al., J Neurosurg 71:105 (1989)). In yet another embodiment, the controlled release system can be placed in the vicinity of the therapeutic target.

[0297] By mixing a polypeptide having the desired purity with an optional "pharmaceutically acceptable" carrier, diluent, excipient or stabilizer commonly used in the art (i.e., buffer, stabilizer, preservative, isotonic agent, nonionic detergent, antioxidant and other various additives), see Remington's Pharmaceutical Sciences, 16th edition, Osol (1980), polypeptide or antibody therapeutic preparations can be prepared as lyophilized preparations or aqueous solutions for storage. These additives are generally non-toxic to the recipient at the dosage and concentration used, and therefore, the excipient, diluent, carrier, etc. are pharmaceutically acceptable.

[0298] An "isolated" or "purified" antibody is substantially free of cellular material, or other contaminating proteins from the culture medium from which the cell or tissue source or protein was derived, or substantially free of chemical precursors or other compounds in the case of chemical synthesis. The phrase "substantially free of cellular material" includes antibody preparations in which the polypeptide / protein is separated from the cellular components of the cells in which the antibody can be isolated or recombinantly produced. Thus, antibodies substantially free of cellular material include antibody preparations having less than about 30%, 20%, 10%, 5%, 2.5% or 1% (dry weight) of contaminating proteins. When the antibody is recombinantly produced, it is also preferably substantially free of culture medium, i.e., the culture medium content is less than about 20%, 10%, 5%, 2.5% or 1% by volume of the protein preparation. When the antibody is chemically synthesized, it is preferably substantially free of chemical precursors or other chemicals and reagents, i.e., the antibody of interest is separated from chemical precursors or other chemicals involved in protein synthesis. Thus, these antibody preparations contain less than about 30%, 20%, 10%, 5% or 1% (dry weight) of chemical precursors or other compounds other than the antibody of interest. In a preferred embodiment of the invention, the antibodies are isolated or purified.

[0299] As used herein, the term "low to undetectable levels of aggregation" refers to a sample containing no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1%, and often no more than 0.5% aggregation (by weight of protein), for example as measured by high performance size exclusion chromatography (HPSEC).

[0300] As used herein, the term "low to undetectable fragmentation levels" refers to a sample containing equal to or greater than 80%, 85%, 90%, 95%, 98% or 99% of total protein, for example, at a single peak as determined by HPSEC, or at two (2) peaks (heavy chain and light chain) as determined by reducing capillary gel electrophoresis (rCGE), and containing no other single peaks greater than 5%, greater than 4%, greater than 3%, greater than 2%, greater than 1%, or greater than 0.5% of total protein, respectively. As used herein, rCGE refers to capillary gel electrophoresis under reducing conditions sufficient to reduce disulfide bonds in the antibody or antibody-type molecule or antibody-derived molecule.

[0301] With respect to liquid formulations containing IL-4 and / or IL-13 antibodies or binding fragments thereof, the terms "stable" and "stable" as used herein refer to the resistance of the antibody or antigen-binding fragment thereof in the formulation to thermal and chemical unfolding, aggregation, degradation or fragmentation under given manufacturing, preparation, transportation and storage conditions. The "stable" formulations of the present invention can maintain a biological activity equal to or greater than 80%, 85%, 90%, 95%, 98%, 99% or 99.5% under given manufacturing, preparation, transportation and storage conditions. The stability of the antibody preparation can be evaluated based on the degree of aggregation, degradation or fragmentation and compared with a reference; the methods used are known to professionals in the art, including but not limited to rCGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and HPSEC.

[0302] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle used together with the therapeutic agent. Such physiological carriers can be sterile liquids, such as water and oil, including oils from petroleum, animal, plant or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a suitable carrier. Saline solutions and aqueous solutions of glucose and glycerol can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. If necessary, the composition may also contain a small amount of wetting agent or emulsifier, or pH buffer. The composition may be in the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release formulation, depot, etc. The composition can be formulated as a suppository with conventional adhesives and carriers such as triglycerides. Oral formulations can contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable carriers are described in "Remington's Pharmaceutical Sciences, Martin". Such compositions will contain an effective amount of the antibody, preferably in purified form, and an appropriate amount of carrier to provide a form suitable for administration to the patient. As is known in the art, the preparation will be constructed in a manner suitable for administration.

[0303] Buffer helps maintain the pH value within a range similar to physiological conditions. Preferably, the buffer is present in a concentration range of about 2 mM to about 50 mM. Suitable buffers for use in the present invention include organic and inorganic acids and salts thereof, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixtures, etc.), and tartrate buffers. In some embodiments, the present invention relates to a buffer solution containing a plurality of buffers, for example, a mixture of fumaric acid and disodium fumarate, a mixture of monosodium fumarate and disodium fumarate, etc.), a gluconate buffer (e.g., a mixture of gluconic acid and sodium gluconate, a mixture of gluconic acid and sodium hydroxide, a mixture of gluconic acid and potassium gluconate, etc.), an oxalate buffer (e.g., a mixture of oxalic acid and sodium oxalate, a mixture of oxalic acid and sodium hydroxide, a mixture of oxalic acid and potassium oxalate, etc.), a lactate buffer (e.g., a mixture of lactic acid and sodium lactate, a mixture of lactic acid and sodium hydroxide, a mixture of lactic acid and potassium lactate, etc.), and an acetate buffer (e.g., a mixture of acetic acid and sodium acetate, a mixture of acetic acid and sodium hydroxide, etc.). Phosphate buffers, carbonate buffers, histidine buffers, triethylamine salts such as Tris, HEPES, and other known such buffers can all be used.

[0304] Preservatives may be added to retard microbial growth and may be added in amounts ranging from 0.2% to 1% (w / v). Preservatives suitable for use in the present invention include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyldimethylbenzyl ammonium chloride, benzyaconium halides (e.g., chlorides, bromides, and iodides), hexadimethoxine, alkylparabens such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0305] The presence of an isotonicifier can ensure the physiological isotonicity of the liquid composition of the present invention, and includes polyols, preferably trivalent or higher trivalent sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol and mannitol. Considering the relative amounts of other ingredients, the polyol can be present in an amount of about 0.1% to about 25% by weight, preferably 1% to 5%.

[0306] Stabilizers refer to a wide range of excipients whose functions range from fillers to additives that dissolve the therapeutic agent or help prevent denaturation or sticking to the container walls. Typical stabilizers can be polyols; amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, arabitol, erythritol, mannitol, sorbitol, xylitol, ribitol, myo-inositol (myoinisitol), galactitol, glycerol, etc., including cyclitols such as inositol; polyethylene glycol; amino acids Acid polymers; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (i.e. <10 residues); proteins such as human serum albumin, fetal bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, carbohydrates, monosaccharides such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose; polysaccharides such as dextran, etc. The stabilizer ranges from 0.1 to 10,000 w / w per active protein portion.

[0307] Other various excipients include fillers (such as starch), chelating agents (such as EDTA), antioxidants (such as ascorbic acid, methionine or vitamin E) and co-solvents.

[0308] Depending on the needs of the specific indications to be treated, the formulations herein may also contain more than one active compound, preferably compounds with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide an immunosuppressant. Such compound molecules are present in combination in an amount effective to achieve the intended purpose.

[0309] The term "surfactant" as used herein refers to an organic substance with an amphiphilic structure, consisting of groups with opposite solubility tendencies, usually an oil-soluble hydrocarbon chain and a water-soluble ionic group. Surfactants can be divided into anionic, cationic and nonionic surfactants according to the charge of the surface active part. Surfactants are often used as wetting agents, emulsifiers, solubilizers and dispersants in a variety of pharmaceutical compositions and biomaterial preparations.

[0310] Nonionic surfactants or detergents (also called "wetting agents") may be added to aid in the dissolution of the therapeutic agent and to protect the therapeutic protein from aggregation caused by oscillation, and to expose the formulation to shear surface stress without causing protein denaturation. Suitable nonionic surfactants include polysorbate (20, 80, etc.), polyoxamer (184, 188, etc.), Polyols and polyoxyethylene sorbitan monoethers ( The nonionic surfactant may be present in the range of about 0.05 mg / ml to about 1.0 mg / ml, preferably about 0.07 mg / ml to about 0.2 mg / ml.

[0311] The term "inorganic salt" as used herein refers to any carbon-free compound formed by replacement of an acid hydrogen or part or all of an acid with a metal or a group acting as a metal, and is often used as a tension-adjusting compound in pharmaceutical compositions and biomaterial preparations. The most common inorganic salts are NaCl, KCl, NaH2PO4, etc.

[0312] The present invention includes liquid formulations having stability at commercial refrigerator and freezer temperatures in a physician's office or laboratory (e.g., about -20°C to about 5°C), as assessed by, for example, high performance size exclusion chromatography (HPSEC), for storage purposes, e.g., about 60 days, about 120 days, about 180 days, about 1 year, about 2 years, or more. The liquid formulations of the present invention also exhibit stability at room temperature for at least a number of hours, e.g., as assessed by HSPEC, e.g., 1 hour, 2 hours, or about 3 hours prior to use.

[0313] The term "small molecule" and similar terms include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and / or organometallic compounds) having a molecular weight of less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[0314] Thus, in the case of cancer, the antibodies of the invention can be administered alone or in combination with other types of cancer treatments, including traditional chemotherapeutic agents (paclitaxel, carboplatin, cisplatin, and doxorubicin), anti-EGFR agents (gefitinib, erlotinib, and cetuximab), anti-angiogenic agents (bevacizumab and sunitinib), and immunomodulators such as interferon alpha and thalidomide.

[0315] As used herein, the term "therapeutic agent" refers to any active agent that is useful for the treatment, management or amelioration of diseases, disorders, conditions, etc. associated with aberrant IL-4 and / or IL-13 metabolism and activity.

[0316] In addition, the antibodies of the invention can be conjugated to a variety of effector molecules, such as heterologous polypeptides, drugs, radionucleotides or toxins, see, for example, WO 92 / 08495; WO 91 / 14438; WO 89 / 12624; US Patent No. 5,314,995; and EPO 396,387. Antibodies and fragments thereof can be conjugated to therapeutic moieties, such as cytotoxins (e.g., cytostatics or cytocides), therapeutic agents, or radioactive metal ions (e.g., alpha emitters such as 213Bi). Cytotoxins or cytotoxic agents include any agent that damages cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin, D), 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and their analogs or homologues.Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, and decarbazine), alkylating agents (e.g., mechlorethamine, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, C) and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin, daunomycin, and doxorubicin), antibiotics (e.g., dactinomycin, actinomycin, bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine).

[0317] Techniques for conjugating such therapeutic moieties to antibodies are well known, see, e.g., Arnon et al., in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. (eds.), p. 243-56 Alan R. Liss (1985); Hellstrom et al., in Controlled Drug Delivery, 2nd ed., Robinson et al., eds., p. 623-53, Marcel Dekker (1987); Thorpe, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al., eds., p. 475-506 (1985); Monoclonal Antibodies For Cancer Detection and Therapy, Baldwin et al., eds., p. 303-16, Academic Press (1985); and Thorpe et al., Immunol Rev 62: 119 (1982). Alternatively, the antibody can be conjugated to a second antibody to form an antibody heteroconjugate, such as a bifunctional antibody, see, eg, US Patent No. 4,676,980.

[0318] The conjugates of the present invention can be used to modify a given biological response, and the therapeutic agent or drug moiety should not be construed as being limited to conventional chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide having a desired biological activity. Such proteins can include, for example, toxins such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator; apoptotic agents such as TNF-α, TNF-β, AIM I (WO 97 / 33899), AIM II (WO 97 / 34911), Fas ligand (Takahashi et al., Int Immunol, 6:1567 (1994)), VEGF (WO 99 / 23105); thrombotic agents; anti-angiogenic agents, such as angiostatin or endostatin; or biological response modifiers, such as lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (GCSF) or other growth factors.

[0319] Preparations for in vivo administration must be sterile. This can be achieved, for example, by filtration through a sterile filtration membrane. For example, the liquid preparations of the present invention can be sterilized by filtration through a 0.2 μm or 0.22 μm filter.

[0320] Sustained release preparations may be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg films or matrices. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate), poly(vinyl alcohol), polylactide (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., injectable microspheres composed of lactic acid-glycolic acid copolymers), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid are able to release molecules for up to 100 days, but certain hydrogels release proteins for shorter periods of time. Depending on the mechanism involved, a rational strategy for stabilization can be developed. For example, if the aggregation mechanism is found to be the formation of intermolecular SS bonds through sulfhydryl-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, freeze drying from acidic solutions, controlling moisture content, using appropriate additives, amino acid substitutions, and developing specific polymer matrix compositions.

[0321] The antibody or variant composition will be formulated, quantified, and administered in a manner consistent with good medical practice. Factors considered in this regard include the specific disorder to be treated, the specific mammal or human to be treated, the clinical condition of the individual patient, the cause of disease, the site of delivery of the active agent, the method of administration, the dosage regimen, and other factors known to medical practitioners. The "therapeutically effective amount" of the antibody or variant to be administered will depend on these considerations and can be the minimum amount necessary to prevent, ameliorate, or treat a certain IL-4 and / or IL-13 mediated disease, condition, or disorder.

[0322] Optionally, the antibody or variant is formulated with one or more active agents currently used to prevent or treat the disorder in question. The effective amount of such other active agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally the same doses used previously and are used by the same route of administration, or are about from 1 to 99% of the doses used previously.

[0323] As used herein, the term "effective amount" refers to an amount of a therapy (e.g., a prophylactic or therapeutic agent) sufficient to reduce the severity and / or shorten the duration of, ameliorate one or more symptoms of, prevent the development of, or cause regression of, a disease mediated by IL-4 and / or IL-13, or to prevent the development, recurrence, onset or progression of a disease mediated by IL-4 and / or IL-13 or one or more symptoms thereof, or to potentiate or improve the prophylactic and / or therapeutic effect of another therapy (e.g., another therapeutic agent) useful for treating a disease mediated by IL-4 and / or IL-13.

[0324] The amount of therapeutic antibody or fragment thereof effective in use or treatment of a particular disease or condition will depend on the nature of the disease or condition and can be determined by standard clinical techniques. When possible, a dose-response curve and the pharmaceutical composition of the present invention can be first derived in vitro. If a suitable animal model system is available, a dose-response curve can also be obtained and used to extrapolate suitable human doses for practicing methods known in the art. However, based on common knowledge in the art, a pharmaceutical composition that effectively promotes the reduction of inflammation, for example, can provide a local therapeutic agent concentration of between about 5 and 20 ng / ml, preferably between about 10 and 20 ng / ml.

[0325] In a preferred embodiment, the aqueous solution of the therapeutic polypeptide, antibody or fragment thereof can be administered by subcutaneous injection. Each dose can range from about 0.5 mg to about 50 mg per kilogram of weight, or more preferably, from about 3 mg to about 30 mg per kilogram of body weight. The dose can be determined empirically for a specific disease, patient population, mode of administration, etc., to practice pharmaceutical methods known in the art.

[0326] The dosage schedule for subcutaneous injections may vary from once weekly to once daily, depending on a number of clinical factors, including disease type, disease severity, and the patient's sensitivity to the therapeutic agent.

[0327] The present invention provides a method for preparing a liquid formulation of an antibody or IL-4 and / or IL-13 binding fragment thereof, comprising concentrating a fraction of a purified antibody to a final concentration of about 15 mg / ml, about 20 mg / ml, about 30 mg / ml, about 40 mg / ml, about 50 mg / ml, about 60 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, about 200 mg / ml, about 250 mg / ml, about 300 mg / ml or more, for example using a precipitate having an appropriate molecular weight (mw) cutoff (e.g., for its F (ab')2 The fragment cutoff value is 30K D For F abThe fragment cut-off value is 10KD) semi-permeable membrane.

[0328] In addition, the present invention also includes stable liquid preparations of the target product with improved in vivo half-life. Therefore, the half-life of the target antibody in a subject (preferably a human) is more than 3 days, more than 7 days, more than 10 days, more than 15 days, more than 25 days, more than 30 days, more than 35 days, more than 40 days, more than 45 days, more than 2 months, more than 3 months, more than 4 months, more than 5 months or longer.

[0329] In order to prolong the serum circulation of antibodies in vivo, a variety of techniques can be used. For example, inert polymer molecules such as high molecular weight polyethylene glycol (PEG) can be conjugated to the N-terminus or C-terminus of the antibody through the site-specific conjugation of PEG or connected to the antibody through the ε amino group present on the lysine residue, wherein a multifunctional linker can be used or not. Linear or branched polymer derivatives that cause minimal loss of biological activity can be used. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure that the PEG molecule is properly conjugated to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate by size exclusion or ion exchange chromatography. Using methods known to professionals in the field, such as immunoassays described herein, the binding activity and in vivo efficacy of PEG-derived antibodies can be determined.

[0330] Antibodies with increased half-life in vivo can also be prepared by introducing one or more amino acid modifications (i.e., substitutions, insertions or deletions) into the IgG constant domain or its F c R binding fragment (such as F e or hinge F e domain fragments), see, e.g., WO 98 / 23289; WO 97 / 34631; and US Patent No. 6,277,375.

[0331] In addition, the antibody can be conjugated to albumin to make the antibody more stable in vivo or have a longer half-life in vivo. These techniques are well known in the art, see, for example, WO 93 / 15199, WO 93 / 15200 and WO 01 / 77137; and EPO 413,622. The antibody can also be modified by, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting groups / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc.

[0332] In one embodiment, the composition is formulated into a pharmaceutical composition suitable for intravenous administration to a human being according to conventional steps. Usually, the composition for intravenous administration is a solution in a sterile isotonic buffered aqueous solution. In necessary situations, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine or other "caine" anesthetics to alleviate the pain at the injection site. Usually, a variety of ingredients are supplied separately or mixed together in the form of a unit dose, for example as a dry lyophilized powder or anhydrous concentrate in a sealed container, the sealed container such as an ampoule or capsule, and the amount of active ingredient is indicated. When the composition is administered by infusion, it can be dispersed in an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided, for example, provided in a kit form so that the composition can be mixed before use.

[0333] The present invention also provides that the liquid formulation of the present invention is contained in a sealed container, such as an ampoule or a sachet, on which the amount of the target product is indicated. The liquid formulation of the present invention can be in a sealed container indicating the amount and concentration of the antibody or antibody fragment. For example, the liquid formulation of the present invention can be supplied in a sealed container having at least 15 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, 150 mg / ml, 200 mg / ml, 250 mg / ml, or 300 mg / ml of IL-4 and / or IL-13 antibody in an amount of 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml or 20 ml.

[0334] A prepared article containing a substance useful for treating the above-mentioned disorders is provided. The prepared article includes a container and a label. Suitable containers include, for example, bottles, vials, syringes and test tubes. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition that can effectively diagnose, prevent or treat IL-4 and / or IL-13-mediated conditions or diseases, and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle). The label on the container or an accompanying label indicates that the composition is used to treat the selected condition. The prepared article may also include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and glucose solution. It may also include other materials required from a commercial and user standpoint, including a buffer, a diluent, a filter, a needle, a syringe and a drug package insert for instructions for use.

[0335] For the benefit of the skilled person, the invention will be illustrated by the following non-limiting examples, which describe some embodiments by which or in which the invention may be implemented. Example

[0336] Example 1: Sequencing of the Fv domain of mouse anti-human IL-13 monoclonal antibody clone B-B13

[0337] The reagents used in the following methods are mouse anti-IL-13 monoclonal antibody clone B-B13 purchased from Cell Sciences, Inc. (Canton, MA, USA). Cell Sciences is the manufacturer of antibody B-B13, Diaclone ( France)'s U.S. distributor.

[0338] The amino acid sequence of anti-IL-13 monoclonal antibody clone B-B13 was determined using a combination of Edman N-terminal sequencing technology and mass spectrometry analysis. The antibody was treated with the following different methods to produce polypeptide or peptide fragments, which were then fractionated using different methods to prepare samples for subsequent Edman N-terminal sequencing and liquid chromatography / mass spectrometry / mass spectrometry (LC-MS / MS) analysis, which was matched with peptides from a relevant protein sequence database.

[0339] SDS-Page of antibodies, with or without pyrogluamino treatment to separate heavy and light chains, followed by blotting onto polyvinylidene fluoride (PVDF) membranes and Edman N-terminal sequencing of the bands.

[0340] The antibodies were partially digested with a specific protease, followed by SDS-Page and blotting onto PVDF membranes and Edman N-terminal sequencing of the bands.

[0341] Limited chemical excision of the entire antibody or heavy and light chain SDS-Page gel bands was performed, followed by SDS-Page and blotting to PVDF membrane and Edman N-terminal sequencing of the bands.

[0342] The whole antibody or the heavy and light chain SDS-Page gel bands were proteolytically digested with specific proteases and analyzed by LC / MS / MS.

[0343] The heavy and light chain SDS-Page gel bands were proteolytically hydrolyzed with specific proteases, followed by fractionation by reverse phase high pressure chromatography (rp-hplc), and each fraction was subjected to Edman N-terminal sequencing and LC / MS / MS analysis.

[0344] The antibody was subjected to limited proteolysis with papain, and the Fd (antibody heavy chain VH-CH1 fragment) gel band was fractionated by SDS-Page, proteolytically hydrolyzed with a specific protease, and reverse-phase high performance liquid chromatography (rp-hplc) was performed, and each fraction was analyzed by Edman N-terminal sequencing and LC / MS / MS.

[0345] Example 2: Fv domain sequencing of mouse anti-human IL-4 monoclonal antibody clone 8D4-8

[0346] Reagents Mouse anti-IL-4 monoclonal antibody clone 8D4-8 was purchased from Biozol diagnostica Vertrieb GmbH (Eching, Germany). Biozol is the German distributor of BioLegend (San Diego, CA, USA), which produces the 8D4-8 antibody.

[0347] The amino acid sequence of mouse anti-IL-4 monoclonal antibody (clone 8D4-8) was determined using a combination of Edman sequencing and mass spectrometry (Pham et al., 2006, Anal. Biochem. 352: 77-86; Roberts et al., 2005, Anal. Chem. 67: 3613-25). Briefly, the antibody was first separated into light and heavy chains, and each chain was then cleaved with a sequence-specific protease or chemical method. The resulting peptides were separated by reverse phase chromatography and analyzed by matrix-assisted laser desorption / ionization mass spectrometry (MALDI) and / or LC-MS / MS. Unique peptides as well as complete heavy and light chains were then subjected to Edman sequencing to accurately determine the sequence of the protein.

[0348] Example 3: Humanization of the Fv domain of mouse anti-human IL-13 monoclonal antibody clone B-B13

[0349] The humanization protocol described here was used to achieve humanization of the B-B13 clone. Six humanization formats were proposed, including mutations in the CDRs to address problematic residues (deamidation sites, solvent exposed methionines, acid labile positions).

[0350] The VL & VH sequences of B-B13 were blasted against the July 2007 version of the Protein Data Bank (PDB). Most similar light and heavy chain amino acid sequences were retrieved. The most homologous variable light chain was found to be 1EGJ. The most homologous variable heavy chain was found to be 1FNS. The 1EGJ & 1FNS structures were used to construct a homology model of the variable domains, which was then energy minimized using standard procedures implemented in the Molecular Operating Environment (MOE). MOE is a comprehensive set of computer-aided drug design software provided by the Chemical Computing group. The 3D homology model of B-B13 was then subjected to 1.7 nanosecond molecular dynamics (MD) calculations in the generalized Born implicit solvent. The root mean square deviation (rmsd) distribution of each B-B13 amino acid was then calculated using the resulting 1,700 MD trajectory snapshots compared to the reference medoid position. Finally, a statistical test was used to compare the root mean square deviation distribution of each amino acid with the global root mean square deviation distribution to determine whether the amino acid is flexible enough from the MD calculation to be considered as interacting with the B cell receptor and thus being responsible for the activation of the immune response. The flexible positions of the mouse B-B13 variable region were compared with the corresponding positions of human antibody sequences downloaded locally from the January 2007 version of the ImMunoGeneTics database. Only those residues with a flexibility greater than three times the average value and some flanking residues that preserved the 3D structure of these flexible residues were retained for the search. The human antibody variable region with the most identical flexible residues was selected, with special consideration given to the CDRs. The positions within the range of 500 mM to 100 mM were selected to replace the flexible residues in the variable region of the mouse B-B13 antibody. Some mutations in the CDRs were also included in the proposed format to avoid problematic residues. The following sequence motifs were studied: Asp-Pro (acidic unstable bond), Asn-X-Ser / Thr (glycosylation), Asn-Gly / Ser / Thr (deamidation site in the exposed region), Met (oxidation in the exposed region). The generated humanized sequences were blasted in the UniProtKB / Swiss-Prot database for sequence similarity, and the confidence level that a reasonable assumption had been made was obtained. It was found that all sequences showed a high degree of similarity with human antibody numbers. In addition, all sequences did not contain any known B- or T-cell epitopes listed in the Immune Epitope Database and Analysis Resource (IEDB database).

[0351] Three forms were proposed for the heavy chain (H1, H2, H3) and three forms were proposed for the light chain (L1, L2 and L3). The three forms of the light chain were obtained from CAA83271.1 (Genebank accession number CAA83271). The L1 form contains 4 mutations. The L2 form contains one additional mutation to remove the DP site (Pro99) in CDR3. Compared to L2, L3 has two additional mutations added to the CDRs, which are 2 putative deamidation sites (N34Q, N96A). The H1, H2 and H3 forms of the heavy chain were obtained from CAC39364.1 (Genebank accession number CAC39364). This template is not the highest scoring template, but it is the highest scoring template that does not contain high homology (>70%) to known immunogen sequences. The H1 form contains 6 mutations and the H2 sequence has two additional mutations added to resolve three deamidation sites (N60A, N73T and N83T). The sequence numbering of the amino acids reflects their natural order in the protein (N-terminus to C-terminus). H3 contains two additional mutations that are thought to improve potency (Y100R & D106K). Six combinations of VL and VH variants are recommended to generate humanized antibodies: VL1xVH1, VL2xVH2, VL1xVH3, VL3xVH1, VL3xVH2 and VL3xVH3. As shown in Table 1, the amino acids in the humanized B-B13 VL and VH variants were changed using the resurfacing techniques given in the detailed description section of this application. The left column shows the original amino acids and their positions in the mouse B-B13 mAb.

[0352] Table 1

[0353]

[0354] Example 4: Humanization of the Fv domain of mouse anti-human IL-4 monoclonal antibody clone 8D4-8

[0355] Humanization of the 8D4-8 clone was achieved using the humanization (resurfacing) techniques described herein above. Two humanized forms were prepared. One form included a mutation in the CDR of the heavy chain that was thought to resolve a problematic residue (an exposed acidic labile position).

[0356] The VL & VH sequences of 8D4-8 were blasted against the July 2007 version of the PDB. Most similar light and heavy chain amino acid sequences were retrieved. The most homologous variable light chain was found to be 1YDJ. The most homologous variable heavy chain was found to be 1IQW. The 1YDG & 1IQW structures were used to build a homology model of the variable domains, which was then energy minimized using standard procedures implemented in MOE. The 3D homology model of 8D4-8 was then subjected to 1.7 nanosecond molecular dynamics (MD) calculations in the generalized Born implicit solvent. The root mean square deviation (rmsd) distribution was then calculated for each 8D4 amino acid compared to the reference medoid position using the resulting 1,700 MD trajectory snapshots. Finally, a statistical test was used to compare the rmsd distribution of each amino acid to the global rmsd distribution to determine whether the amino acid was sufficiently flexible from the MD calculations to be considered as potentially interacting with the B cell receptor and thus responsible for the activation of an immune response. The flexible positions of the mouse 8D4-8 variable region were compared with the corresponding positions of human antibody sequences downloaded from the ImMunoGeneTics database in January 2007. Only residues with a flexibility greater than three times the average and some flanking residues that preserved the 3D structure of these flexible residues were retained for the search. The human antibody variable region with the most identical flexible residues was selected, with special consideration given to residues located in the CDRs. The positions within the range were selected to replace the flexible residues in the variable region of the mouse 8D4-8 antibody. Finally, some additional mutations were made to avoid problematic residues. The following sequence motifs were studied: Asp-Pro (acidic unstable bond), Asn-X-Ser / Thr (glycosylation), Asn-Gly / Ser / Thr (deamidation site in exposed regions), Met (oxidation in exposed regions). The only problematic residue found was the DP site in the heavy chain CDR2. The generated humanized sequences were blasted against the UniProtKB / Swiss-Prot database for sequence similarity, and a confidence level was obtained that a reasonable assumption had been made. All sequences showed a high degree of similarity to many human antibodies. In addition, all sequences did not contain any known B cell or T cell epitopes listed in the IEDB database.

[0357] It is suggested that two forms (H1, H2) are obtained for the heavy chain and one form (L1) is obtained for the light chain. The L1 form of the light chain is obtained from BAC01676.1 (Genebank accession number BAC01676). The L1 form contains 3 mutations. The H1 and H2 forms of the heavy chain are obtained from BAC02418.1 (Genebank accession number BAC02418). The H1 form contains 9 mutations and the H2 form contains an additional mutation to remove the DP site (Pro53) in CDR2. Two combinations, VL1xVH1 and VL1xVH2, were prepared.

[0358] Table 2 shows the amino acid changes made to the humanized 8D4-8 VL and VH variants using humanization (resurfacing) techniques. The left column shows the original amino acids and their positions in the murine 8D4-8 mAb.

[0359] Table 2

[0360]

[0361] Example 5: Cloning and Generation of Chimeric Anti-IL-13 Clone B-B13 Monoclonal Antibody, Chimeric Anti-IL-4 Clone 8D4-8 Monoclonal Antibody, and Humanized Variants

[0362] The amino acid sequences of the variable heavy and light chains of the anti-IL-13 clone B-B13 and the anti-IL-4 clone 8D4-8 were reverse translated into nucleotide sequences and generated separately using a modified overlap extension PCR (OE-PCR) protocol described by Young L. and Dong Q. (Nucl. Acids Res. (2004), 32(7), e59). The PCR products were cloned into 4-TOPO, and sequenced with M13 forward and M13 reverse primers. The variable domains were fused to the constant heavy chain (IGHG1, Genebank accession number Q569F4) or light chain (IGKC) Genebank accession number Q502W4), digested with NheI and HindIII, and each domain was ligated to the NheI / HindIII site of the episomal expression vector pXL, an analog of the pTT vector described in Durocher et al. (2002), Nucl. Acids Res. 30 (2), E9, thereby generating chimeric B-B13 heavy and light chains and chimeric 8D4-8 heavy and light chains mammalian expression plasmids.

[0363] Expression clones encoding humanized variants of anti-IL-13 clone B-B13 and anti-IL-4 clone 8D4-8 were also synthetically generated by overlap extension PCR (OE-PCR) based on the proposed amino acid exchanges of the original sequences.

[0364] The expression plasmids encoding the heavy and light chains of the antibody were propagated in E. coli DH5a. The plasmids used for transfection were prepared from E. coli using the Qiagen EndoFree Plasmid Mega Kit.

[0365] For transfection, 3 x 10 cells / mL were cultured in 100 mL volume of serum-free FreeStyle medium (Invitrogen) in a 500 mL shake flask. 5 HEK293 FreeStyle cells (Invitrogen) were seeded at 10 cells / mL. Cells were cultured in an incubator at 37°C with 8% CO2 humidified air on an orbital shaker platform rotating at 110 rpm.

[0366] Three days after inoculation, the cells were counted using a CASY electronic cell counter ( The number of viable cells and total cells was determined by ELISA System GmbH. Cells with a viability greater than 90% were used for transfection at a cell density of 1-1.5 x 10 6 cells / mL. 100mL of cells were cultured in a mixture of heavy and light chain expression plasmids (5x10 -7 Transfection was performed using FugeneHD (Roche) in 500 mL shake flasks containing 1 μg DNA / cell under the conditions specified by the manufacturer, with a DNA:FugeneHD ratio of 2:7. The transfected cells were cultured in a 37°C incubator (8% CO2) on an orbital shaker platform rotating at 110 rpm for 7 days.

[0367] Goat anti-human IgG (Fc specific) [NatuTecA80-104A] was coated on a Nunc F96-MaxiSorp-Immuno plate. The antibody was diluted to 10ug / ml in carbonate coating buffer (50mM sodium carbonate pH 9.6) and 50uL was dispensed into each well. The plate was sealed with tape and stored overnight at 4°C. The plate was washed three times with wash buffer (PBS pH 7.4, 0.1% Tween20). 150uL of blocking solution (1% BSA / PBS) was dispensed into each well to cover the plate. After 1 hour at room temperature, wash three times with wash buffer. Add 100uL of sample or standard (ranging from 1500ng / ml to 120ng / ml) and leave at room temperature for 1 hour. Wash the plate three times with wash buffer. 100uL goat anti-human IgG-FC–HRP conjugate [NatuTec A80-104P-60] diluted 1:10.000 in incubation solution (0.1% BSA, PBS pH 7.4, 0.05% Tween20) was added. After 1 hour incubation at room temperature, the plate was washed three times with wash buffer. 100uL ABTS substrate (10mg ABTS tablet (Pierce34026) dissolved in ml of 0.1M Na2HPO4, 0.05M citric acid solution, pH 5.0. 10uL 30% H2O2 / 10ml substrate buffer was added before use) was dispensed into each well and allowed to develop color. After color development (approximately 10 to 15 minutes), 50uL of 1% SDS solution was added to stop the reaction. The plate was read at A405.

[0368] Protein in protein A ( HiTrap TM Protein A HP Columns , GE Life Sciences) by affinity chromatography. After elution with 100mM acetate buffer pH 3.5 containing 100mM NaCl, the monoclonal antibody was formulated in PBS solution and filtered through 0.22μm. The protein concentration was determined by measuring the absorbance at 280nm. Each batch was analyzed on an Agilent 2100 bioanalyzer under reducing and non-reducing conditions using the Protein200Plus LabChip kit to determine the purity and molecular weight of each subunit and monomer.

[0369] Example 6: Characterization of humanized anti-IL-13 clone B-B13 variants and humanized anti-IL-4 clone 8D4-8 variants

[0370] Recombinant human IL-13 and IL-4 reagents were purchased from Chemicon (USA). Biacore kinetic analysis was performed as follows.

[0371] The purified antibodies were characterized in detail kinetically on a Biacore 3000 (GE Healthcare) using surface plasmon technology. A capture assay was used that used a species-specific antibody (e.g., human-Fc specific MAB 1302, Chemicon) to capture and orient the antibody of interest. The capture antibody was immobilized on a research-grade CM5 chip (GE Life Science) via primary amines (10,000 RU) using standard procedures. The antibody analyzed was captured and the RU value was adjusted to result in a maximum of 30 RU analyte binding at a flow rate of 10 μl / min. The binding kinetics were measured relative to recombinant IL-4 and IL-13 over a range of HBS EP concentrations between 0 and 50 nM (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) at a flow rate of 30 μl / min. The chip surface was regenerated with 10 mM glycine pH 2.5. Kinetic parameters were analyzed and calculated in the BIA Evaluation Program package using a flow cell without captured antibody as a reference. To study additive binding of two antigens, a wizard-driven co-injection approach was applied, where one antigen was injected immediately followed by an injection of the IL-13 / IL-4 antigen mixture.

[0372] The biological activity of the antibodies of the invention was measured by measuring the inhibition of IL-4 or IL-13 mediated cell proliferation in TF-1 cells. Briefly, Applicants stimulated the growth of TF-1 cells with IL-4 or IL-13. TF-1 is a cell line that is dependent on cytokines for growth and is responsive to many cytokines, including IL-4 and IL-13. The induced growth (compared to baseline conditions in the absence of cytokines) then represents the biological activity of IL-4 or IL-13. Anti-IL-4, anti-IL-13 and bispecific anti-IL-4 / IL-13 antibodies were shown to block IL-4 or IL-13 induced growth of TF-1 cells. In addition, bispecific anti-IL-4 / IL-13 antibodies have been shown to block TF-1 cell proliferation induced by combined IL-4 and IL-13 stimulation. The blocking effect was measured in a dose-dependent manner to generate an IC50 (antibody concentration at 50% inhibition) as the neutralizing ability of the antibody to its target, i.e., IL-4 or IL-13. The details of the method used are described in more detail below.

[0373] TF-1 cells (ATCC, CRL-2003) were maintained in complete medium (DMEM with high glucose, 25 mM Hepes buffer and glutamine, 10% FBS, 1x P / S, 1 mM sodium pyruvate) with freshly added hGM-CSF (final concentration 4 ng / ml). 24 hours before IL-13 (15 ng / ml) or IL-4 (1 ng / ml) treatment. Cells were seeded at 0.05x106 / ml in complete medium without hGM-CSF in 96-well plates. Serial dilutions of antibodies with the corresponding cytokines were preincubated at 37°C for 30 minutes before addition to the cells. Cells were cultured for 72 hours (37°C, 5% CO2). MTS / PMS solution of cellTiter96Aqueous was added. Cells were then incubated for 3 hours. After this period, the absorbance was recorded at 490 nm using a culture plate reader. IC50 values ​​were calculated using Speed ​​software.

[0374] The binding kinetics and neutralization activities of the humanized B-B13 variants are shown in Table 3 (nt, not measured).

[0375] Table 3

[0376]

[0377] A humanized B-B13 variant, huB-B13 VL3xVH2, has significantly higher affinity than the original mouse B-B13 (13-fold) and chimeric B-B13 (6-fold). When these humanized anti-IL-13 antibodies are used to treat asthma patients, the improved affinity can lead to increased efficacy and effectiveness. In addition, when used in humans, humanized antibodies can have reduced immunogenicity compared to mouse antibodies or chimeric antibodies.

[0378] The binding kinetics and neutralization activities of the humanized 8D4-8 variants are shown in Table 4.

[0379] Table 4

[0380]

[0381] A humanized 8D4-8 variant, hu8D4-8 VL1xVH1, has significantly higher affinity than the original murine 8D4-8 (11-fold) and chimeric 8D4-8 (2-fold). The improved affinity may lead to increased potency and efficacy when this humanized anti-IL-4 antibody is used to treat asthma patients. In addition, when used in humans, humanized antibodies may have reduced immunogenicity compared to murine or chimeric antibodies.

[0382] Example 7: Cloning and production of humanized anti-IL-4 / IL-13 bispecific antibodies

[0383] The form used to express bispecific antibodies (BsAb) is an IgG variant of the dual-domain double-head form described in US5,989,830. In this form, the IgG molecule is extended by another variable domain of the second antibody at the N-terminus of its corresponding heavy chain and light chain. Therefore, the obtained IgG molecule is a heterotetramer consisting of two heavy chains and two light chains. The heavy chain consists of two variable heavy domains (VH1-VH2), from two different antibodies connected together by a connector, and the connector consists of ten amino acids (G4S) 2, and is fused to the IgG4 constant domain. The light chain consists of two variable light domains (VL1-VL2), from two different antibodies connected together by a connector, and the connector consists of ten amino acids (G4S) 2, and is fused to the constant κ region.

[0384] The sequences of the variable heavy and light domains of the 8D4-8 variant were generated by PCR at their respective encoding (G4S)2-( GGA TCC) A BamHI restriction site was introduced into the 5' end of one part of -8D4-8 ( GGA TCC The 3' sequence of the 8D4-8 humanized variant VH ends with an ApaI restriction site (encoding the first amino acid of the CH1 domain) for subsequent fusion to the IGHG4 sequence (Q569F4 with a terminal Lys deletion and double mutations of S241P and L248E). The 3'-end of VL8D4-8 ends with a BsiWI restriction site encoding the first two amino acids of the constant kappa chain for subsequent fusion to IGKC (Genebank accession number Q502W4).

[0385] The sequences of the variable heavy and light domains of the B-B13 variant were generated by PCR in their respective encodings (G4S)2-(B-B13)-(GGA GGC GGA GGG TCC GGA GGC GGA GGA TCC A BamHI restriction site was introduced at the 3'-end of one portion of the B-B13 variant (SEQ ID NO:7). The VH and VL sequences of the B-B13 variant were generated at their respective 5'-ends by a NheI restriction site, followed by an ATG start codon and a leader peptide of the coding sequence.

[0386] The VH of B-B13 and 8D4-8 were fused together through their BamHI sites in a (G4S)2 linker. The VL of B-B13 and 8D4-8 were fused to each other through their BamHI sites in a (G4S)2 linker. Thus, the resulting tandem of heavy and light chains had the following composition.

[0387] Bispecific antibody heavy chain: NheI-leader peptide-VH-B-B13-(G4S)2-VH 8D4-8-ApaI.

[0388] Bispecific antibody light chain: NheI-leader peptide-VL-B-B13-(G4S)2-VL 8D4-8-BsiWI.

[0389] All PCR intermediate fragments were cloned into 4-TOPO and sequenced using M13 forward and M13 reverse primers.

[0390] After sequence confirmation, the heavy chain tandem was fused to the IGHG4 sequence through its ApaI site, and the variable light chain tandem was fused to IGKC through its BsiWI site. The resulting dual-domain heavy and light chains were digested with NheI and HindIII, and each was ligated into the NheI / HindIII sites of the episomal gene expression vector pXL, generating plasmids for mammalian expression of the TBTI heavy and light chains, respectively.

[0391] Four humanized bispecific anti-IL-4 / anti-IL-13 constructs were generated based on the combination of humanized VH and VL formats of B-B13 and 8D4-8 as shown in Table 5 below.

[0392] Table 5

[0393] Bispecific anti-IL-4 / anti-IL-13Ab Anti-IL-13Fv Anti-IL-4 Fv huTBTI3_1_1 B-B13 VL3xVH2 8D4-8 VL1xVH2 huTBTI3_2_1 B-B13 VL3xVH2 8D4-8 VL1xVH1 huTBTI3_1_2 B-B13 VL2xVH2 8D4-8 VL1xVH2 huTBTI3_2_2 B-B13 VL2xVH2 8D4-8 VL1xVH1

[0394] Example 8: Characterization of humanized bispecific antibodies

[0395] Binding and neutralization assays were performed as described in the Examples above.

[0396] The binding kinetics of the four humanized anti-IL-4 / IL-13 antibody variants are given in Table 6. All four bispecific antibody constructs bound to IL-4 and IL-13 with high affinity.

[0397] Table 6

[0398]

[0399] The neutralizing activities of the humanized anti-IL-4 / IL-13 bispecific antibody variants are summarized in Table 7. huTBTI3-1_1 and huTBTI3-2_1 completely neutralized IL-13 or IL-4-induced TF-1 cell proliferation, with IC50 values ​​shown below.

[0400] Table 7

[0401] Antibody IC50 (nM) in IL-13 assay IC50 (nM) in IL-4 assay huTBTI3-1_1 3.7 1.7 huTBTI3-2_1 4.1 0.32

[0402] It is well known that mutant IL-13 alleles are associated with asthma at high frequencies (Heinzmann A. et al., 2000, Hum Mol Genet 9, 4, pp. 549-559). Therefore, the binding kinetics of bispecific antibodies to mutant IL-13 proteins (human IL-13R112Q variant, PeproTech, Rocky Hill, NJ, USA) were studied. The results showed that the binding of huTBTI3-1_1 and huTBTI3-2_1 to IL-13 variants was similar to that to wild-type IL-13.

[0403] Table 8 shows the binding kinetics of humanized anti-IL-4 / IL-13 molecules to mutated IL-13 proteins.

[0404] Table 8

[0405]

Claims

1. An isolated humanized antibody or an antibody fragment thereof that specifically binds to IL-13, wherein the antibody or the antibody fragment comprises a light chain variable domain and a heavy chain variable domain, wherein the CDR1, CDR2 and CDR3 of the light chain are composed of the amino acid sequences RASESVDSYGQSYMH (SEQ ID NO: 8), LASNLES (SEQ ID NO: 9) and QQNAEDSRT (SEQ ID NO: 10), respectively, and the CDR1, CDR2 and CDR3 of the heavy chain are composed of the amino acid sequences GFSLTDSSIN (SEQ ID NO: 11), DGRID (SEQ ID NO: 12) and DGYFPYAMDF (SEQ ID NO: 13), respectively, and wherein the antibody fragment thereof is selected from the group consisting of: F ab 、F ab’ 、F (ab’)2 , sF v and scAb.

2. The antibody or antibody fragment thereof according to claim 1, wherein the light chain variable domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1, and the heavy chain variable domain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

2.

3. The antibody or antibody fragment thereof according to claim 1 or 2, wherein the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 1, and the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:

2. 4 . The antibody or antibody fragment according to claim 1 or 2 , wherein the antibody is a bispecific antibody or a bispecific antibody fragment thereof.

5. The antibody or antibody fragment of claim 4, wherein the bispecific antibody specifically binds to IL-13 and IL-4.

6. A pharmaceutical composition comprising the antibody or antibody fragment according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

7. Use of the antibody or antibody fragment thereof according to any one of claims 1 to 5 in the preparation of a medicament for treating an IL-13-mediated disease, wherein the IL-13-mediated disease is asthma.

8. A nucleic acid molecule encoding the antibody or antibody fragment according to any one of claims 1 to 5.

9. A method for preparing the antibody or antibody fragment according to any one of claims 1 to 5, comprising expressing the nucleic acid according to claim 8 in a host cell.

Images