Anti-IL-9 antibodies and methods of use thereof

Abstract

The present disclosure provides isolated antibodies that specifically bind to IL-9 (e.g., human IL-9 or murine IL-9). Also provided are pharmaceutical compositions that include these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating subjects with these antibodies.

Description

[Technical field]

[0001] (1. Field) The present disclosure relates to anti-IL-9 antibodies and methods of use thereof. [Background technology]

[0002] (2.Background) The protein interleukin-9 (IL-9) is a cytokine secreted by several different immune cells, including Th9 cells, type 2 innate lymphoid cells (ILC2), Th17 cells, mast cells, osteoblasts, NKT cells, and memory B cells. Binding of IL-9 to its receptors, including IL-9Rα, activates the associated Janus kinase (JAK)1 and JAK3, leading to activation of signal transducers and activators of transcription (STAT)1, STAT3, or STAT5 pathways, the mitogen-activated protein (MAP) kinase pathway, and the insulin-related substance (IRS) pathway.

[0003] This wide range of cellular sources implies a complex system of IL-9 expression, suggesting the involvement of IL-9 in many physiological conditions and diseases. Increased IL-9 signaling has been found to be involved in several inflammatory and autoimmune diseases, including asthma, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and inflammatory bowel disease. Furthermore, high levels of IL-9 signaling have also been found to promote the survival and proliferation of cancer cells, including melanoma and blood cancers, such as lymphoma and Hodgkin's disease.

[0004] Therefore, therapeutic agents designed to antagonize IL-9 activity are highly desirable. Summary of the Invention

[0005] (3. Overview) The present disclosure provides antibodies that specifically bind to IL-9 (e.g., human IL-9) and antagonize IL-9 activity. Also provided are pharmaceutical compositions that include these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating subjects with these antibodies.

[0006] 1. An isolated antibody that specifically binds to human IL-9, comprising a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1 comprises the amino acid sequence set forth in SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, or 122; CDRH2 comprises the amino acid sequence set forth in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, or 123; The antibody, wherein RH3 comprises the amino acid sequence set forth in SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, or 124; CDRL1 comprises the amino acid sequence set forth in SEQ ID NO: 22, 25, 28, 31, 33, 36, 39, or 125; CDRL2 comprises the amino acid sequence set forth in SEQ ID NO: 23, 26, 29, 34, 37, 40, or 126; and CDRL3 comprises the amino acid sequence set forth in SEQ ID NO: 24, 27, 30, 32, 35, 38, 41, or 127.

[0007] In one aspect, the present disclosure provides an isolated antibody that specifically binds to human IL-9, (a) a VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence of SEQ ID NO: 42, 43, 44, 45, 46, 47, 48, 49, or 128; and / or (b) a VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequences of SEQ ID NOs: 50, 51, 52, 53, 54, 55, 56, or 129. The antibody comprises:

[0008] In embodiments, CDRH1, CDRH2 and CDRH3 comprise the CDRH1, CDRH2 and CDRH3 amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3; 4, 5, and 6; 7, 8, and 9; 10, 11, and 12; 13, 14, and 15; 16, 17, and 18; 19, 20, and 21; or 122, 123, and 124, respectively.

[0009] In embodiments, CDRL1, CDRL2 and CDRL3 comprise the CDRL1, CDRL2 and CDRL3 amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24; 25, 26, and 27; 28, 29, and 30; 31, 29, and 32; 33, 34, and 35; 36, 37, and 38; 39, 40, and 41; or 125, 126, and 127, respectively.

[0010] In embodiments, the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 1, 2, 3, 22, 23, and 24; 4, 5, 6, 25, 26, and 27; 7, 8, 9, 28, 29, and 30; 7, 8, 9, 31, 29, and 32; 10, 11, 12, 33, 34, and 35; 13, 14, 15, 36, 37, and 38; 16, 17, 18, 39, 40, and 41; 19, 20, 21, 36, 37, and 38; or 122, 123, 124, 125, 126, and 127, respectively.

[0011] In embodiments, the antibody comprises a VH amino acid sequence of SEQ ID NO: 42, 43, 44, 45, 46, 47, 48, 49, or 128. In embodiments, the amino acid sequence of the VH consists of the amino acid sequence of SEQ ID NO: 42, 43, 44, 45, 46, 47, 48, 49, or 128.

[0012] In embodiments, the antibody comprises a VL amino acid sequence of SEQ ID NO: 50, 51, 52, 53, 54, 55, 56, or 129. In embodiments, the amino acid sequence of the VL consists of the amino acid sequence of SEQ ID NO: 50, 51, 52, 53, 54, 55, 56, or 129.

[0013] In one aspect, the disclosure provides an isolated antibody that specifically binds to human IL-9, the antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 42, 43, 44, 45, 46, 47, 48, 49, or 128, and a VL comprising the amino acid sequence of SEQ ID NO: 50, 51, 52, 53, 54, 55, 56, or 129.

[0014] In embodiments, the amino acid sequence of the VH consists of the amino acid sequence of SEQ ID NO: 42, 43, 44, 45, 46, 47, 48, 49, or 128, and the amino acid sequence of the VL consists of the amino acid sequence of SEQ ID NO: 50, 51, 52, 53, 54, 55, 56, or 129.

[0015] In an embodiment, the antibody comprises the VH and VL amino acid sequences of SEQ ID NOs: 42 and 51; 43 and 55; 44 and 54; 45 and 53; 46 and 50; 47 and 52; 48 and 50; 49 and 56; or 128 and 129. In an embodiment, the VH and VL amino acid sequences consist of the amino acid sequences of SEQ ID NOs: 42 and 51; 43 and 55; 44 and 54; 45 and 53; 46 and 50; 47 and 52; 48 and 50; 49 and 56; or 128 and 129, respectively.

[0016] In one aspect, the disclosure provides an isolated antibody that specifically binds to human IL-9, the antibody binding to at least residue R91 of human IL-9 upon binding to IL-9. In an aspect, the disclosure provides an isolated antibody that specifically binds to one or more amino acid(s) of human IL-9 selected from the group consisting of R84, Y85, P86, L87, I88, F89, S90, R91, and K94. In an aspect, the disclosure provides an isolated antibody that specifically binds to one or more amino acid(s) of human IL-9 selected from the group consisting of L87, I88, R91, K94, S95, and V98. In an aspect, the disclosure provides an isolated antibody that specifically binds to one or both amino acid(s) of human IL-9 selected from the group consisting of I88 and R91. In an aspect, the disclosure provides an isolated antibody that specifically binds to one or more amino acid(s) of mouse IL-9 selected from the group consisting of R84, P87, V88, H90, R91, R94, I95, V98, and L99.

[0017] In embodiments, the antibody comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0018] In an embodiment, the antibody comprises a heavy chain constant region that is a mutant of a wild-type heavy chain constant region, and the mutant heavy chain constant region binds to FcγR with a higher affinity than the wild-type heavy chain constant region binds to FcγR. In an embodiment, the FcγR is FcγRIIB or FcγRIIIA.

[0019] In an embodiment, the amino acid at position 297 of the heavy chain constant region is A or Q according to the EU numbering system. In an embodiment, the amino acids at positions 234 and 235 of the heavy chain constant region are both A according to the EU numbering system. In an embodiment, the amino acids at positions 433, 434, and 436 of the heavy chain constant region are K, F, and Y, respectively, according to the EU numbering system. In an embodiment, the amino acids at positions 252, 254, and 256 of the heavy chain constant region are Y, T, and E, respectively, according to the EU numbering system. In an embodiment, the amino acids at positions 428 and 434 of the heavy chain constant region are L and S, respectively, according to the EU numbering system. In an embodiment, the amino acids at positions 309, 311, and 434 of the heavy chain constant region are D, H, and S, respectively, according to the EU numbering system.

[0020] In one embodiment, the present disclosure provides an isolated antibody that cross-competes with an antibody disclosed herein for binding to human IL-9. In one embodiment, the present disclosure provides an isolated antibody that binds to the same epitope of human IL-9 as an antibody disclosed herein.

[0021] In one embodiment, the antibody inhibits the binding of human IL-9 to human IL-9Rα.In one embodiment, the antibody binds to human IL-9 with a KD of less than 1 nM.In one embodiment, the antibody is bispecific.In one embodiment, the antibody is conjugated to a cytotoxic agent, a cytostatic agent, a toxin, a radionuclide, or a detectable label.

[0022] In one aspect, the disclosure provides an isolated polynucleotide encoding the VH and / or VL, or the heavy and / or light chains, of an isolated antibody disclosed herein.

[0023] In one aspect, the disclosure provides a vector comprising a polynucleotide disclosed herein.

[0024] In one aspect, the present disclosure provides: (a) a polynucleotide disclosed herein; (b) a vector disclosed herein; (c) a polynucleotide encoding the VH and VL, or heavy and light chains, of an isolated antibody disclosed herein; (d) a vector comprising a polynucleotide encoding the VH and VL, or heavy and light chains, of an isolated antibody disclosed herein; (e) a first polynucleotide encoding the VH or heavy chain of an isolated antibody disclosed herein, and a second polynucleotide encoding the VL or light chain of an isolated antibody disclosed herein; or (f) a first vector comprising a first polynucleotide encoding the VH or heavy chain of an isolated antibody disclosed herein, and a second vector comprising a second polynucleotide encoding the VL or light chain of an isolated antibody disclosed herein. A recombinant host cell comprising the

[0025] In one aspect, the disclosure provides a pharmaceutical composition comprising an isolated antibody disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, or a host cell disclosed herein, and a pharma- ceutical acceptable carrier or excipient.

[0026] In one aspect, the disclosure provides a method of producing an isolated antibody, the method comprising culturing a host cell disclosed herein under suitable conditions such that the polynucleotide is expressed to produce the isolated antibody.

[0027] In one aspect, the disclosure provides a method of producing an isolated antibody, comprising: (a) a first polynucleotide encoding the VH of an antibody disclosed herein, and a second polynucleotide encoding the VL of an antibody disclosed herein; or (b) a first polynucleotide encoding a heavy chain of an antibody disclosed herein, and a second polynucleotide encoding a light chain of an antibody disclosed herein. in a cell under suitable conditions such that the polynucleotide is expressed, and producing the antibody.

[0028] In one aspect, the disclosure provides a method of antagonizing the interaction of human or mouse IL-9 with an IL-9 receptor in a subject, the method comprising administering to the subject an effective amount of an isolated antibody disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein.

[0029] In one aspect, the disclosure provides a method of treating an inflammatory disease in a subject, the method comprising administering to the subject an effective amount of an isolated antibody disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein.

[0030] In one aspect, the disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an isolated antibody disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein.

[0031] In one aspect, the disclosure provides a method of treating an autoimmune disease in a subject, the method comprising administering to the subject an effective amount of an isolated antibody disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein.

[0032] In embodiments, the isolated antibody, polynucleotide, vector, host cell, or pharmaceutical composition is administered systemically, intravenously, subcutaneously, intratumorally, or delivered to a tumor-draining lymph node. In embodiments, the method further comprises administering to the subject an additional therapeutic agent. [Brief description of the drawings]

[0033] (4. BRIEF DESCRIPTION OF THE DRAWINGS) [Figure 1] 1A and 1B are graphs showing the activity of anti-IL9 mAbs. Fig. 1A is a graph showing the neutralizing activity of an initial batch of anti-IL9 mAbs. Fig. 1B is a graph showing the neutralizing activity of a second batch of anti-IL9 mAbs.

[0034] [Diagram 2] FIG. 2 is a graph showing the affinity of anti-IL9 mab by Biolayer Interferometry (BLI) analysis.

[0035] [Diagram 3] FIG. 3 is a schematic showing the in vivo experimental design for testing anti-IL-9 mAbs in a mouse model of asthma.

[0036] [Figure 4] Figures 4A-F depict Fab:IL-9 complexes. Figure 4A depicts Fab 6D3:hIL-9 complex. Figure 4B depicts Fab 6E2:hIL-9 complex. Figure 4C depicts Fab 7D6:hIL-9 complex. Figure 4D is an overlay of Fab 6D3:hIL-9 complex, Figure 4E is an overlay of Fab 6E2:hIL-9 complex, and Figure 4F is an overlay of Fab 7D6:hIL-9 complex. Figures 4D, 4E, and 4F are each overlays of the Fab:hIL-9 complex and the hIL-9:hIL-9Rα complex based on a structural superposition of hIL-9 in front and top views of the binding of the C helix by the Fab. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] (5. Detailed Description) The present disclosure provides isolated anti-IL-9 antibodies. Also provided are pharmaceutical compositions that include these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating subjects with these antibodies.

[0038] (5.1 Definition) As used herein, the term "IL-9" refers to interleukin-9, which is encoded by the IL9 gene in humans and mice. As used herein, the term "human IL-9" or "mouse IL-9" refers to the IL-9 protein encoded by the wild-type IL9 gene (e.g., GenBank™ Accession Nos. NM_000590.2 (human) or NM_008373.2 (mouse)). An exemplary amino acid sequence of a human IL-9 protein is provided as SEQ ID NO:57. An exemplary sequence of a mouse IL-9 protein is provided as SEQ ID NO:138. Table 1. Exemplary IL-9 Amino Acid Sequences [Table 1]

[0039] As used herein, the terms "antibody" and "antibodies" include full length antibodies, antigen-binding fragments of full length antibodies, and molecules comprising the CDR, VH region, and / or VL region of an antibody. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelized antibodies, affibodies, Fab fragments, F(ab') 2Antibodies include, but are not limited to, antibodies, fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and antigen-binding fragments of any of the above. In certain embodiments, the antibodies described herein refer to polyclonal antibody populations. Antibodies include any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY) and any class (e.g., IgG, IgE, IgM, IgD, IgA, or IgY) of immunoglobulin molecule. 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , or IgA 2 ), or any subclass (e.g., IgG 2 a or IgG 2 In certain embodiments, the antibodies described herein can be IgG antibodies, or antibodies of a class thereof (e.g., human IgG 1 or IgG 4 ) or subclass. In embodiments, the antibody is a humanized monoclonal antibody. In embodiments, the antibody is a human monoclonal antibody.

[0040] A "multispecific antibody" is an antibody that specifically binds to two or more different antigens or two or more different regions of the same antigen (e.g., a bispecific antibody). Multispecific antibodies include bispecific antibodies that contain two different antigen-binding sites (excluding the Fc region). Multispecific antibodies include, for example, recombinantly produced antibodies, human antibodies, humanized antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing two heavy chain molecules and two light chain molecules, antibody light chain monomers, heteroconjugate antibodies, linked single chain antibodies or linked single chain Fvs (scFvs), camelized antibodies, affibodies, linked Fab fragments, F(ab') 2 Multispecific antibodies can include any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY) and any class (e.g., IgG, IgE, IgM, IgD, IgA, or IgY) of immunoglobulin molecule. 1 , IgG 2 , IgG 3, IgG 4 , IgA 1 , or IgA 2 ), or any subclass (e.g., IgG 2 a or IgG 2 In embodiments, the multispecific antibodies described herein can be of the IgG antibody or class thereof (e.g., human IgG 1 , IgG 2 , or IgG 4 ) or a subclass.

[0041] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen-binding sites found within the variable regions of heavy and light chain polypeptides. These particular regions are described, for example, by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest (1991), by Chothia et al., J. Mol. Biol. 196:901-917 (1987), and by MacCallum et al., J. Mol. Biol. 262:732-745 (1996), all of which are fully incorporated herein by reference, and in which the definitions include overlapping or subsets of amino acid residues when compared to each other. In some embodiments, the term "CDR" refers to the CDRs defined by MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A., Protein Sequence and Structure Analysis of Antibody Variable Domains, in Antibody Engineering, Kontermann and Dubel (eds.), Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In some embodiments, the term "CDR" refers to the CDRs defined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest (1991). In some embodiments, the heavy chain CDRs and light chain CDRs of an antibody are defined using different rules.In certain embodiments, the heavy and / or light chain CDRs are defined by performing a structural analysis of the antibody to identify residues in the variable region(s) that are predicted to contact the epitope region of the target molecule (e.g., human and / or mouse IL-9). CDRH1, CDRH2, and CDRH3 refer to the heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 refer to the light chain CDRs.

[0042] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. A variable region usually refers to a portion of an antibody, generally a light or heavy chain portion, usually about the amino-terminal 110-120 amino acids or 110-125 amino acids in the mature heavy chain and about 90-115 amino acids in the mature light chain, which vary widely in sequence between antibodies and are used in the binding and specificity of a particular antibody to its particular antigen. The sequence variability is concentrated in regions called complementarity determining regions (CDRs), while the more highly conserved regions in the variable region are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or mouse CDRs and human framework regions (FRs). In an embodiment, the variable region is a primate (e.g., non-human primate) variable region. In an embodiment, the variable region comprises rodent or mouse CDRs and a primate (e.g., non-human primate) framework region (FR).

[0043] As used herein, the terms "VH" and "VL" refer to antibody heavy and light chain variable regions, respectively, as described in Kabat et al., (1991) Sequences of protein of immunological interest (NIH Publication No. 91-3242, Bethesda), which is incorporated herein by reference in its entirety.

[0044] As used herein, the term "constant region" is common in the art. The constant region is the portion of an antibody that is not directly involved in binding an antibody to an antigen but can exhibit various effector functions, such as interaction with Fc receptors, e.g., the carboxyl-terminal portion of the light and / or heavy chains.

[0045] As used herein, the term "heavy chain" when used in reference to an antibody refers to an antibody of the IgG subclass, e.g., IgG 1 , IgG 2 , IgG 3 , and IgG 4 can refer to any of the different types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant region, which give rise to antibodies of the IgA, IgD, IgE, IgG, and IgM classes, respectively.

[0046] As used herein, the term "light chain" when used in reference to an antibody can refer to any of the distinct types, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant region. Light chain amino acid sequences are well known in the art. In an embodiment, the light chain is a human light chain.

[0047] As used herein, the terms "specifically bind", "specifically recognize", "immunospecifically bind", and "immunospecifically recognize" are similar terms in the context of antibodies and refer to a molecule that binds to an antigen (e.g., an epitope or immune complex) as such binding is understood by those of skill in the art. For example, a molecule that specifically binds to an antigen may generally bind to other peptides or polypeptides with lower affinity, as determined, for example, by immunoassays, Biacore™, KinExA 3000 instruments (Sapidyne Instruments, Boise, ID), or other assays known in the art. In embodiments, a molecule that specifically binds to an antigen has a K that is lower than the K that the molecule nonspecifically binds to another antigen.A At least 2 log (e.g., 10 log), 2.5 log, 3 log, 4 log or more greater than K A binds to the antigen.

[0048] As used herein, the term "EU numbering system" refers to the EU numbering rules for antibody constant regions as described in Edelman GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of protein of immunological interest, US Dept. Health and Human Services, 5th ed., 1991, each of which is incorporated herein by reference in its entirety.

[0049] As used herein, the terms "treat," "treating," and "treatment" refer to therapeutic or prophylactic measures as described herein. Methods of "treatment" utilize administration of an antibody to a subject having or susceptible to a disease or disorder to prevent, cure, delay, reduce the severity, or ameliorate one or more symptoms of the disease or disorder, or a recurrent disease or disorder, or to prolong the survival of the subject beyond that expected in the absence of such treatment.

[0050] As used herein, the term "effective amount" in the context of administration of a therapeutic agent to a subject refers to the amount of the therapeutic agent that achieves a desired prophylactic or therapeutic effect.

[0051] As used herein, the term "subject" includes any human or non-human animal. In some embodiments, the subject is a human or non-human mammal. In some embodiments, the subject is a human.

[0052] As used herein with respect to an antibody or polynucleotide, the term "isolated" refers to an antibody or polynucleotide that has been separated from one or more contaminants (e.g., polypeptides, polynucleotides, lipids, or sugars, etc.) that are present in the natural source of the antibody or polynucleotide. All instances of "isolated antibodies" described herein are further contemplated as antibodies that may, but do not have to be isolated. All instances of "isolated polynucleotides" described herein are further contemplated as polynucleotides that may, but do not have to be isolated. All instances of "antibodies" described herein are further contemplated as antibodies that may, but do not have to be isolated. All instances of "polynucleotides" described herein are further contemplated as polynucleotides that may, but do not have to be isolated.

[0053] The determination of "percent identity" between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90: 5873-5877, each of which is fully incorporated herein by reference. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al. (1990) J Mol Biol 215: 403, which is fully incorporated herein by reference. BLAST nucleotide searches can be performed, for example, with the NBLAST nucleotide program parameters set to score=100, word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed, for example, with XBLAST program parameters set to score 50, word length = 3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul SF et al. (1997) Nuc Acids Res 25: 3389-3402, which is incorporated herein by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterative search that detects distant relationships between molecules (ibid.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov).Another non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0054] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. When determining percent identity, typically only exact matches are counted.

[0055] (5.2 Anti-IL-9 antibody) In one aspect, the disclosure provides antibodies that specifically bind to IL-9 (e.g., human IL-9 or mouse IL-9). The amino acid sequences of the CDRs and VH / VL sequences of exemplary antibodies that specifically bind to IL-9 are set forth in Tables 2 and 3, respectively. Table 2. Amino acid sequences of the CDRs of exemplary anti-IL-9 antibodies [Table 2] Table 3. VH / VL amino acid sequences of exemplary anti-IL-9 antibodies [Table 3] TIFF2024522213000004.tif155170Table 4. VH and VL framework (FR) sequences of exemplary anti-IL-9 antibodies [Table 4]

[0056] In embodiments, the disclosure provides an isolated antibody that specifically binds to IL-9 (e.g., human IL-9 or murine IL-9), the antibody comprising a CDRH1, a CDRH2, and a CDRH3 listed in Table 2. In embodiments, the antibody comprises a CDRH1 listed in Table 2. In embodiments, the antibody comprises a CDRH2 listed in Table 2. In embodiments, the antibody comprises a CDRH3 listed in Table 2.

[0057] In embodiments, the disclosure provides an isolated antibody that specifically binds to IL-9 (e.g., human IL-9 or murine IL-9), the antibody comprising a VH domain that comprises one, two, or all three of the CDRs of a VH domain set forth in Table 3. In embodiments, the antibody comprises a CDRH1 of a VH domain set forth in Table 3. In embodiments, the antibody comprises a CDRH2 of a VH domain set forth in Table 3. In embodiments, the antibody comprises a CDRH3 of a VH domain set forth in Table 3.

[0058] In embodiments, the disclosure provides an isolated antibody that specifically binds to IL-9 (e.g., human IL-9 or murine IL-9), the antibody comprising a CDRL1, a CDRL2, and a CDRL3 as listed in Table 2. In embodiments, the antibody comprises a CDRL1 as listed in Table 2. In embodiments, the antibody comprises a CDRL2 as listed in Table 2. In embodiments, the antibody comprises a CDRL3 as listed in Table 2.

[0059] In embodiments, the disclosure provides an isolated antibody that specifically binds to IL-9 (e.g., human IL-9 or murine IL-9), the antibody comprising a VL domain that comprises one, two, or all three of the CDRs of a VL domain disclosed in Table 3. In embodiments, the antibody comprises a CDRL1 of a VL domain described in Table 3. In embodiments, the antibody comprises a CDRL2 of a VL domain described in Table 3. In embodiments, the antibody comprises a CDRL3 of a VL domain described in Table 3.

[0060] The individual CDRs of the antibodies disclosed herein may be determined according to any CDR numbering scheme known in the art.

[0061] In embodiments, one or more CDRs of an antibody disclosed herein can be determined with reference to Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest (1991), each of which is incorporated herein by reference in its entirety.

[0062] In embodiments, the present disclosure provides antibodies that specifically bind IL-9 (e.g., human IL-9 or murine IL-9) and comprise the CDRs of an antibody disclosed in Table 2 or Table 3 herein, as determined by the Kabat numbering scheme.

[0063] In embodiments, one or more CDRs of an antibody disclosed herein can be determined according to the Chothia numbering scheme, which refers to the location of the immunoglobulin structural loops (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226, all of which are incorporated herein by reference in their entireties).

[0064] In embodiments, the present disclosure provides antibodies that specifically bind IL-9 (e.g., human IL-9 or murine IL-9) and comprise the CDRs of an antibody disclosed in Table 2 or Table 3 herein, as determined by the Chothia numbering system.

[0065] In an embodiment, one or more CDRs of an antibody disclosed herein can be determined according to MacCallum RM et al., (1996) J Mol Biol 262: 732-745, which is incorporated herein by reference in its entirety. See also, for example, Martin A., Antibody Engineering, "Protein Sequence and Structure Analysis of Antibody Variable Domains," Kontermann and Dubel (eds.), Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001), which is incorporated herein by reference in its entirety.

[0066] In embodiments, the present disclosure provides antibodies that specifically bind IL-9 (e.g., human IL-9 or murine IL-9) and comprise the CDRs of an antibody disclosed in Table 2 or Table 3 herein, as determined by the MacCallum numbering system.

[0067] In embodiments, the CDRs of the antibodies disclosed herein can be determined in accordance with the IMGT numbering system described in Lefranc MP, (1999) The Immunologist 7: 132-136; Lefranc MP et al., (1999) Nucleic Acids Res 27: 209-212, each of which is incorporated by reference in its entirety; and Lefranc MP et al., (2009) Nucleic Acids Res 37: D1006-D1012.

[0068] In embodiments, the present disclosure provides antibodies that specifically bind IL-9 (e.g., human IL-9 or murine IL-9) and comprise the CDRs of an antibody disclosed in Table 2 or Table 3 herein, as determined by the IMGT numbering system.

[0069] In embodiments, the CDRs of the antibodies disclosed herein can be determined according to the AbM numbering scheme, which refers to the AbM hypervariable regions, which represent a compromise between the Kabat CDRs and the Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.), which is incorporated herein by reference in its entirety.

[0070] In embodiments, the present disclosure provides antibodies that specifically bind IL-9 (e.g., human IL-9 or murine IL-9) and comprise the CDRs of an antibody disclosed in Table 2 or Table 3 herein, as determined by the AbM numbering scheme.

[0071] In embodiments, the CDRs of the antibodies disclosed herein can be determined according to the AHo numbering system described in Honegger and Pluckthun A, J. Mol. Biol. 309:657-670 (2001), which is incorporated herein by reference in its entirety.

[0072] In embodiments, the present disclosure provides antibodies that specifically bind IL-9 (e.g., human IL-9 or murine IL-9) and comprise the CDRs of an antibody disclosed in Table 2 or Table 3 herein, as determined by the AHo numbering system.

[0073] In embodiments, the individual CDRs of the antibodies disclosed herein are each independently determined according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the multispecific molecule, which identifies residues in the variable region(s) that are predicted to contact the epitope region of IL-9.

[0074] In embodiments, the disclosure provides an antibody that specifically binds IL-9 (e.g., human IL-9 or murine IL-9), comprising a VH comprising the amino acid sequence of the CDRH1, CDRH2, and CDRH3 regions of the VH set forth in SEQ ID NO: 42, 43, 44, 45, 46, 47, 48, 49, or 128, and a VL comprising the amino acid sequence of the CDRL1, CDRL2, and CDRL3 regions of the VL set forth in SEQ ID NO: 50, 51, 52, 53, 54, 55, 56, or 129, wherein each CDR is determined according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or independently by structural analysis of the multispecific molecule, wherein the structural analysis identifies residues in the variable region(s) that are predicted to contact an epitope region of IL-9 (e.g., human IL-9 or murine IL-9).

[0075] In embodiments, the disclosure provides an isolated antibody that specifically binds to IL-9 (e.g., human IL-9 or murine IL-9), comprising a VH comprising the CDRH1, CDRH2 and CDRH3 amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3; 4, 5, and 6; 7, 8, and 9; 10, 11, and 12; 13, 14, and 15; 16, 17, and 18; 19, 20, and 21; or 122, 123, and 124, respectively.

[0076] In an embodiment, the present disclosure provides an isolated antibody that specifically binds to IL-9 (e.g., human IL-9 or murine IL-9), comprising a VL comprising the CDRL1, CDRL2 and CDRL3 amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24; 25, 26, and 27; 28, 29, and 30; 31, 29, and 32; 33, 34, and 35; 36, 37, and 38; 39, 40, and 41; or 125, 126, and 127, respectively.

[0077] In embodiments, the disclosure provides an isolated antibody that specifically binds IL-9 (e.g., human IL-9 or murine IL-9), comprising a VH comprising CDRH1, CDRH2, and CDRH3 regions, and a VL comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions are set forth in SEQ ID NOs: 1, 2, 3, 22, 23, and 24, respectively; 4, 5, 6, 25, 26, and 27; 7, 8, 9, 28, 29, and 30; 7, 8, 9, 31, 29, and 32; 10, 11, 12, 33, 34, and 35; 13, 14, 15, 36, 37, and 38; 16, 17, 18, 39, 40, and 41; 19, 20, 21, 36, 37, and 38; or 122, 123, 124, 125, 126, and 127.

[0078] In embodiments, the disclosure provides an isolated antibody that specifically binds IL-9 (e.g., human IL-9 or murine IL-9) and comprises a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 42, 43, 44, 45, 46, 47, 48, 49, or 128. In embodiments, the disclosure provides an isolated antibody that specifically binds IL-9 (e.g., human IL-9 or murine IL-9) and comprises a VH comprising an amino acid sequence set forth in SEQ ID NO: 42, 43, 44, 45, 46, 47, 48, 49, or 128. In embodiments, the amino acid sequence of the VH consists of the amino acid sequence set forth in SEQ ID NO: 42, 43, 44, 45, 46, 47, 48, 49, or 128.

[0079] In embodiments, the disclosure provides an isolated antibody that specifically binds IL-9 (e.g., human IL-9 or murine IL-9) and comprises a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 50, 51, 52, 53, 54, 55, 56, or 129. In embodiments, the disclosure provides an isolated antibody that specifically binds IL-9 (e.g., human IL-9 or murine IL-9) and comprises a VL comprising an amino acid sequence set forth in SEQ ID NO: 50, 51, 52, 53, 54, 55, 56, or 129. In embodiments, the amino acid sequence of the VL consists of the amino acid sequence set forth in SEQ ID NO: 50, 51, 52, 53, 54, 55, 56, or 129.

[0080] In embodiments, the present disclosure provides a method for the production of a method for the production of IL-9 (e.g., human IL-9 or murine IL-9) that specifically binds to IL-9 and has a homology of at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) to an amino acid sequence set forth in SEQ ID NO: 42, 43, 44, 45, 46, 47, 48, 49, or 128. and a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 50, 51, 52, 53, 54, 55, 56, or 129. In embodiments, the present disclosure provides an isolated antibody that specifically binds IL-9 (e.g., human IL-9 or murine IL-9), and comprises a VH comprising the amino acid sequence of SEQ ID NO: 42, 43, 44, 45, 46, 47, 48, 49, or 128, and a VL comprising the amino acid sequence of SEQ ID NO: 50, 51, 52, 53, 54, 55, 56, or 129. In embodiments, the amino acid sequence of the VH consists of the amino acid sequence set forth in SEQ ID NO: 42, 43, 44, 45, 46, 47, 48, 49, or 128, and the amino acid sequence of the VL consists of the amino acid sequence set forth in SEQ ID NO: 50, 51, 52, 53, 54, 55, 56, or 129.

[0081] In embodiments, the disclosure provides an isolated antibody that specifically binds IL-9 (e.g., human IL-9 or murine IL-9) and comprises the VH and VL amino acid sequences set forth in SEQ ID NOs: 42 and 51; 43 and 55; 44 and 54; 45 and 53; 46 and 50; 47 and 52; 48 and 50; 49 and 56; or 128 and 129. In embodiments, the VH and VL amino acid sequences consist of the amino acid sequences set forth in SEQ ID NOs: 42 and 51; 43 and 55; 44 and 54; 45 and 53; 46 and 50; 47 and 52; 48 and 50; 49 and 56; or 128 and 129, respectively.

[0082] In embodiments, the disclosure provides isolated antibodies that cross-compete for binding to IL-9 (e.g., human IL-9 or murine IL-9) with an antibody comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 42 and 51; 43 and 55; 44 and 54; 45 and 53; 46 and 50; 47 and 52; 48 and 50; 49 and 56; or 128 and 129.

[0083] In embodiments, the disclosure provides isolated antibodies that bind to an epitope of IL-9 (e.g., an epitope of human IL-9 or murine IL-9) that is identical to or overlaps with an antibody described herein, e.g., an antibody comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 42 and 51; 43 and 55; 44 and 54; 45 and 53; 46 and 50; 47 and 52; 48 and 50; 49 and 56; or 128 and 129.

[0084] In embodiments, the disclosure provides an isolated antibody that binds to an epitope that includes at least residue R91 of IL-9 (e.g., an epitope of human IL-9 or murine IL-9). In embodiments, the isolated antibody binds to one or more residues of the C helix of human or murine IL-9 (amino acids 84-102 of IL-9). The amino acid sequence of the C helix of human IL-9 is: [ka] and the amino acid sequence of the C helix of mouse IL-9 is [ka] In an embodiment, the isolated antibody binds to one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) amino acid(s) of human IL-9 selected from the group consisting of R84, Y85, P86, L87, I88, F89, S90, R91, and K94. In an embodiment, the isolated antibody binds to one or more (e.g., 2, 3, 4, 5, or 6) amino acid(s) of human IL-9 selected from the group consisting of L87, I88, R91, K94, S95, and V98. In an embodiment, the isolated antibody binds to one or both amino acid(s) of human IL-9 selected from the group consisting of I88 and R91. In embodiments, the isolated antibody binds to one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) amino acid(s) of mouse IL-9 selected from the group consisting of R84, P87, V88, H90, R91, R94, I95, V98, and L99. In embodiments, the isolated antibody also binds to cynomolgus IL-9.

[0085] In embodiments, the epitope of an antibody can be determined by, for example, NMR spectroscopy, surface plasmon resonance (BIAcore®), X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). In the case of X-ray crystallography, crystallization can be achieved using any of the methods known in the art (e.g., Giege R et al. (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, all of which are fully incorporated herein by reference). Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques and improved using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; e.g., Meth Enzymol (1985) vols. 114 & 115, Wyckoff HW et al. (eds.); U.S. Patent Application No. 2004 / 0014194) and BUSTER (see Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, Carter CW (eds.); Roversi P et al. (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323, all of which are incorporated herein by reference in their entireties). Mutagenesis mapping studies can be accomplished using any method known to one of skill in the art.For example, see Champe M et al., (1995) supra, and Cunningham BC & Wells JA, (1989) supra, for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques. In an embodiment, the epitope of the antibody is determined using alanine scanning mutagenesis studies. Furthermore, antibodies that recognize and bind to the same or overlapping epitopes of IL-9 (e.g., human IL-9 or mouse IL-9) can be identified using routine techniques such as immunoassays, i.e., competitive binding assays, by showing the ability of one antibody to block the binding of another antibody to a target antigen. Competitive binding assays can also be used to determine whether two antibodies have similar binding specificity for an epitope. Competitive binding can be determined in an assay in which the immunoglobulin under test inhibits the specific binding of a reference antibody to a common antigen, such as IL-9 (e.g., human IL-9 or mouse IL-9). There are many types of competitive binding assays, for example: solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see Stahli C et al. (1983) Methods Enzymol 9: 242-253); solid-phase direct biotin-avidin EIA (see Kirkland TN et al. (1986) J Immunol 137: 3614-9); solid-phase direct label assays, solid-phase direct label sandwich assays (see Harlow E & Lane D (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct label RIA using I-125 label (see Morel GA et al. (1988) Mol Immunol 25(1): 7-15); solid-phase direct biotin-avidin EIA (Cheung RC et al. (1990) Virology 176:546-52); and direct label RIA (Moldenhauer G et al., (1990) Scand J Immunol 32:77-82), all of which are incorporated herein by reference in their entireties.Typically, such assays involve the use of purified antigen (e.g., IL-9, e.g., human IL-9 or mouse IL-9) bound to a solid surface or cells bearing either of these unlabeled test immunoglobulins and a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess. Typically, the competing antibody, when present in excess, will inhibit specific binding of the reference substance or antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more. Competitive binding assays can be configured in a number of different formats, using either labeled antigen or labeled antibody. In a common version of this assay, the antigen is immobilized on a 96-well plate. The ability of unlabeled antibodies to block binding of the labeled antibodies to the antigen is then measured using radioactive or enzymatic labels. For further details, see, e.g., Wagener C et al. (1983) J Immunol 130: 2308-2315; Wagener C et al. (1984) J Immunol Methods 68: 269-274; Kuroki M et al. (1990) Cancer Res 50: 4872-4879; Kuroki M et al. (1992) Immunol Invest 21: 523-538; Kuroki M et al. (1992) Hybridoma 11: 391-407 and Antibodies: A Laboratory Manual, Ed Harlow E & Lane D (eds.), supra, pp. 386-389, all of which are incorporated herein by reference in their entireties.

[0086] In an embodiment, the antibody inhibits the binding of human IL-9 to human IL-9Rα. In an embodiment, the binding of human IL-9 to human IL-9Rα is reduced by 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more in the presence of the antibody compared to the binding of human IL-9 to human IL-9Rα in the absence of the antibody.

[0087] In an embodiment, the antibody disclosed herein is conjugated to a cytotoxic agent, a cytostatic agent, a toxin, a radionuclide, or a detectable label. In an embodiment, the cytotoxic agent is capable of inducing death or destruction of a cell that comes into contact with it. In an embodiment, the cytostatic agent is capable of preventing or substantially reducing the proliferation and / or inhibiting the activity or function of a cell that comes into contact with it. In an embodiment, the cytotoxic agent or cytostatic agent is a chemotherapeutic agent. In an embodiment, the radionuclide is an isotope 3 H, 14 C. 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 67 Cu, 90 Y, 99 Tc, 111 In, 117 Lu, 121 I, 124 I, 125 I, 131 I, 198 Au, 211 At, 213 Bi, 225 Ac, and 186 Re. In embodiments, the detectable label comprises a fluorescent moiety or a click chemistry handle.

[0088] Any immunoglobulin (Ig) constant region can be used in the antibodies disclosed herein. In embodiments, the Ig region can be a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class of immunoglobulin molecule (e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 ), or any subclass (e.g., IgG 2 a and IgG 2 b).

[0089] In embodiments, one, two, or more mutations (e.g., amino acid substitutions) are made in the Fc region (e.g., the CH2 domain (human IgG 1 (residues 231-340 of human IgG 1 The amino acid sequence is introduced into the ribosome (residues 341-447 of the present invention) and / or into the hinge region (residues 216-230 numbered according to the EU numbering system).

[0090] In an embodiment, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of an antibody described herein to alter (e.g., increase or decrease) the number of cysteine ​​residues in the hinge region, e.g., as described in U.S. Patent No. 5,677,425, which is incorporated herein by reference in its entirety. The number of cysteine ​​residues in the hinge region may be altered, for example, to facilitate assembly of the light and heavy chains or to alter (e.g., increase or decrease) the stability of the antibody.

[0091] In an embodiment, one, two or more amino acid mutations (e.g., substitutions, insertions or deletions) are introduced into the IgG constant region, or its FcRn-binding fragment (preferably, Fc or hinge-Fc fragment) to alter (e.g., decrease or increase) the half-life of the antibody in vivo. For examples of mutations that may alter (e.g., decrease or increase) the half-life of the antibody in vivo, see, for example, International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375 and 6,165,745, all of which are fully incorporated herein by reference. In certain embodiments, one, two or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into the IgG constant region, or an FcRn-binding fragment thereof (preferably, an Fc or hinge-Fc fragment) to decrease the half-life of the antibody in vivo. In other embodiments, one, two or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into the IgG constant region, or an FcRn-binding fragment thereof (preferably, an Fc or hinge-Fc fragment) to increase the half-life of the antibody in vivo. In embodiments, the antibody comprises a second constant (CH2) domain numbered according to the EU numbering system (human IgG 1 Residues 231 to 340 of human IgG 1 In some embodiments, the IgG of the antibody described herein may have one or more amino acid mutations (e.g., substitutions) at residues 341 to 447 of the antibody. 1The constant region of contains a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU numbering system. See U.S. Patent No. 7,658,921, which is incorporated herein by reference in its entirety. This type of mutant IgG, termed the "YTE mutant," has been shown to have a four-fold increase in half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281: 23514-24, which is incorporated herein by reference in its entirety). In certain embodiments, the antibody comprises an IgG constant region comprising one, two, three or more amino acid substitutions at amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU numbering system.

[0092] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are made in the Fc region (e.g., the CH2 domain (e.g., human IgG) of an antibody described herein to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activating Fc receptor) on the surface of an effector cell. 1 Residues 231-340 of human IgG 1 The Fc region of an antibody is introduced into the Fc receptor (residues 341-447 of the Fc region of the antibody) and / or into the hinge region (residues 216-230 numbered according to the EU numbering system). Mutations in the Fc region of an antibody that reduce or increase the affinity of the antibody for the Fc receptor, and techniques for introducing such mutations into an Fc receptor or a fragment thereof, are known to those of skill in the art. Examples of mutations in the Fc receptor of an antibody that can alter the affinity of the antibody for the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109: 6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, all of which are incorporated herein by reference in their entirety.

[0093] In one embodiment, the antibody comprises a heavy chain constant region that is a mutant of a wild-type heavy chain constant region, and the mutant heavy chain constant region binds to FcγRIIB with a higher affinity than the wild-type heavy chain constant region binds to FcγRIIB. In one embodiment, the mutant heavy chain constant region is a mutant human heavy chain constant region, such as a mutant human IgG 1 , mutant human IgG 2 or mutant human IgG 4 Heavy chain constant region. In certain embodiments, the variant human IgG heavy chain constant region comprises one or more of the following amino acid mutations according to the EU numbering system: G236D, P238D, S239D, S267E, L328F, and L328E. In certain embodiments, the variant human IgG heavy chain constant region comprises a set of amino acid mutations selected from the group consisting of S267E and L328F; P238D and L328E; P238D and one or more substitutions selected from the group consisting of E233D, G237D, H268D, P271G, and A330R; P238D, E233D, G237D, H268D, P271G, and A330R; G236D and S267E; S239D and S267E; V262E, S267E, and L328F; and V264E, S267E, and L328F according to the EU numbering system. In embodiments, FcγRIIB is expressed on a cell selected from the group consisting of macrophages, monocytes, B cells, dendritic cells, endothelial cells, and activated T cells.

[0094] In an embodiment, one, two or more amino acid substitutions are introduced into the Fc region of the IgG constant region to alter the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 239, 243, 267, 292, 297, 300, 318, 320, 322, 328, 330, 332, and 396 numbered according to the EU numbering system can be replaced with different amino acid residues such that the affinity of the antibody to the effector ligand is altered but the antigen binding ability of the parent antibody is retained. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260, each of which is incorporated herein by reference in its entirety. In some embodiments, deletion or inactivation of constant region domains (by point mutation or other means) can reduce Fc receptor binding of circulating antibodies, thereby increasing tumor localization. For example, see U.S. Patent Nos. 5,585,097 and 8,591,886, each of which is incorporated herein by reference in its entirety, for a description of mutations that delete or inactivate constant regions, thereby increasing tumor localization. In embodiments, one or more amino acid substitutions can be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites on the Fc region, thereby reducing Fc receptor binding (see, e.g., Shields RL et al., (2001) J Biol Chem 276: 6591-604, incorporated herein by reference in its entirety).In various embodiments, one or more of the following mutations in the constant region of the antibodies described herein may occur: an N297A substitution; an N297Q substitution; an L234A substitution; an L234F substitution; an L235A substitution; an L235F substitution; an L235V substitution; an L237A substitution; an S239D substitution; an E233P substitution; an L234V substitution; an L235A substitution; a C236 deletion; a P238A substitution; an S239D substitution; an F243L substitution; a D265A substitution; an S267E substitution; an L328F substitution; an R292P substitution; an Y300L substitution; an A327Q substitution; a P329A substitution; an A330L substitution; an I332E substitution; or a P396L substitution, as numbered according to the EU numbering system.

[0095] In some embodiments, a mutation selected from the group consisting of D265A, P329A, and combinations thereof, numbered according to the EU numbering system, may occur in the constant region of the antibody described herein. In some embodiments, a mutation selected from the group consisting of L235A, L237A, and combinations thereof, numbered according to the EU numbering system, may occur in the constant region of the antibody described herein. In some embodiments, a mutation selected from the group consisting of S267E, L328F, and combinations thereof, numbered according to the EU numbering system, may occur in the constant region of the antibody described herein. In some embodiments, a mutation selected from the group consisting of S239D, I332E, optionally A330L, and combinations thereof, numbered according to the EU numbering system, may occur in the constant region of the antibody described herein. In some embodiments, a mutation selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, and combinations thereof, numbered according to the EU numbering system, may occur in the constant region of the antibody described herein. In certain embodiments, a mutation selected from the group consisting of S267E, L328F, and combinations thereof, numbered according to the EU numbering system, may occur in the constant region of an antibody described herein.

[0096] In embodiments, the antibodies described herein are IgG1 with the N297Q or N297A amino acid substitutions as numbered according to the EU numbering system. 1In certain embodiments, the antibody described herein comprises an IgG1 constant region having a mutation selected from the group consisting of D265A, P329A, and combinations thereof, as numbered according to the EU numbering system. 1 In another embodiment, the antibody described herein comprises an IgG1 constant region having a mutation selected from the group consisting of L234A, L235A, and combinations thereof, as numbered according to the EU numbering system. 1 In another embodiment, the antibody described herein comprises an IgG1 constant region having a mutation selected from the group consisting of L234F, L235F, N297A, and combinations thereof, as numbered according to the EU numbering system. 1 In one embodiment, the constant region of human IgG is numbered according to the EU numbering system. 1 The amino acid residues in the constant regions of the antibodies described herein at positions corresponding to positions L234, L235, and D265 in the heavy chain are not L, L, and D, respectively. This approach is described in detail in International Publication WO 14 / 108483, which is incorporated herein by reference in its entirety. In an embodiment, human IgG1 is numbered according to the EU numbering system. 1 The amino acids corresponding to positions L234, L235, and D265 in the heavy chain are F, E, and A; or A, A, and A, respectively.

[0097] In an embodiment, according to the EU numbering system, the amino acids at positions 433, 434, and 436 of the heavy chain constant region are K, F, and Y, respectively. In an embodiment, according to the EU numbering system, the amino acids at positions 252, 254, and 256 of the heavy chain constant region are Y, T, and E, respectively. In an embodiment, according to the EU numbering system, the amino acids at positions 428 and 434 of the heavy chain constant region are L and S, respectively. In an embodiment, according to the EU numbering system, the amino acids at positions 309, 311, and 434 of the heavy chain constant region are D, H, and S, respectively.

[0098] In embodiments, one or more amino acids selected from amino acid residues 329, 331, and 322 of the constant region of the antibody described herein, numbered according to the EU numbering system, can be replaced with different amino acid residues to alter the antibody's C1q binding and / or reduce or eliminate complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 (Idusogie et al.), which is incorporated herein by reference in its entirety. In embodiments, one or more amino acid residues within amino acid positions 231-238 in the N-terminal region of the CH2 domain of the antibody described herein, numbered according to the EU numbering system, are altered, thereby altering the antibody's ability to fix complement. This approach is described further in International Publication WO 94 / 29351, which is incorporated herein by reference in its entirety. In embodiments, the Fc region of an antibody described herein may be any of the following positions numbered according to the EU numbering system: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 372, 376, 378, 380, 383, 385, 386, 389, 390, 392, 393, 394, 395, 3 The antibody may be modified to enhance its ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase its affinity for Fcγ receptors by mutating (e.g., introducing amino acid substitutions) one or more amino acids at position 8, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439. This approach is further described in International Publication WO 00 / 42072, which is incorporated herein by reference in its entirety.

[0099] In embodiments, any of the constant region mutations or modifications described herein may be introduced into one or both heavy chain constant regions of an antibody described herein that has two heavy chain constant regions.

[0100] In embodiments, the disclosure provides isolated antibodies that specifically bind to IL-9 (e.g., human IL-9 or murine IL-9) and function as antagonists (e.g., reduce or inhibit IL-9 activity).

[0101] In embodiments, the disclosure provides isolated antibodies that specifically bind IL-9 (e.g., human IL-9 or murine IL-9) and reduce or inhibit IL-9 (e.g., human IL-9 or murine IL-9) activity by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% relative to IL-9 (e.g., human IL-9 or murine IL-9) activity when not including the antibody or when including an unrelated antibody (e.g., an antibody that does not specifically bind IL-9), when assessed by methods described herein and / or known to one of skill in the art. In embodiments, the disclosure provides isolated antibodies that specifically bind IL-9 (e.g., human IL-9 or murine IL-9) and reduce or inhibit IL-9 (e.g., human IL-9 or murine IL-9) activity by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more, relative to IL-9 (e.g., human IL-9 or murine IL-9) activity when not including the antibody or when including an unrelated antibody (e.g., an antibody that does not specifically bind IL-9), when assessed by methods described herein and / or known to one of skill in the art. Non-limiting examples of IL-9 (e.g., human IL-9 or mouse IL-9) activity can include IL-9 (e.g., human IL-9 or mouse IL-9) signal transduction, binding of IL-9 (e.g., human IL-9 or mouse IL-9) to its receptor (e.g., IL-9Rα); IL-9 (e.g., human IL-9 or mouse IL-9)-induced cell proliferation. In embodiments, the reduction of IL-9 (e.g., human IL-9 or mouse IL-9) activity is assessed as described in the Examples.

[0102] In embodiments, the present disclosure provides compounds having a dissociation constant (K DThe present invention provides isolated antibodies that specifically bind to IL-9 (e.g., human IL-9 or murine IL-9) at an antibody level of 0.1-0.25 μM.

[0103] 5.3 Pharmaceutical Compositions Provided herein are compositions comprising an isolated anti-IL-9 antibody disclosed herein having a desired degree of purity in a physiologically acceptable carrier, excipient, or stabilizer (see, e.g., Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (about 10 proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN. (商標) , PLURONICS (商標) , or polyethylene glycol (PEG).

[0104] In an embodiment, the pharmaceutical composition comprises an isolated anti-IL-9 antibody disclosed herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharma- ceutically acceptable carrier. In an embodiment, the pharmaceutical composition comprises an isolated anti-IL-9 antibody disclosed herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharma- ceutically acceptable carrier. In an embodiment, the antibody is the only active ingredient contained in the pharmaceutical composition. In an embodiment, the present disclosure provides a pharmaceutical composition comprising an isolated anti-IL-9 antibody disclosed herein for use as a medicament. In another embodiment, the present disclosure provides a pharmaceutical composition for use in the treatment of inflammatory disease or cancer.

[0105] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharma- ceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, and dextrose and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrates can be added to parenteral preparations packaged in multidose containers, including phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN® 80). Sequestering or chelating agents for metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

[0106] The pharmaceutical composition may be formulated for any route of administration to a subject. Specific examples of routes of administration include nasal, oral, pulmonary, transdermal, intradermal, and parenteral administration. Parenteral administration, characterized by either subcutaneous, intramuscular, or intravenous injection, is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for dissolution or suspension in liquid prior to injection, or as emulsions. Injectables, solutions, and emulsions also include one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the pharmaceutical composition to be administered can also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other such agents, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0107] Preparations for parenteral administration of antibodies include sterile, ready-to-inject solutions, sterile, dry soluble products ready for combination with a solvent immediately prior to use, including subcutaneous tablets, e.g., lyophilized powders, sterile, ready-to-inject suspensions, sterile, dry insoluble products ready for combination with a vehicle immediately prior to use, and sterile emulsions. Liquids can be either aqueous or non-aqueous.

[0108] If administered intravenously, suitable carriers include saline or phosphate buffered saline (PBS), as well as solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

[0109] Topical mixtures containing the antibody are prepared as described for local and systemic administration. The resulting mixture may be a solution, suspension, emulsion, etc., and may be formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, wash, spray, suppository, bandage, skin patch, or any other formulation suitable for topical administration.

[0110] The isolated anti-IL-9 antibodies disclosed herein can be formulated as aerosols for local application, such as by inhalation (see, for example, U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for the delivery of steroids useful in the treatment of inflammatory diseases, particularly asthma, and are incorporated herein by reference in their entirety). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a superfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such cases, the particles of the formulation, in certain embodiments, have a diameter of less than 50 microns, and in certain embodiments, have a diameter of less than 10 microns.

[0111] The isolated anti-IL-9 antibodies disclosed herein can be formulated for local or topical application, e.g., topical application to the skin and mucosa, e.g., in the eye, in the form of gels, creams, and lotions, as well as application to the eye or intracisternal or intrathecal application. Transdermal delivery, and topical administration to the eye or mucosa, or for inhalation therapy, are contemplated. Nasal solutions of the antibodies can also be administered alone or in combination with other pharma- ceutically acceptable excipients.

[0112] Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those of skill in the art and can be used to administer antibodies. For example, such patches are disclosed in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, all of which are incorporated herein by reference in their entireties.

[0113] In an embodiment, the pharmaceutical compositions comprising the antibodies described herein are lyophilized powders that can be reconstituted as liquids, emulsions, and other mixtures for administration. They can also be reconstituted and formulated as solids or gels. The lyophilized powders are prepared by dissolving the antibodies described herein, or a pharma- ceutically acceptable derivative thereof, in a suitable solvent. In an embodiment, the lyophilized powders are sterile. The solvent can contain excipients that improve the stability or other pharmacological components of the powder or the reconstituted solution prepared from the powder. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or another suitable agent. The solvent can also contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffers known to those skilled in the art at about neutral pH in some embodiments. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those skilled in the art provides the desired formulation. In an embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial contains a single or multiple doses of the compound. The lyophilized powder can be stored under appropriate conditions, for example, at about 4°C to room temperature. Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount depends on the compound selected. Such amounts can be determined empirically.

[0114] The isolated anti-IL-9 antibodies disclosed herein, and other compositions provided herein, can also be formulated to target specific tissues, receptors, or other parts of the body of the subject to be treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods are contemplated herein for use in the compositions. For non-limiting examples of targeting methods, see, e.g., U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874, all of which are incorporated herein by reference in their entireties. In embodiments, the antibodies described herein are targeted to tumors.

[0115] Formulations to be used for in vivo administration can be sterilized, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0116] (5.4 Instructions and Use) In one embodiment, the present disclosure provides a method of treating a subject with the anti-IL-9 antibodies disclosed herein. Any disease or disorder in a subject that would benefit from reduced IL-9 (e.g., human IL-9 or mouse IL-9) function can be treated with the isolated anti-IL-9 antibodies disclosed herein. In an embodiment, the disease or disorder is an inflammatory disease or disorder, an autoimmune disease or disorder, or a cancer.

[0117] In embodiments, inflammatory diseases or disorders that can be treated by the methods disclosed herein include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, osteoarthritis, spondyloarthropathy (e.g., psoriatic arthritis, ankylosing spondylitis, Reiter's syndrome (reactive arthritis), inflammatory osteolysis, Wilson's disease, and chronic inflammation resulting from chronic viral or bacterial infections.

[0118] In embodiments, autoimmune diseases or disorders that can be treated by the methods disclosed herein include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune disease of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (IDP), and / or other autoimmune disorders. TP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic-related Arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff man syndrome, systemic lupus erythematosus, lupus erythematosus, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis syndromes such as dermatitis herpetiformis, vasculitis, vitiligo, and Wegener's granulomatosis.

[0119] Cancers that can be treated with the isolated anti-IL-9 antibodies or pharmaceutical compositions disclosed herein include, but are not limited to, solid tumors, hematological cancers (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma), and metastatic lesions. In certain embodiments, the cancer is a solid tumor. Examples of solid tumors include malignant tumors, e.g., sarcomas and carcinomas, e.g., adenocarcinomas of various organ systems, e.g., those affecting the lung, breast, ovary, lymphocyte, gastrointestinal tract (e.g., colon), anus, genital and genitourinary tract (e.g., kidney, urothelium, bladder cells, prostate), pharynx, CNS (e.g., brain, neuronal or glial cells), head and neck, skin (e.g., melanoma), and pancreas, as well as adenocarcinomas, including malignant tumors such as colon cancer, rectal cancer, renal cell carcinoma, liver cancer, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), cancer of the small intestine, and cancer of the esophagus. The cancer can be early stage, intermediate stage, late stage, or metastatic cancer.

[0120] In embodiments, the cancer is selected from lung cancer (e.g., lung adenocarcinoma or non-small cell lung cancer (NSCLC) (e.g., NSCLC of squamous and / or non-squamous histology, or NSCLC adenocarcinoma)), melanoma (e.g., advanced melanoma), kidney cancer (e.g., renal cell carcinoma), liver cancer (e.g., hepatocellular carcinoma), myeloma (e.g., multiple myeloma), prostate cancer, breast cancer (e.g., breast cancer that does not express one, two or all of estrogen receptors, progesterone receptors, or Her2 / neu, e.g., triple-negative breast cancer), ovarian cancer, colorectal cancer, pancreatic cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), anal cancer, gastroesophageal cancer (e.g., esophageal squamous cell carcinoma), mesothelioma, nasopharyngeal carcinoma, thyroid cancer, cervical cancer, epithelial carcinoma, peritoneal cancer, or lymphoproliferative disorders (e.g., post-transplant lymphoproliferative disorders).

[0121] In an embodiment, the cancer is a blood cancer, such as leukemia, lymphoma, or myeloma. In an embodiment, the cancer is a leukemia, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute myeloblastic leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia (CLL), or hairy cell leukemia. In embodiments, the cancer is a lymphoma, e.g., B cell lymphoma, diffuse large B cell lymphoma (DLBCL), activated B cell-like (ABC) diffuse large B cell lymphoma, germinal center B cell (GCB) diffuse large B cell lymphoma, mantle cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, relapsed non-Hodgkin lymphoma, refractory non-Hodgkin lymphoma, relapsed follicular non-Hodgkin lymphoma, Burkitt lymphoma, small lymphocytic lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, or extranodal marginal zone lymphoma.

[0122] 5.5 Polynucleotides, Vectors, and Methods of Antibody Generation In aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding an antibody or portion thereof, or fragment thereof (e.g., VL and / or VH; and light chain and / or heavy chain) described herein that specifically binds to an IL-9 (e.g., human IL-9 or murine IL-9) antigen, as well as vectors comprising such polynucleotides, e.g., vectors for recombinant expression in host cells (e.g., E. coli and mammalian cells). Provided herein are polynucleotides comprising a nucleotide sequence encoding the heavy and / or light chains of the antibodies provided herein, as well as vectors comprising such polynucleotide sequences, e.g., expression vectors for their efficient expression in host cells, e.g., mammalian cells.

[0123] As used herein, an "isolated" polynucleotide or nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule (e.g., mouse or human). Furthermore, an "isolated" nucleic acid molecule, e.g., a cDNA molecule, can be substantially free of other cellular material, or other cellular material or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. For example, the term "substantially free" includes preparations of polynucleotides or nucleic acid molecules that have less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (particularly less than about 10%) of other materials, e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors and / or other chemicals. In an embodiment, the nucleic acid molecule(s) encoding the antibodies described herein are isolated or purified.

[0124] In aspects, provided herein are antibodies that specifically bind to an IL-9 (e.g., human IL-9 or murine IL-9) polypeptide and comprise an amino acid sequence described herein, as well as polynucleotides comprising a nucleotide sequence encoding an antibody that competes (e.g., in a dose-dependent manner) with such an antibody for binding to an IL-9 (e.g., human IL-9 or murine IL-9) polypeptide or binds to the same epitope as such an antibody.

[0125] In an aspect, provided herein is a polynucleotide comprising a nucleotide sequence encoding a light chain or a heavy chain of an antibody described herein. The polynucleotide may comprise a nucleotide sequence encoding a light chain comprising the VL FRs and CDRs of an antibody described herein (see, e.g., Tables 2 and 3), or a nucleotide sequence encoding a heavy chain comprising the VH FRs and CDRs of an antibody described herein (see, e.g., Tables 2 and 3). In an embodiment, the polynucleotide encodes the VH, VL, heavy chain, and / or light chain of an antibody described herein. In an embodiment, the polynucleotide encodes a first VH and a first VL of an antibody described herein. In an embodiment, the polynucleotide encodes a second VH and a second VL of an antibody described herein. In an embodiment, the polynucleotide encodes a first heavy chain and a first light chain of an antibody described herein. In an embodiment, the polynucleotide encodes a second heavy chain and a second light chain of an antibody described herein. In an embodiment, the polynucleotide encodes a VH and / or a VL, or a heavy chain and / or a light chain of an isolated antibody described herein.

[0126] Also provided herein are polynucleotides encoding isolated anti-IL-9 antibodies that are optimized, for example, by codon / RNA optimization, exchange with a heterologous signal sequence, and removal of mRNA instability elements. Methods for generating optimized nucleic acids encoding isolated anti-IL-9 antibodies or fragments thereof (e.g., light chain, heavy chain, VH domain, or VL domain) for recombinant expression by introducing codon changes and / or removing inhibitory regions in the mRNA can be performed by correspondingly adapting the optimization methods described in, for example, U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, all of which are fully incorporated herein by reference. For example, potential splice sites and instability elements (e.g., A / T or A / U rich elements) within the RNA can be mutated without altering the amino acids encoded by the nucleic acid sequence to increase the stability of the RNA for recombinant expression. The modification may take advantage of, for example, the degeneracy of the genetic code, which uses alternative codons for the same amino acid. In an embodiment, it may be desirable to modify one or more codons to encode a conservative mutation, for example, a similar amino acid having a similar chemical structure and properties and / or function as the original amino acid. Such methods may increase the expression of the isolated anti-IL-9 antibody or fragment thereof by at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more, compared to the expression of the isolated anti-IL-9 antibody encoded by a non-optimized polynucleotide.

[0127] In an embodiment, an optimized polynucleotide sequence encoding an isolated anti-IL-9 antibody or fragment thereof (e.g., VL domain and / or VH domain) described herein can hybridize to an antisense (e.g., complementary) polynucleotide of a non-optimized polynucleotide sequence encoding an isolated anti-IL-9 antibody or fragment thereof (e.g., VL domain and / or VH domain) described herein. In an embodiment, an optimized nucleotide sequence encoding an isolated anti-IL-9 antibody or fragment thereof described herein hybridizes under high stringency conditions to an antisense polynucleotide of a non-optimized polynucleotide sequence encoding an isolated anti-IL-9 antibody or fragment thereof described herein. In an embodiment, an optimized nucleotide sequence encoding an isolated anti-IL-9 antibody or fragment thereof described herein hybridizes under high stringency, medium stringency, or lower stringency hybridization conditions to an antisense polynucleotide of a non-optimized nucleotide sequence encoding an isolated anti-IL-9 antibody or fragment thereof described herein. Information regarding hybridization conditions has been described, see, eg, US Patent Application Publication No. 2005 / 0048549 (eg, paragraphs 72-73), which is incorporated herein by reference.

[0128] The polynucleotides can be obtained by any method known in the art, and the nucleotide sequence of the polynucleotides can be determined by the method. The nucleotide sequences encoding the antibodies described herein, such as those described in Tables 2 and 3, and modified versions of these antibodies, can be determined using methods well known in the art, i.e., nucleotide codons known to encode specific amino acids are assembled in such a way as to generate nucleic acids encoding the antibodies. Such polynucleotides encoding the antibodies can be assembled from chemically synthesized oligonucleotides (e.g., those described in Kutmeier G et al., (1994), BioTechniques 17: 242-6, incorporated herein by reference in its entirety), which briefly includes the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of the oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0129] Alternatively, polynucleotides encoding the antigen-binding regions of the antibodies described herein can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from a hybridoma cell producing the antibody of interest. Using such PCR amplification methods, nucleic acid comprising sequences encoding the light and / or heavy chains of the antibody can be obtained. Using such PCR amplification methods, nucleic acid comprising sequences encoding the variable light and / or variable heavy chain regions of the antibody can be obtained. The amplified nucleic acid can be cloned into a vector for expression in a host cell and for further cloning.

[0130] If a clone containing a nucleic acid encoding a particular antigen-binding region or antibody is not available, but the sequence of the antigen-binding region or antibody molecule is known, nucleic acid encoding an immunoglobulin can be chemically synthesized or obtained from a suitable source (e.g., any tissue or cell expressing the antibody, e.g., an antibody cDNA library or a cDNA library generated from hybridoma cells selected to express an antibody described herein, or nucleic acid isolated from the cells, preferably polyA+RNA) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning, e.g., using oligonucleotide probes specific for the particular gene sequence to identify a cDNA clone from a cDNA library that encodes the antibody. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method well known in the art.

[0131] DNA encoding the isolated anti-IL-9 (e.g., human IL-9 or murine IL-9) antibodies described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of an anti-IL-9 (e.g., human IL-9 or murine IL-9) antibody). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells that do not otherwise produce immunoglobulin protein, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from CHO GS System™ (Lonza)), or myeloma cells, to obtain synthesis of the anti-IL-9 antibody in the recombinant host cells.

[0132] To generate a complete antibody or antigen-binding region, the VH or VL sequence can be amplified in a scFv clone using PCR primers that contain the VH or VL nucleotide sequence, a restriction site, and a flanking sequence to protect the restriction site. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a heavy chain constant region, such as human gamma 1 or human gamma 4 constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a light chain constant region, such as human kappa or lambda constant region. In one embodiment, the vector for expressing the VH or VL domain contains an EF-1α promoter, a secretion signal, a cloning site for the variable region, a constant region, and a selection marker such as neomycin. The VH and VL domains can also be cloned into one vector that expresses the necessary constant region. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into a cell line using techniques known to those skilled in the art to generate a stable or transient cell line expressing a full-length antibody, such as IgG.

[0133] The DNA can also be modified, for example, by substituting human heavy and light chain constant region coding sequences for the murine sequences, or by covalently joining all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0134] Also provided are polynucleotides that hybridize to polynucleotides encoding the antibodies described herein under high stringency, medium stringency or lower stringency hybridization conditions. In embodiments, the polynucleotides described herein hybridize to polynucleotides encoding the VH and / or VL domains provided herein under high stringency, medium stringency or lower stringency hybridization conditions.

[0135] Hybridization conditions are described in the art and are known to those skilled in the art. For example, hybridization under stringent conditions can include hybridization to filter-bound DNA in 6× sodium chloride / sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC / 0.1% SDS at about 50-65° C.; hybridization under highly stringent conditions can include hybridization to filter-bound nucleic acid in 6×SSC at about 45° C., followed by one or more washes in 0.1×SSC / 0.2% SDS at about 68° C. Hybridization under other stringent hybridization conditions is known to those of skill in the art and is described, for example, in Current Protocols in Molecular Biology, Vol. I, edited by Ausubel FM et al., 1989, Green Publishing Associates and John Wiley & Sons, Inc., New York, pages 6.3.1 to 6.3.6 and 2.10.3, which is incorporated herein by reference in its entirety.

[0136] In aspects, provided herein are cells (e.g., host cells) that express (e.g., recombinantly) an antibody described herein that specifically binds IL-9 (e.g., human IL-9 or murine IL-9), as well as associated polynucleotides and expression vectors. Provided herein are vectors (e.g., expression vectors) that include a polynucleotide that includes a nucleotide sequence or fragment encoding an anti-IL-9 antibody, for recombinant expression in a host cell, preferably a mammalian cell (e.g., a CHO cell). Also provided herein are host cells that include such vectors for recombinantly expressing an anti-IL-9 antibody (e.g., a human or humanized antibody) described herein. In aspects, provided herein are methods of producing an antibody described herein, comprising expressing the antibody from a host cell.

[0137] Recombinant expression of an antibody described herein (e.g., a full-length antigen-binding region of an antibody described herein or a heavy and / or light chain of the antibody) that specifically binds to IL-9 (e.g., human IL-9 or murine IL-9) generally involves the construction of an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide encoding an antibody molecule described herein, an antibody heavy and / or light chain, or a fragment thereof (e.g., a heavy and / or light chain variable region) is obtained, a vector for the production of the antibody molecule can be generated by recombinant DNA technology using techniques well known in the art. Thus, described herein are methods for preparing a protein by expressing a polynucleotide containing a nucleotide sequence encoding an antibody or antibody fragment (e.g., a light or heavy chain). Methods well known to those skilled in the art can be used to construct expression vectors containing sequences encoding an antibody or antibody fragment (e.g., a light or heavy chain) and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vitro genetic recombination. Also provided are replicable vectors comprising a nucleotide sequence encoding an antibody molecule described herein, an antibody heavy or light chain, an antibody heavy or light chain variable region or fragment thereof, or a heavy or light chain CDR, operably linked to a promoter. Such vectors can include, for example, a constant region of an antibody molecule (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036; and U.S. Patent No. 5,122,464, which are incorporated herein by reference in their entireties), and an antibody variable region can be cloned into such a vector for expression of an entire heavy chain, an entire light chain, or both the entire heavy and light chains.

[0138] In an embodiment, the vector comprises a polynucleotide encoding the VH, VL, heavy chain, and / or light chain of an antibody described herein. In another embodiment, the vector comprises a polynucleotide encoding the VH and VL of an antibody described herein. In another embodiment, the vector comprises a polynucleotide encoding the heavy and light chains of an antibody described herein.

[0139] The expression vector can be introduced into a cell (e.g., a host cell) by conventional techniques and the resulting cells can then be cultured by conventional techniques to produce an antibody or fragment thereof described herein. Thus, provided herein is a host cell containing a polynucleotide encoding an antibody or fragment thereof described herein, or a heavy or light chain thereof, or a fragment thereof, or a single chain antibody described herein, operably linked to a promoter for expression of such sequences in the host cell.

[0140] In an embodiment, the host cell comprises a polynucleotide encoding the VH and VL of the isolated antibody described herein. In another embodiment, the host cell comprises a vector comprising a polynucleotide encoding the VH and VL of the isolated antibody described herein. In another embodiment, the host cell comprises a first polynucleotide encoding the VH of the isolated antibody described herein and a second polynucleotide encoding the VL of the isolated antibody described herein. In another embodiment, the host cell comprises a first vector comprising a first polynucleotide encoding the VH of the isolated antibody described herein and a second vector comprising a second polynucleotide encoding the VL of the isolated antibody described herein.

[0141] In embodiments, the heavy chain / heavy chain variable region expressed by a first host cell associates with a light chain / light chain variable region of a second host cell to form an anti-IL-9 (e.g., human IL-9 or mouse IL-9) antibody described herein. In embodiments, provided herein is a population of host cells comprising such a first host cell and such a second host cell.

[0142] In embodiments, provided herein is a population of vectors comprising a first vector comprising a polynucleotide encoding the light chain / light chain variable region of an anti-IL-9 (e.g., human IL-9 or murine IL-9) antibody described herein, and a second vector comprising a polynucleotide encoding the heavy chain / heavy chain variable region of an anti-IL-9 (e.g., human IL-9 or murine IL-9) antibody described herein.

[0143] A variety of host-expression vector systems can be used to express the antibody molecules described herein (see, e.g., U.S. Patent No. 5,807,715, which is incorporated herein by reference in its entirety). Such host-expression systems are vehicles in which coding sequences of interest can be produced and subsequently purified, but also cells which, when transformed or transfected with the appropriate nucleotide coding sequences, are capable of expressing the antibody molecules described herein in situ. These include microorganisms, such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequences; yeast (e.g., Saccharomyces and Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequences; insect cell systems, for example, infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequences; plant cell systems (e.g., green algae, such as the green alga Chlamydomonas reinhardtii) infected with recombinant viral expression vectors (e.g., Cauliflower Mosaic Virus, CaMV; Tobacco Mosaic Virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody coding sequences. reinhardtii); or mammalian cell lines (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells) harboring a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter; a vaccinia virus 7.5K promoter). In an embodiment, the cells for expression of the antibodies described herein are Chinese Hamster Ovary (CHO) cells, e.g., CHO cells from the CHO GS System™ (Lonza).In an embodiment, the heavy and / or light chains of the antibody produced by the CHO cells may have an N-terminal glutamine or glutamic acid residue substituted with pyroglutamic acid. In an embodiment, the cells for expression of the antibodies described herein are human cells, e.g., human cell lines. In an embodiment, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In an embodiment, bacterial cells, e.g., E. coli, or eukaryotic cells (e.g., mammalian cells), especially for expression of complete recombinant antibody molecules, are used for expression of recombinant antibody molecules. Mammalian cells, e.g., CHO cells, in combination with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus, are an effective expression system for antibodies (Foecking MK & Hofstetter H (1986) Gene 45: 101-5; and Cockett MI et al. (1990) Biotechnology 8(7): 662-7, each of which is incorporated herein by reference in its entirety). In embodiments, the antibodies described herein are produced by CHO cells or NS0 cells. In embodiments, expression of a nucleotide sequence encoding an antibody described herein that specifically binds IL-9 (e.g., human IL-9 or mouse IL-9) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0144] In bacterial systems, several expression vectors can be advantageously selected depending on the intended use for the expressed antibody molecule. For example, if a large amount of such an antibody is to be produced, a vector that directs the expression of a high level of a fusion protein product that is easily purified for the production of a pharmaceutical composition of the antibody molecule may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794); pIN vector (Inouye S & Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24: 5503-5509), in which coding sequences can be individually ligated and inserted into the vector in frame with the lac Z coding region to produce a fusion protein, all of which are fully incorporated herein by reference. For example, pGEX vectors can be used to express heterologous polypeptides as fusion proteins with glutathione 5-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0145] In an insect system, for example, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. Coding sequences can be cloned individually into non-essential regions of the virus (e.g., the polyhedrin gene) and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0146] In mammalian host cells, several virus-based expression systems are available. When adenovirus is used as an expression vector, the coding sequence of interest can be ligated to the adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., regions El or E3) results in recombinant viruses that are viable and capable of expressing molecules in infected hosts (see, e.g., Logan J & Shenk T, (1984) PNAS 81(12): 3655-9, which is incorporated herein by reference in its entirety). Specific initiation signals may also be required for efficient translation of the inserted antibody coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bitter G et al., (1987) Methods Enzymol. 153:516-544, incorporated herein by reference in its entirety).

[0147] Furthermore, a host cell strain can be chosen which modulates the expression of the inserted sequences or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. An appropriate cell line or host system can be chosen to ensure the correct modification and processing of the expressed heterologous protein. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10 and HsS78Bst cells. In embodiments, the anti-IL-9 (e.g., human IL-9 or mouse IL-9) antibodies described herein are produced in mammalian cells, e.g., CHO cells.

[0148] In an embodiment, the antibody described herein has reduced or no fucose content. Such antibodies can be produced by those skilled in the art using techniques known to those skilled in the art. For example, the antibody can be expressed in cells that are defective or lack the ability to fucosylate. In one example, a cell line that knocks out both alleles of α1,6-fucosyltransferase can be used to produce antibodies with reduced fucose content. The Potelligent® system (Lonza) is an example of such a system that can be used to produce antibodies with reduced fucose content.

[0149] For long-term, high-yield production of recombinant proteins, stable expressing cells can be generated. For example, cell lines can be engineered that stably express the anti-IL-9 (e.g., human IL-9 or mouse IL-9) antibodies described herein. In embodiments, the cells provided herein stably express the light chain / light chain variable region and heavy chain / heavy chain variable region that assemble to form an antigen-binding region, or the antibody described herein.

[0150] In some embodiments, rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer sequences, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the heterologous DNA / polynucleotide, engineered cells can be allowed to grow in rich medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows the cells to stably integrate the plasmid into their chromosomes and grow to form foci that can be further cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express anti-IL-9 (e.g., human IL-9 or murine IL-9) or fragments thereof as described herein. Such engineered cell lines can be particularly useful in screening and evaluating compositions that interact directly or indirectly with antibody molecules.

[0151] Several selection systems can be used in tk-, hgprt- or aprt- cells, respectively, including, but not limited to, the herpes simplex virus thymidine kinase gene (Wigler M et al. (1977) Cell 11(1): 223-32), the hypoxanthine guanine phosphoribosyltransferase gene (Szybalska EH & Szybalski W (1962) PNAS 48(12): 2026-2034), and the adenine phosphoribosyltransferase gene (Lowy I et al. (1980) Cell 22(3): 817-23), all of which are incorporated herein by reference in their entireties. In addition, antimetabolite resistance has been associated with the following genes: dhfr, which confers resistance to methotrexate (Wigler M et al. (1980) PNAS 77(6): 3567-70; O'Hare K et al. (1981) PNAS 78: 1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); and neo, which confers resistance to the aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan RC (1993) Science 260: 926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62: 191-217; Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5): 211-5); and hygro, which confers resistance to hygromycin (Santerre RF et al., (1984) Gene 30(1-3): 147-56), all of which are incorporated herein by reference in their entireties.The desired recombinant clones can be selected by routine application of methods commonly known in the field of recombinant DNA technology, such as those described, for example, in Ausubel FM et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Chapters 12 and 13 of Dracopoli NC et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbere-Garapin F et al. (1981) J Mol Biol 150: 1-14, all of which are incorporated herein by reference in their entireties.

[0152] The expression level of an antibody molecule can be increased by vector amplification (for a review, see Bebbington CR & Hentschel CCG, The use of vector based on gene amplification for the expression of cloned genes in mammalian cells, Vol. 3 (Academic Press, New York, 1987), which is incorporated herein by reference in its entirety). If the marker in the vector system is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the gene of interest, protein production will also increase (Crouse GF et al., (1983) Mol Cell Biol 3: 257-66, which is incorporated herein by reference in its entirety).

[0153] A host cell can be co-transfected with two or more expression vectors described herein, where a first vector encodes a heavy chain derived polypeptide and a second vector encodes a light chain derived polypeptide. The two vectors can contain identical selectable markers that allow for equal expression of heavy and light chain polypeptides. A host cell can be co-transfected with different amounts of two or more expression vectors. For example, a host cell can be transfected with any one of the following ratios of the first expression vector and the second expression vector: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

[0154] Alternatively, a single vector can be used that can encode and express both heavy and light chain polypeptides. In such circumstances, the light chain should be placed before the heavy chain to avoid an excess of non-toxic heavy chain (Proudfoot NJ (1986) Nature 322: 562-565; and Kohler G (1980) PNAS 77: 2197-2199, each of which is incorporated herein by reference in its entirety). The coding sequences for the heavy and light chains can comprise cDNA or genomic DNA. The expression vector can be monocistronic or polycistronic. Polycistronic nucleic acid constructs can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes per nucleotide sequence, or in the range of 2-5, 5-10, or 10-20 genes per nucleotide sequence. For example, a bicistronic nucleic acid construct can include, in the following order: a promoter, a first gene (e.g., the heavy chain of an antibody described herein), and a second gene (e.g., the light chain of an antibody described herein). In such an expression vector, transcription of both genes can be driven by a promoter, but translation of mRNA from the first gene can be by a cap-dependent scanning mechanism and translation of mRNA from the second gene can be by a cap-independent mechanism, e.g., by an IRES.

[0155] Once an antibody molecule described herein is produced by recombinant expression, it can be purified by any method known in the art for the purification of immunoglobulin molecules, such as, for example, by chromatography (e.g., ion exchange, affinity, particularly affinity for a specific antigen followed by Protein A and sizing column chromatography), centrifugation, differential solubility, or any other standard protein purification technique. Further, the antibodies described herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0156] In embodiments, the antibodies described herein are isolated or purified. In embodiments, an isolated antibody is an antibody that is substantially free of other antibodies having antigen specificity different from the isolated antibody. For example, in certain embodiments, preparations of antibodies described herein are substantially free of cellular material and / or chemical precursors. The term "substantially free of cellular material" includes preparations of antibodies in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, antibodies that are substantially free of cellular material include preparations of antibodies having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins") and / or variants of antibodies, e.g., different post-translationally modified forms of antibodies or other different types of antibodies (e.g., antibody fragments). When the antibody is recombinantly produced, it is also usually substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation.When the antibody is produced by chemical synthesis, it is usually substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals involved in the synthesis of the protein.Thus, such preparations of the antibody have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest.In an embodiment, the antibody described herein is isolated or purified.

[0157] Anti-IL-9 (e.g., human IL-9 or murine IL-9) antibodies or fragments thereof can be produced by any method known in the art for the synthesis of proteins or antibodies, for example, by chemical synthesis or by recombinant expression techniques. The methods described herein utilize, unless otherwise indicated, conventional techniques of molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields, which are within the capabilities of one of ordinary skill in the art. These techniques are described in the references cited herein and are fully explained in the literature.See, e.g., Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annually revised); Current Protocols in Immunology, John Wiley & Sons (1987 and annually revised); Gait (ed.) (1984), Oligonucleotide Synthesis: A Practical Approach, IRL Press; See Eckstein (eds.) (1991), Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al. (eds.) (1999), Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press, all of which are incorporated herein by reference in their entireties.

[0158] In embodiments, the antibodies described herein are prepared, expressed, generated, or isolated by any means, including, for example, synthetically, by genetically engineering the creation of DNA sequences. In certain embodiments, such antibodies comprise sequences (e.g., DNA sequences or amino acid sequences) that do not naturally occur within the antibody germline repertoire of an animal or mammal (e.g., human) in vivo.

[0159] In one aspect, provided herein is a method of making an anti-IL-9 (e.g., human IL-9 or mouse IL-9) antibody, comprising culturing a cell or host cell described herein. In an embodiment, the method is performed in vitro. In an aspect, provided herein is a method of making an anti-IL-9 (e.g., human IL-9 or mouse IL-9) antibody, comprising expressing (e.g., recombinantly expressing) the antibody using a cell or host cell described herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody described herein). In an embodiment, the cell is an isolated cell. In an embodiment, an exogenous polynucleotide has been introduced into the cell. In an embodiment, the method further comprises purifying the antibody obtained from the cell or host cell.

[0160] In an embodiment, the isolated antibody is generated by expressing a polynucleotide encoding the VH and VL of the antibody described herein in a cell under suitable conditions for the polynucleotide to be expressed to generate the antibody. In another embodiment, the isolated antibody is generated by expressing a polynucleotide encoding the heavy and light chains of the antibody described herein in a cell under suitable conditions for the polynucleotide to be expressed to generate the antibody. In an embodiment, the isolated antibody is generated by expressing a first polynucleotide encoding the VH of the antibody described herein and a second polynucleotide encoding the VL of the antibody described herein in a cell under suitable conditions for the polynucleotide to be expressed to generate the antibody. In an embodiment, the isolated antibody is generated by expressing a first polynucleotide encoding the heavy chain of the antibody described herein and a second polynucleotide encoding the light chain of the antibody described herein in a cell under suitable conditions for the polynucleotide to be expressed to generate the antibody.

[0161] Methods for generating polyclonal antibodies are known in the art (see, e.g., Chapter 11 in Short Protocols in Molecular Biology, (2002) 5th ed., Ausubel FM et al. (eds.), John Wiley and Sons, New York, which is incorporated herein by reference in its entirety).

[0162] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma technology, including techniques known in the art and taught in, for example, Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling GJ et al., Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety. The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. For example, monoclonal antibodies can be recombinantly produced from host cells that exogenously express an antibody described herein or a fragment thereof, such as the light chain and / or the heavy chain of such an antibody.

[0163] In embodiments, a "monoclonal antibody" as used herein is an antibody produced by a single cell (e.g., a hybridoma or a recombinant antibody-producing host cell), which specifically binds to anti-IL-9 (e.g., human IL-9 or mouse IL-9) as determined, for example, by ELISA or other antigen-binding or competitive binding assays known in the art or in the examples provided herein. In embodiments, the monoclonal antibody may be a chimeric or humanized antibody. In embodiments, the monoclonal antibody is a monovalent antibody or a multivalent (e.g., bivalent) antibody. In embodiments, the monoclonal antibody is a monospecific or multispecific antibody (e.g., bispecific antibody). The monoclonal antibodies described herein can be produced by hybridoma methods, e.g., as described in Kohler G & Milstein C (1975) Nature 256: 495, which is incorporated herein in its entirety, or can be isolated from phage libraries, e.g., using techniques as described herein. Other methods for preparation of clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art (see, e.g., Chapter 11 in Short Protocols in Molecular Biology, 5th ed. (2002), Ausubel FM et al., supra).

[0164] As used herein, an antibody binds an antigen multivalently (e.g., bivalently) if the antibody comprises at least two (e.g., two or more) monovalent binding regions, each of which can bind to an epitope on the antigen. Each monovalent binding region can bind to the same or different epitopes on the antigen.

[0165] Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. For example, in the hybridoma method, a mouse or other suitable host animal, such as a sheep, goat, rabbit, rat, hamster, or macaque, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization (e.g., IL-9). Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding JW (ed.), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986), incorporated herein by reference in its entirety). Additionally, animals can be immunized using RIMMS (repetitive immunization multiple sites) technology (Kilpatrick KE et al., (1997) Hybridoma 16:381-9, incorporated herein by reference in its entirety).

[0166] In an embodiment, a mouse (or other animal, e.g., rat, monkey, donkey, pig, sheep, hamster, or dog) can be immunized with an antigen (e.g., IL-9), and when an immune response is detected, e.g., an antibody specific to the antigen is detected in the mouse serum, the mouse spleen is removed, and splenocytes are isolated. The splenocytes are then fused by well-known techniques with any suitable myeloma cells, e.g., cells from cell line SP20 available from American Type Culture Collection (ATCC®) (Manassas, VA), to form hybridomas. Hybridomas are selected and cloned by limiting dilution. In an embodiment, lymph nodes of the immunized mouse are harvested and fused with NS0 myeloma cells.

[0167] The hybridoma cells thus prepared are seeded and grown in an appropriate culture medium, preferably containing one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

[0168] In an embodiment, myeloma cells are utilized that fuse efficiently, support stable high-level production of antibodies by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these myeloma cell lines are NS0 cell lines, or those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, CA, USA, and mouse myeloma cell lines such as SP-2 or X63-Ag8.653 cells available from the American Type Culture Collection, Rockville, MD, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor D, (1984) J Immunol 133: 3001-5; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), each of which is incorporated herein by reference in its entirety).

[0169] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies against IL-9 (e.g., human IL-9 or murine IL-9). The binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by methods known in the art, such as immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0170] After hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding JW (ed.), Monoclonal Antibodies: Principles and Practice, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI 1640 medium. Additionally, hybridoma cells can be grown in vivo as ascites tumors in an animal.

[0171] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0172] The antibodies described herein include, for example, antibody fragments that recognize IL-9 (e.g., human IL-9 or mouse IL-9) and can be generated by any technique known to those of skill in the art. For example, the Fab and F(ab') fragments described herein can be used to identify IL-9 or human IL-9. 2 Fragments may be purified using papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 F(ab') fragments can be generated by proteolytic cleavage of immunoglobulin molecules using enzymes such as Fab' (Fab') and Fab' (Fab')'s. Fab fragments correspond to one of the two identical arms of an antibody molecule and contain an intact light chain paired with the VH and CH1 domains of the heavy chain.2 The fragment contains the two antigen-binding arms of an antibody molecule linked by disulfide bonds in the hinge region.

[0173] Additionally, the antibodies described herein can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or mouse cDNA libraries of diseased tissues). The DNA encoding the VH and VL domains are recombined with an scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated into E. coli, and the E. coli is infected with helper phage. The phages used in these methods are typically filamentous phages, including fd and M13, and the VH and VL domains are typically recombinantly fused to either phage gene III or gene VIII. Phage expressing antigen-binding regions that bind to a particular antigen can be selected or identified with the antigen, for example, with labeled antigen, or with antigen bound or captured to a solid surface or bead.Examples of phage display methods that can be used to generate the antibodies described herein include those described in Brinkman U et al. (1995) J Immunol Methods 182: 41-50; Ames RS et al. (1995) J Immunol Methods 184: 177-186; Kettleborough CA et al. (1994) Eur J Immunol 24: 952-958; Persic L et al. (1997) Gene 187: 9-18; Burton DR & Barbas CF (1994) Advan Immunol 57: 191-280; PCT Application PCT / GB91 / 001134; International Publication Nos. WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO Nos. WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, WO 95 / 20401, and WO 97 / 13844; as well as those disclosed in U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108, all of which are incorporated herein by reference in their entireties.

[0174] As described in the above references, after phage selection, for example as described below, the antibody coding region from the phage can be isolated and used to generate complete antibodies, including human antibodies, or any other desired antigen-binding fragment, for expression in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria. Fab, Fab' and F(ab') fragments can be isolated using methods known in the art, such as those disclosed in PCT Publication WO 92 / 22324; Mullinax RL et al., (1992) BioTechniques 12(6): 864-9; Sawai H et al., (1995) Am J Reprod Immunol 34: 26-34; and Better M et al., (1988) Science 240: 1041-1043. 2 Techniques are also available for the recombinant production of antibody fragments, such as the antibody fragments ##STR00011## all of which are incorporated herein by reference in their entireties.

[0175] In some embodiments, to generate a complete antibody, a PCR primer containing a VH or VL nucleotide sequence, a restriction site, and adjacent sequences to protect the restriction site can be used to amplify the VH or VL sequence from a template, e.g., an scFv clone. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a VH constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a VL constant region, e.g., a human kappa or lambda constant region. The VH and VL domains can also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into a cell line using techniques known to those skilled in the art to generate a stable or transient cell line expressing a full-length antibody, e.g., an IgG.

[0176] A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody can contain the variable region of a mouse or rat monoclonal antibody fused to the constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison SL (1985) Science 229: 1202-7; Oi VT & Morrison SL (1986) BioTechniques 4: 214-221; Gillies SD et al. (1989) J Immunol Methods 125: 191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415, all of which are fully incorporated herein by reference.

[0177] A humanized antibody can bind to a given antigen and comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin (e.g., a murine immunoglobulin). In certain embodiments, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin. The antibody can also comprise the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Humanized antibodies can be of any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, as well as IgG. 1 , IgG 2 , IgG 3 , and IgG 4Humanized antibodies can be selected from any isotype, including, but not limited to, IgG, IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18, IgG19, IgG20, IgG210, IgG220, IgG230, IgG240, IgG250, IgG251, IgG260, IgG270, IgG280, IgG290, IgG300, IgG310, IgG320, IgG330, IgG410, IgG420, IgG430, IgG440, IgG450, IgG460, IgG470, IgG480, IgG49 ... 969-973), chain shuffling (U.S. Pat. No. 5,565,332), as well as methods such as those described in, for example, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication WO 93 / 17105; Tan P et al. (2002) J Immunol 169: 1119-25; Caldas C et al. (2000) Protein Eng. 13(5): 353-60; Morea V et al. (2000) Methods 20(3): 267-79; Baca M et al. (1997) J Biol Chem 272(16): 10678-84; Roguska MA et al. (1996) Protein Eng 9(10): 895 904; Couto JR et al. (1995) Cancer Res. 55 (23 Supp): 5973s-5977s; Couto JR et al. (1995) Cancer Res 55(8): 1717-22; Sandhu JS (1994) Gene 150(2): 409-10 and Pedersen JT et al. (1994) J Mol Biol 235(3): 959-73, all of which are fully incorporated herein by reference. See also U.S. Patent Application Publication No. US 2005 / 0042664 A1 (February 24, 2005), which is fully incorporated herein by reference.

[0178] Methods for making multispecific antibodies (e.g., bispecific antibodies) have been described, see, e.g., U.S. Patent Nos. 7,951,917; 7,183,076; 8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992, and 8,586,713, all of which are incorporated by reference in their entireties.

[0179] Bispecific, bivalent antibodies, and methods for making the same, are described, for example, in U.S. Patent Nos. 5,731,168, 5,807,706, 5,821,333, and U.S. Patent Application Publication Nos. 2003 / 020734 and 2002 / 0155537; each of which is incorporated herein by reference in its entirety. Bispecific, tetravalent antibodies, and methods for making the same, are described, for example, in International Application Publication Nos. WO 02 / 096948 and WO 00 / 44788, the disclosures of both of which are incorporated herein by reference in their entireties. See generally, International Application Publication Nos. WO 93 / 17715, WO 92 / 08802, WO 91 / 00360, and WO 92 / 05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; and 5,601,819; and Kostelny et al., J. Immunol. 148:1547-1553 (1992), each of which is incorporated by reference in its entirety.

[0180] The bispecific antibodies described herein can be produced, for example, according to the DuoBody technology platform (Genmab A / S) described in International Publications WO 2011 / 131746, WO 2011 / 147986, WO 2008 / 119353, and WO 2013 / 060867, and in Labrijn AF et al., (2013) PNAS 110(13): 5145-5150. The DuoBody technology can be used to combine a first monospecific antibody comprising two heavy chains and two light chains, or half of a first antigen-binding region, with a second monospecific antibody comprising two heavy chains and two light chains, or half of a second antigen-binding region. The resulting heterodimer contains one heavy and one light chain from the first antibody, or a first antigen-binding region, paired with one heavy and one light chain from the second antibody, or a second antigen-binding region. If both monospecific antibodies, or antigen-binding regions, recognize different epitopes on different antigens, the resulting heterodimer is a bispecific antibody.

[0181] The DuoBody technology requires that each of the monospecific antibodies or antigen-binding regions comprises a heavy chain constant region with a single point mutation in the CH3 domain. The point mutation results in stronger interactions between the CH3 domains in the resulting bispecific antibody than between the CH3 domains of either of the monospecific antibodies or antigen-binding regions. The single point mutation in each monospecific antibody or antigen-binding region is, for example, residue 366, 368, 370, 399, 405, 407, or 409 in the CH3 domain of the heavy chain constant region, numbered according to the EU numbering system, as described in International Publication WO 2011 / 131746. Furthermore, the single point mutation is located at a different residue in one monospecific antibody or antigen-binding region compared to the other monospecific antibody or antigen-binding region. For example, one monospecific antibody, or antigen-binding region, may contain the mutation F405L (i.e., a phenylalanine to leucine mutation at residue 405), while the other monospecific antibody, or antigen-binding region, may contain the mutation K409R (i.e., a lysine to arginine mutation at residue 409), numbered according to the EU numbering system. The heavy chain constant region, or antigen-binding region, of the monospecific antibody may be an IgG 1 , IgG 2 , IgG 3 , or IgG 4 Isotype (e.g., human IgG 1 isotype), and the bispecific antibodies generated by DuoBody technology can retain Fc-mediated effector functions.

[0182] Another method to generate bispecific antibodies is called the "knobs-into-holes" strategy (see, for example, International Publication WO 2006 / 028936). In this technique, mispairing of Ig heavy chains is reduced by mutating selected amino acids that form the interface of the CH3 domain of IgG. At positions in the CH3 domain where the two heavy chains directly interact, an amino acid with a small side chain (hole) is introduced into the sequence of one heavy chain, and an amino acid with a large side chain (knob) is introduced into the corresponding interacting residue position on the other heavy chain. In some embodiments, the compositions of the invention have immunoglobulin chains whose CH3 domains have been modified by mutating selected amino acids that interact at the interface between the two polypeptides to preferentially form bispecific antibodies. Bispecific antibodies are antibodies that are directed against two polypeptides of the same subclass (e.g., IgG 1 or IgG 3 ) or different subclasses (e.g., IgG 1 and IgG 3 , or IgG 3 and IgG 4 ) immunoglobulin chains.

[0183] Bispecific antibodies may optionally be 4 and IgG 1 , IgG 4 and IgG 2 , IgG 4 and IgG 2 , IgG 4 and IgG 3 , or IgG 1 and IgG 3 Such heterodimeric heavy chain antibodies can be made, for example, with a heterodimer of the heavy chain of human IgG, such that heterodimeric heavy chain formation is favored. 4 and IgG 1 or IgG 3 These can be routinely engineered by modifying selected amino acids that form the interface of the CH3 domain in.

[0184] In embodiments, an antibody described herein that binds to the same epitope of IL-9 (e.g., human IL-9 or mouse IL-9) as the anti-IL-9 antibody described herein is a human antibody. In embodiments, an antibody described herein that competitively (e.g., dose-dependently) blocks any one of the antibodies described herein from binding to IL-9 (e.g., human IL-9 or mouse IL-9) is a human antibody. Human antibodies can be generated using any method known in the art. For example, transgenic mice that are incapable of expressing functional endogenous immunoglobulins but can express human immunoglobulin genes can be used. In particular, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be made non-functional separately or simultaneously with the introduction of the human immunoglobulin loci by homologous recombination. In particular, J HHomozygous deletion of the region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to generate homozygous offspring expressing human antibodies. The transgenic mice are immunized in the usual manner with a selected antigen, e.g., all or a portion of an antigen (e.g., IL-9). Monoclonal antibodies against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice are rearranged during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, such technology can be used to generate therapeutically useful IgG, IgA, IgM, and IgE antibodies. For an overview of this technology for generating human antibodies, see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93, which is incorporated herein by reference in its entirety. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies, as well as protocols for producing such antibodies, see, e.g., International Publication Nos. WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735; 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, and 5,939,598, all of which are incorporated herein by reference in their entireties. An example of a mouse capable of producing human antibodies is the Xenomouse mouse. (商標) (Abgenix; U.S. Patent Nos. 6,075,181 and 6,150,184), the HuAb-Mouse (商標) (Medarex / Gen Pharm; U.S. Patent Nos. 5,545,806 and 5,569,825), the Trans Chromo Mouse (商標) (Kirin) and the KM Mouse (商標) (Medarex / Kirin), all of which are incorporated herein by reference in their entireties.

[0185] Human antibodies that specifically bind IL-9 (e.g., human IL-9 or murine IL-9) can be made by a variety of methods known in the art, including the phage display methods described above, using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887, 4,716,111, and 5,885,793; and International Publication Nos. WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741, all of which are incorporated herein by reference in their entireties.

[0186] In some embodiments, human antibodies can be produced using mouse-human hybridomas. For example, Epstein-Barr Virus (EBV)-transformed human peripheral blood lymphocytes can be fused with mouse myeloma cells to produce mouse-human hybridomas that secrete human monoclonal antibodies, and these mouse-human hybridomas can be screened to determine which lymphocytes secrete human monoclonal antibodies that specifically bind to a target antigen (e.g., IL-9). Such methods are known and described in the art, see, for example, Shinmoto H et al., (2004) Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14: 27-31, each of which is incorporated herein by reference in its entirety.

[0187] (5.6 kit) Also provided is a kit comprising one or more antibodies, or pharmaceutical compositions or conjugates thereof, as described herein. In an embodiment, provided herein is a pharmaceutical pack or kit comprising one or more containers filled with one or more of the components of the pharmaceutical compositions described herein, such as one or more antibodies as provided herein. In an embodiment, the kit contains the pharmaceutical compositions described herein and any prophylactic or therapeutic agent, such as those described herein. In an embodiment, the kit may contain, for example, a T cell mitogen, such as phytohemagglutinin (PHA) and / or phorbol myristate acetate (PMA), or a TCR complex stimulating antibody, such as an anti-CD3 antibody and an anti-CD28 antibody. Such container(s) may optionally be associated with a notice in the form prescribed by a government agency regulating the manufacture, use, or sale of drugs or biological products, which notice indicates the approval by the agency of the manufacture, use, or sale for human administration.

[0188] Also provided are kits that can be used in the above methods. In embodiments, the kits comprise, in one or more containers, an antibody, preferably a purified antibody, as described herein. In embodiments, the kits described herein contain a substantially isolated IL-9 (e.g., human IL-9 or mouse IL-9) antigen as a control. In embodiments, the kits described herein further comprise a control antibody that does not react with the IL-9 (e.g., human IL-9 or mouse IL-9) antigen. In embodiments, the kits described herein contain one or more elements for detecting the binding of an antibody to an IL-9 (e.g., human IL-9 or mouse IL-9) antigen (e.g., the antibody can be conjugated to a detectable substrate, e.g., a fluorescent compound, an enzymatic substrate, a radioactive compound, or a luminescent compound, or a second antibody that recognizes the first antibody can be conjugated to a detectable substrate). In embodiments, the kits provided herein can comprise a recombinantly produced or chemically synthesized IL-9 (e.g., human IL-9 or mouse IL-9) antigen. The IL-9 (e.g., human IL-9 or mouse IL-9) antigen provided in the kit can also be bound to a solid support. In an embodiment, the detection means of the above kit comprises a solid support to which the IL-9 (e.g., human IL-9 or mouse IL-9) antigen is bound. Such a kit can also comprise a non-bound reporter-labeled anti-human antibody or anti-mouse / rat antibody. In this embodiment, the binding of the antibody to the IL-9 (e.g., human IL-9 or mouse IL-9) antigen can be detected by the binding of said reporter-labeled antibody. In one embodiment, the present invention relates to the use of the kit of the present invention for in vitro assay and / or detection of IL-9 (e.g., human IL-9 or mouse IL-9) antigen in a biological sample. EXAMPLES

[0189] 6. Working Examples The examples in this section (ie, Section 6) are offered by way of illustration and not by way of limitation.

[0190] 6.1 Example 1: Generation of neutralizing IL-9 monoclonal antibodies A. Llama immunization and library construction Llamas, housed outdoors in accordance with French animal welfare legislation, were immunized intramuscularly with recombinant human IL-9 or murine IL-9 (R&D Systems) and boosted weekly for 6 weeks. Briefly, each llama (total of 4) received 40 μg of IL-9 buffered in phosphate-buffered saline (PBS) and mixed with incomplete Freund's adjuvant (Sigma-Aldrich) for the first 2 weeks and 20 μg of IL-9 for the remaining 4 weeks. Fab library generation was performed using the SIMPLE antibody platform as previously described (see WO2010 / 001251, the contents of which are incorporated herein in their entirety). Five days after the last immunization, 400 mL of blood containing peripheral blood lymphocytes was collected from the llama, purified by centrifugation on a Ficoll-Paque gradient, and used for the extraction of total RNA. Total RNA was then converted to random-primed cDNA using reverse transcriptase, and gene sequences encoding the VH-CH1 region and the VL-CL domains (kappa and lambda) of llama IgG1 were isolated by PCR and subcloned into the phagemid vector pCB3, which allows the expression of recombinant antibodies as Fab fragments fused to the phage pIII envelope protein.

[0191] B. Selection of Fabs that Bind to IL-9 E. coli TG1 (Netherlands Culture Collection of Bacteria) was transformed with the recombinant phagemids to generate Fab-expressing phage libraries (one lambda and one kappa library per immunized llama). The resulting Fab-expressing phages, with diversities ranging from 108 to 109, were then adsorbed to immobilized recombinant biotinylated IL-9 and eluted with trypsin as previously described (De Haard et al. (1999) Journal of Biological Chemistry, 274: 18218-30). Three rounds of selection were performed to enrich for phages expressing IL-9-specific Fabs. Finally, TG1 E. coli were infected with the selected phages and individual colonies were isolated. Secretion of Fabs into the periplasm of E. coli strain TG1 was induced under low glucose concentrations (0.1% w / v) using isopropyl β-D-1-thiogalactopyranoside (Sigma-Aldrich) and the Fab-containing periplasmic fraction was collected.

[0192] C. Screening, Characterization, and Generation of Fabs Binding of the Fabs (periplasmic extracts) to their respective mouse or human targets was determined by surface plasmon resonance (SPR) using a Biacore 3000 instrument (GE Healthcare). IL-9 was immobilized on a carboxymethyl dextran sensor chip (CM-5) using amine coupling in sodium acetate buffer (GE Healthcare). Fab-containing periplasmic extracts were loaded at a flow rate of 30 μL / min. Fab binding and off-rates were measured over 90 seconds (Table 5). Binding clones were sequenced and VHs were grouped into families. From this selection, 11 different families were identified for human IL-9 and 10 for mouse IL-9. In addition, the ability of the antibodies to compete for binding of human or mouse IL-9 to human or rat IL-9R was also tested on the Biacore 3000. For this assay, human or rat IL-9R was coated at high density onto a carboxymethyl dextran sensor chip (CM-5). A pre-prepared mixture of periplasmic extract (Fab) and IL-9 was then injected. IL-9 did not bind to the coated receptor, indicating that Fab binding was competing with IL-9R binding. Table 5. Off-rates of Fab from periplasmic extracts [Table 5] (nb = not bound)

[0193] D. Generation, Purification, and Characterization of Monospecific Abs The eight most prevalent (k off s -1The cDNAs encoding the VH and VL (lambda or kappa) domains of the neutralizing hIL-9 specific Fabs (those with the lowest potency: 7D6, 8C3, 6C4, 6E2, 7A4, 6D3, 6F2, and 8G3) were selected and re-engineered as full IgGs. Full IgGs were cloned into two separate pUPE mammalian expression vectors, one containing cDNAs encoding the CH1, CH2, and CH3 domains of human IgG1 containing mutations that disrupt Fc receptor-mediated Ab effector functions, the other containing the CL domain (lambda or kappa). For the anti-mIL-9 Fabs, only the VH and VL of the most potent one (35D8) were recloned as full mIgG2a. Antibodies were produced by transient transfection of mammalian cells and purified by protein A affinity chromatography as previously described (Basilico et al. (2014) Journal of Clinical Investigation 124:3172).

[0194] The CDR, VH and VL sequences of the selected antibodies are shown in Tables 2-4 above.

[0195] 6.2. Example 2: In Vitro Characterization of IL-9 mAbs IL-9 mAbs were tested for their ability to bind to their respective targets in vitro and inhibit cellular effects mediated by IL-9 signaling.

[0196] A. Inhibition of IL-9-induced Baf3hIL9RA6 cell proliferation The neutralizing activity of IL-9 mAbs was assessed in an in vitro cell assay using Baf3hIL9RA6 cells, which proliferate in response to IL-9.

[0197] Human IL-9 SN baculo (50 U / mL) was incubated with eight different concentrations of IL-9 mAb (ng / ml) for 30 min. Then, 3,000 Baf3h9RA6 cells were added, and after 3 days, hexosaminidase substrate was added for 2 h 30 min and hexosaminidase activity was measured. As shown in Figure 1A, IL-9-specific mAb potently inhibited human IL-9-induced Baf3hIL9RA6 cell proliferation. Specifically, hIL-9 mAb blocked cell proliferation induced by human IL-9 with IC50s ranging from 61 pM to 6.3 nM. To confirm the high potency of some antibodies, new batches of antibodies were generated and tested again (Figure 1B). High potency of anti-hIL9 antibodies 6E2 and 6D3 (IC50 of 30.85 pM and 58.35 pM, respectively) was observed, while a lower potency of 7D6 (2.11 nM) was also observed.

[0198] Mouse IL-9 mAbs were also tested for potency in vitro. Mouse IL-9 SN baculo (20 U / ml) was incubated with eight different concentrations of anti-IL9 (ng / ml) for 30 min. 3,000 TS1 cells were then added and hexosaminidase activity was measured after 3 days. Hexosaminidase substrate was added 2 h 30 min before the measurement. As shown in Figure 1A, antibody 35D8 neutralized mIL9 with a potency of 46 pM. The very high potency of the mIL9 antibodies was confirmed, with an IC50 of 20.06 pM for the new antibody batch (mIgG1-N297A) (Figure 1B).

[0199] B. Affinity of Neutralizing IL-9 mAbs Biolayer interferometry (BLI) experiments were used to analyze the affinity of three anti-hIL-9 antibodies (6E2, 6D3, and 7D6) and one anti-mIL-9 antibody (35D8) (Figure 2). BLI experiments with antagonist antibodies were performed in kinetics buffer (PBS, 0.1% (w / v) BSA, 0.02% (v / v) Tween20) at 298K using an Octet Red 96 machine (Sartorius). Anti-hIgG Fc capture (AHC) or anti-mIgG Fc capture sensor (Sartorius) was functionalized with IL-9 mAb. The functionalized chip was then immersed in different concentrations of hIL-9 or mIL-9 (both from R&D systems). A non-functionalized chip was used as a negative control in a double-reference setup. After subtraction of the control sensorgrams, a 1:1 binding model was fitted to the obtained data. Data analysis was performed using data analysis software 9.0.0.14 (Sartorius) (Table 6). The results show that the anti-hIL-9 antibody had a strong affinity for hIL-9, and the anti-mIL-9 antibody 35D8 had a strong affinity for mIL-9. Table 6. Kinetic properties of IL-9 mAbs [Table 6]

[0200] 6.3 Example 3: In vivo characterization of anti-IL-9 mAbs in a mouse model of asthma Patients with acute asthma who suffer from uncontrolled disease despite maximal corticosteroid therapy require further treatment with biologics. Type 2 helper T cells (Th2) are the key cell type driving asthma pathology. However, in addition to Th2 cells, their natural counterparts, namely group 2 innate lymphoid cells (ILC2), are also thought to play a major role, especially in patients with acute asthma due to steroid resistance.

[0201] ILC2s are characterized by high expression of IL-9, which has been shown to promote ILC2 proliferation in an autocrine manner, therefore blocking IL-9 with antibodies may provide a solution for patients with acute ILC2-driven asthma.

[0202] The antibodies are tested in vivo using an ILC2 / IL-9-dependent mouse asthma model. In this mouse model, recombinant IL-33 is administered to the lungs of C57BL / 6J mice to activate ILC2 (Du et al., 2020). Anti-IL-9 mAb is administered intraperitoneally at a dose of 200 μg for three consecutive days (n=6 per group) (Figure 3). On the same day, mice are lightly anesthetized with isoflurane (2.5% in air) and challenged intratracheally with 150 ng of rIL-33. IL-33 challenge is performed at least 4 hours after anti-IL9 mAb administration to ensure adequate biodistribution of the mAb. On day 4, mice are sacrificed, bronchoalveolar lavage (BAL) is performed, and whole lungs are removed. The removed lungs are digested with RPMI containing 10% FCS + Liberase 1 / 50 and DNAse 1 / 1000 to obtain a single cell suspension. Bronchoalveolar lavage (BAL) fluid and cellular fractions of lung samples will be further analyzed by flow cytometry using a flow panel designed to analyze eosinophilia and ILC2 activation markers.

[0203] 6.4 Example 4: Structural analysis of conjugated anti-IL-9 antibodies The structure of the Fab:IL-9 complex was determined by X-ray crystallography. The first Fab:hIL-9 complex (Fab 6D3:hIL-9) crystals yielded a data set with a resolution of 1.7 Å (Table 7). The map obtained after stepwise molecular replacement with a model of the Fab allowed to build hIL-9 de novo with electron density without any trends. Regarding the Fab:hIL-9 interface, Fab 6D3 targets hIL-9 mainly by binding the C helix and the first half of the A helix with a polar footprint covering an interface area of ​​750.9 Å2 (Figure 4A and Table 8). Two Arg-Asp interactions influence the specificity of the interaction (Figure 4A). This includes Arg91 in IL-9, which is also involved in the interaction with hIL-9Ra.

[0204] Next, two more Fab:hIL-9 complexes were structurally determined (Figures 4B and 4C and Table 7). Fab 6E2 binds hIL-9 with an average interface area of ​​987.6 Å2. The cytokine is positioned with its A helix between the light and heavy chains of the Fab (Figure 4B and Table 8). The light chain of Fab 6E2 specifically interacts with the C helix by coordinating Arg91 of the cytokine via Asp31 and Asp49 (Figure 4B). Another Asp on the light chain, Asp95, interacts with the main chain of Leu24 located at the tip of helix A. The heavy chain of Fab 6E2 joins this interaction site by engaging with the A helix and part of the D helix. Furthermore, Fab 7D6 binds primarily to the A helix, with the heavy chain wrapping around the first half of the helix and the light chain wrapping around the second half, creating an interface area of ​​862.8 Å (Figure 4C and Table 8). Even though this interaction primarily wraps around the A helix, the light chain still engages Arg91 through an interaction with the main chain of Lys94 on the light chain (Figure 4C).

[0205] Thus, all Fab:hIL-9 complexes engage Arg91 of IL-9 in a specific interaction.

[0206] Next, to understand how the antibodies inhibit the binding of hIL-9 to its receptor and the different effectiveness of the three antibodies in inhibiting the IL-9 signaling pathway, the structure of the binary hIL-9:hIL-9Ra complex was superimposed with the structure of the Fab:hIL-9 complex based on the superposition of hIL-9 in each structure (Figure 4D). All three Fabs, and therefore the corresponding full antibodies, partially overlap the binding site of the receptor, thus sterically hindering the binding of IL-9 to the receptor.

[0207] Antibody 7D6 inhibits IL-9 poorly in cell proliferation assays, whereas 6E2 performs very well (IC50 = 2.1 nM vs. 30.8 pM) (Table 6). As the large difference in potency cannot be explained by only a slight difference in affinity (KD = 1.8 nM for 7D6 vs. 0.24 nM for 6E2), the epitopes of these Fabs were further analyzed. Both Fab fragments extensively cover the A helix. However, their orientation on hIL-9 is perpendicular to each other. This allows Fab 6E2 to retain hIL-9 in the gap between the light and heavy chains and also covers the C helix to a large extent (Figures 4B and 4D and Table 9). On the other hand, Fab 7D6 barely covers the C helix (Figures 4C and 4D and Table 9). This availability of the C helix may create an opening for hIL-9Rα to engage hIL-9 via the C helix and force dissociation of Fab from hIL-9.

[0208] Further analysis of the epitope of Fab 6D3 was also performed. This antibody is slightly less potent than antibody 6E2 in inhibiting IL-9 signaling in cell reporter assays (Table 6). The structure of Fab6D3:hIL-9 shows that Fab 6D3 binds mainly through the C-helix, leaving the A-helix almost completely available (Figures 4A and 4D). This indicates that the availability of the A-helix is ​​not sufficient for the receptor hIL-9Rα to efficiently bind hIL-9 and remove the Fab. Fab 6D3 does not completely cover the hIL-9Rα binding site on the C-helix, which could explain the difference in efficacy between mAb 6E2 and mAb 6D3 (Figure 4D).

[0209] These results indicate that the efficacy of antibodies in inhibiting hIL-9:hIL-9Rα interaction and the hIL-9 signaling pathway correlates with the ability of the antibody to cover the hIL-9Rα binding site on the C-helix of hIL-9. This indicates that the C-helix is ​​important in the interaction of hIL-9 with hIL-9Rα. Therefore, an efficient therapeutic neutralizing agent (e.g., anti-IL-9 antibody) needs to target this C-helix since it is the main entry point of hIL-9Rα on hIL-9. Table 7: Crystallographic data and refinement statistics [Table 7] Table 8. Summary of buried residues and hydrogen bonds at the interaction interface. Buried residues and hydrogen bonds at the interaction interface analyzed by PISA. For the Fab:h / mIL-9 complex, only the interface between Fab and h / mIL-9 was analyzed. [Table 8] TIFF2024522213000012.tif126170TIFF2024522213000013.tif226170Table 9. Antibody heavy and light chain interactions with buried IL-9 residues [Table 9]

[0210] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to be included within the scope of the appended claims.

[0211] All references (e.g., publications or patents or patent applications) cited in this specification are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated herein by reference in its entirety for all purposes.

[0212] Other embodiments are within the scope of the following claims.

Claims

1. An isolated antibody that specifically binds to human or mouse IL-9, wherein said antibody has, respectively, SEQ ID NO (a) 1, 2, 3, 22, 23, and 24; (b) 4, 5, 6, 25, 26, and 27; (c) 7, 8, 9, 28, 29, and 30; (d) 7, 8, 9, 31, 29, and 32; (e) 10, 11, 12, 33, 34, and 35; (f) 13, 14, 15, 36, 37, and 38; (g) 16, 17, 18, 39, 40, and 41; (h) 19, 20, 21, 36, 37, and 38; or (i) 122, 123, 124, 125, 126, and 127 and comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences set forth therein.

2. The isolated antibody according to claim 1, wherein said antibody comprises the VH amino acid sequence and the VL amino acid sequence of SEQ ID NOs 42 and 51; 43 and 55; 44 and 54; 45 and 53; 46 and 50; 47 and 52; 48 and 50; 49 and 56; or 128 and 129, respectively.

3. The isolated antibody according to claim 2, wherein the amino acid sequences of said VH and VL consist of the amino acid sequences of SEQ ID NOs 42 and 51; 43 and 55; 44 and 54; 45 and 53; 46 and 50; 47 and 52; 48 and 50; 49 and 56; or 128 and 129, respectively.

4. The antibody is human IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 The isolated antibody according to claim 1, comprising a heavy chain constant region selected from the group consisting of

5. The isolated antibody according to claim 1, wherein said antibody comprises a heavy chain constant region that is a variant of the wild-type heavy chain constant region, and wherein said variant heavy chain constant region binds to FcγR with a higher affinity than the wild-type heavy chain constant region that binds to FcγR.

6. The isolated antibody according to claim 5, wherein said FcγR is FcγRIIB or FcγRIIIA.

7. (i) the amino acid at position 297 of said heavy chain constant region is A or Q according to the EU numbering system, or (ii) the amino acids at positions 234 and 235 of said heavy chain constant region are both A according to the EU numbering system, or (iii) the amino acids at positions 433, 434, and 436 of said heavy chain constant region are K, F, and Y, respectively, according to the EU numbering system, or (iv) the amino acids at positions 252, 254, and 256 of said heavy chain constant region are Y, T, and E, respectively, according to the EU numbering system, or (v) the amino acids at positions 428 and 434 of said heavy chain constant region are L and S, respectively, according to the EU numbering system, or (vi) The isolated antibody according to claim 4, wherein the amino acids at positions 309, 311, and 434 of the heavy chain constant region are D, H, and S, respectively, according to the EU numbering system.

8. The isolated antibody according to claim 1, wherein the antibody inhibits the binding of human IL-9 to human IL-9Rα.

9. The antibody binds to human IL-9 with a K D of less than 1 nM, the isolated antibody according to claim 1.

10. The isolated antibody according to claim 1, wherein the antibody is bispecific.

11. The isolated antibody according to claim 1, wherein the isolated antibody is conjugated to a cytotoxic agent, a cell division inhibitor, a toxin, a radionuclide, or a detectable label.

12. An isolated polynucleotide encoding VH and / or VL, or a heavy chain and / or a light chain, of the isolated antibody according to any one of claims 1 to 11.

13. A vector comprising the polynucleotide according to claim 12.

14. A recombinant host cell comprising the vector according to claim 13.

15. A pharmaceutical composition comprising the isolated antibody according to any one of claims 1 to 11.

16. A method for generating an isolated antibody, comprising culturing the host cell according to claim 14 under appropriate conditions such that the polynucleotide is expressed to generate the isolated antibody.

17. A method for generating an isolated antibody, (a) a first polynucleotide encoding VH of the antibody according to any one of claims 1 to 11, and a second polynucleotide encoding VL of the antibody according to any one of claims 1 to 11; or (b) a first polynucleotide encoding the heavy chain of the antibody according to any one of claims 1 to 11, and a second polynucleotide encoding the light chain of the antibody according to any one of claims 1 to 11 are expressed intracellularly under appropriate conditions such that the polynucleotide is expressed to generate the antibody.

18. A pharmaceutical composition for treating an inflammatory disease, cancer, or autoimmune disease in a subject, comprising the isolated antibody according to any one of claims 1 to 11.

19. The pharmaceutical composition according to claim 18, wherein the isolated antibody, polynucleotide, vector, host cell, or pharmaceutical composition is for systemic, intravenous, subcutaneous, or intratumoral administration, or for delivery to tumor-draining regional lymph nodes.

20. Use of the isolated antibody according to any one of claims 1 to 11 in the manufacture of a medicament for the treatment of an inflammatory disease, cancer or autoimmune disease in a subject.

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