BR122024010248A2 - BINDING MOLECULE THAT BITS TO CCR9, THERAPEUTIC AGENT, METHOD FOR ASSESSING THE DEPLETION OF CCR9-EXPRESSING CELLS BY A BINDING MOLECULE, ISOLATED POLYNUCLEOTIDE, VECTOR, HOST CELL, METHOD FOR PRODUCTION OF A BINDING MOLECULE, PHARMACEUTICAL COMPOSITION, METHOD FOR DETECTING THE PRESENCE OF A CCR9 POLYPEPTIDE IN A SAMPLE, USES AND KITS - Google Patents

Abstract

"BINDING MOLECULE THAT BIDS TO CCR9, THERAPEUTIC AGENT, METHOD FOR ASSESSING THE DEPLETION OF CELLS EXPRESSING CCR9 BY A BINDING MOLECULE, ISOLATED POLYNUCLEOTIDE, VECTOR, HOST CELL, METHOD FOR PRODUCING A BINDING MOLECULE, PHARMACEUTICAL COMPOSITION, METHOD FOR DETECTING THE PRESENCE OF A CCR9 POLYPEPTIDE IN A SAMPLE, USES AND KITS". The invention relates to binding molecules, such as antibodies, that bind to the chemokine receptor CCR9. More particularly, the invention relates to the treatment of diseases or conditions mediated by CCR9, such as inflammatory bowel disease (IBD), and methods for detecting CCR9, which utilize the binding molecules of the invention.

Description

1. FIELD OF INVENTION

[001] The present invention relates to binding molecules, such as antibodies, that bind to the chemokine receptor CCR9. More particularly, the invention relates to the treatment of diseases or conditions mediated by CCR9, such as inflammatory bowel disease (IBD), and methods for the detection of CCR9, which utilize the binding molecules of the invention.

2. BACKGROUND OF THE INVENTION

[002] IBD is a group of chronic disorders of the gastrointestinal tract characterized pathologically by intestinal inflammation and epithelial injury. Two forms of IBD, Crohn's disease and ulcerative colitis, are associated with marked morbidity and can have a major impact on an individual's quality of life and ability to work [1]. These morbidities highlight the need for improved therapies for IBD.

[003] Therapeutic interventions for IBD are tailored to address symptomatic response and subsequent tolerance to intervention. Current therapies include aminosalicylates, corticosteroids, and antibodies. However, despite these treatments, it is estimated that surgical interventions will be required in up to two-thirds of patients with Crohn's disease during their lifetime [2]. Clinical management of Crohn's disease has historically employed a stepwise approach, starting with steroids, immunosuppressants (thiopurines and methotrexate), and/or aminosalicylates, although evidence for the efficacy of the latter is very limited [3]. No licensed therapy achieves durable remission in the majority of patients.

[004] Targeting anti-CCR9 antibodies has been proposed as a mechanism for cancer treatment. Aberrant expression of CCR9 in ovarian carcinomas, prostate cancer, breast cancer, and melanomas correlates with in vitro invasiveness in response to CCL25. Engagement of CCL25 increases cell survival and resistance to apoptosis. Somovilla-Crespo et al. 2018 [4] and WO2015075269 A1 describe mouse anti-CCR9 antibodies 91R (mouse anti-human CCR9 IgG2b) and 92R (mouse anti-human CCR9 IgG2a).

[005] Therefore, there is a need for an improved drug to treat or prevent such disorders. The present invention solves one or more of the above-mentioned problems.

3. SUMMARY OF THE INVENTION

[006] The inventors have discovered that the chemokine receptor, CCR9, is expressed at high levels in the colon and ileum of patients with IBD, where it is co-expressed with the inflammatory cytokines IFN-Y, IL-4, and IL-17. The inventors have successfully generated binding molecules that bind to CCR9-expressing cells, inhibit the interaction between CCR9 and its natural ligand CCL25, and prevent CCL25-mediated internalization of CCR9. Advantageously, the binding molecules can induce the death of the CCR9-expressing cells to which they bind, for example, by mediating an antibody-dependent cell-mediated cytotoxicity (ADCC) against the CCR9-expressing cells to which they bind. The development of a binding molecule that inhibits the binding of CCL25 to CCR9 and induces the death of a CCR9-expressing cell to which it binds is particularly advantageous for individuals with IBD.

[007] Thus, the invention relates to binding molecules that bind to CCR9. The binding molecules of the invention preferably inhibit the binding of CCL25 to CCR9. The binding molecules of the invention are preferably capable of inducing the death of CCR9-expressing cells to which they bind. For example, the binding molecules may be capable of mediating an ADCC response against the cells to which they bind. The binding molecules of the invention are preferably capable of preventing or inhibiting the migration of CCR9-expressing cells to which they bind. For example, the binding molecules may prevent the migration of CCR9-expressing cells in response to CCL25 or the binding molecules may be capable of preventing the migration of CCR9-expressing cells from the periphery into the intestine of a subject.

[008] In one aspect, the invention provides a binding molecule that binds to CCR9 and comprises a heavy chain variable region (VH) having a set of CDRs HCDR1, HCDR2, and HCDR3 and a light chain variable region (VL) having a set of CDRs LCDR1, LCDR2, and LCDR3, wherein (a) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 4 (RSSQSLVHX1NX2NTYLH wherein XI and X2 are any amino acid), LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; (b) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, and HCDR3 of SEQ ID NO: 9, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO: 12; or (c) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 13, HCDR2 of SEQ ID NO: 14, and HCDR3 of SEQ ID NO: 15, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 16, LCDR2 of SEQ ID NO: 17, and LCDR3 of SEQ ID NO: 18; or wherein any one or more of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprises 1, 2 or 3 amino acid substitutions compared to said sequences.

[009] The binding molecule of the invention may comprise a VH region amino acid sequence comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2 and HCDR3 of SEQ ID NO: 3, and a VL region amino acid sequence comprising LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6, and LCDR1 having the amino acid sequence selected from: SEQ ID NO: 4, SEQ ID NO: 19 (RSSQSLVHX1NX2NTYLH wherein XI of SEQ ID NO: 19 is P or S and X2 of SEQ ID NO: 19 is R, T or G), SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22.

[0010] The binding molecules of the invention preferably comprise an immunoglobulin Fc domain, or a fragment thereof that retains the ability to bind to one or more Fc receptors. For example, the binding molecule may comprise a human IgG1 Fc domain or fragment thereof. The binding molecules of the invention are preferably afucosylated. For example, the binding molecules may be afucosylated at amino acid position 297. The binding molecule of the invention may be present in a composition comprising multiple copies of said binding molecule, wherein at least 50%, 75%, 90%, 95%, 98%, 99% or 100% of the copies of the binding molecule in the composition are afucosylated.

[0011] In another aspect, the invention provides a binding molecule that binds to CCR9, wherein the binding molecule does not compete for binding to CCR9 with another binding molecule of the invention, such as does not compete for binding to CCR9 with a binding molecule as described above, such as does not compete for binding to CCR9 with a binding molecule as described herein for use in therapy. The binding molecule of this aspect can be used to determine the abundance of CCR9+ cells in a sample that has been contacted with a binding molecule of the invention.

[0012] Likewise, the invention provides a method for assessing the depletion of CCR9-expressing cells by a binding molecule of the invention, the method comprising: (i) contacting said binding molecule with a population of cells, wherein the population of cells comprises CCR9-expressing cells and immune effector cells, under conditions suitable to allow antibody-dependent cell-mediated cytotoxicity by the effector cells; (ii) contacting said population of cells with a binding molecule that binds to CCR9 and that does not compete for binding to CCR9 with the binding molecule of step (i); (iii) detecting CCR9-expressing cells in the population of cells that are bound by the binding molecule of (ii); (iv) comparing the amount of CCR9-expressing cells detected in step (iii) with the amount of CCR9-expressing cells in the original cell population used in step (i), and thereby determining the amount of CCR9-expressing cells that were depleted in step (i).

[0013] The invention also provides an isolated polynucleotide encoding any binding molecule of the invention. Also provided is a vector comprising a polynucleotide of the invention operatively associated with a promoter. Also provided is a vector comprising a polynucleotide encoding the VH region of a binding molecule of the invention and a polynucleotide encoding the VL region of a binding molecule of the invention, wherein said polynucleotides are operatively associated with one or more promoters.

[0014] The invention provides a host cell comprising a polynucleotide of the invention or a vector of the invention. Also provided is a method of producing a binding molecule of the invention, comprising expressing a polynucleotide of the invention or a vector of the invention in a host cell.

[0015] The invention provides a pharmaceutical composition comprising the binding molecule of the invention and a pharmaceutically acceptable carrier or diluent.

[0016] The invention also relates to the use of binding molecules of the invention in medicine, such as a binding molecule of the invention for use in therapy. The invention relates to the treatment of a CCR9-mediated disease or condition in a subject, by a method comprising administering to the subject an effective amount of a binding molecule of the invention or a composition of the invention. The invention also relates to the treatment of a CCR9-mediated disease or condition in a subject, by a method comprising administering to the subject an effective amount of a binding molecule that binds to CCR9, wherein said binding molecule is capable of (i) inhibiting the binding of CCL25 to CCR9, and (ii) mediating antibody-dependent cell-mediated cytotoxicity against a CCR9-expressing cell to which it binds.

[0017] The binding molecules described herein can also be used to detect CCR9. For example, the invention provides a method for detecting the presence of a CCR9 polypeptide in a sample, comprising: (a) contacting a sample with a binding molecule that binds to CCR9, such as any binding molecule of the invention or binding molecule as described herein, to provide a binding molecule-antigen complex; (b) detecting the presence or absence of said binding molecule-antigen complex; (c) wherein the presence of the binding molecule-antigen complex confirms the presence of a CCR9 polypeptide.

[0018] The present invention relates to improved anti-CCR9 antibodies and their use in the treatment of inflammatory bowel disease. The present invention particularly relates to a humanized afucosylated monoclonal antibody that specifically binds to the C-C motif chemokine receptor 9 (CCR9). Through its Fc receptor functionality, binding of the antibody to the CCR9 receptor initiates an ADCC response resulting in the depletion of CCR9-expressing cell populations. The antibodies of the invention are humanized, CDR-optimized. The antibodies are afucosylated without any loss of potency.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1: CCR9 expression in various cell types.

[0020] Figure 1A: CCR9 expression on CD4+ cells from cynomolgus and human blood cells, mesenteric lymph node (cynomolgus only), colon, ileum and thymus; Figure 1B: CCR9 expression on B cells in the ileum (left) and colon (right) of healthy and IBD patients; Figure 1C: CCR9 expression on effector T cells and Tregs from the colon or ileum of healthy or IBD patients.

[0021] Figure 2: The association of CCR9 with pro-inflammatory cytokines in CD4+ T cells.

[0022] Figure 2A: Expression of IFN-Y, IL-4 and IL-17 in CCR9- and CCR9+ cells; Figure 2B: Relative change in the level of cytokine expression assessed in Figure 2A between CD4+CCD9- cells and CD4+CCR9+ cells.

[0023] Figure 3: Aligned amino acid sequences of the variable regions of nine anti-CCR9 antibodies

[0024] Figure 3A: VH region; Figure 3B: VL region. The locations of the CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) are indicated by underline. The locations of the framework (FW) regions are also indicated.

[0025] Figure 4: The aligned VH and VL regions of antibody AB1020243, a humanized form of that antibody (AB1020243-fgl), and two CDR-optimized forms of the antibody (243LO0326 and 243LO0331)

[0026] Figure 5: Binding of anti-CCR9 antibodies to cells expressing or not expressing CCR9

[0027] Figure 5A: Binding to Molt 4 cell line; Figure 5B: Binding to Molt 4 and Jurkat cell lines

[0028] Figure 6: Binding of AB1020243 to different forms of CCR9 in HEK cells

[0029] As a control, binding was assessed in HEK cells not transfected with CCR9 (parental HEK) and binding was also assessed in HEK cells overexpressing human CCR9A (Hu A HEK), human CCR9B (Hu B HEK), cynomolgus CCR9 (Cyno HEK), mouse CCR9 (mouse HEK) and rat CCR9 (rat HEK).

[0030] Figure 7: Activation of NK cells by afucosylated anti-CCR9 antibodies preincubated with CCR9A-expressing HEK cells (top) or Molt 4 cells (bottom).

[0031] Figure 8: The effects of anti-CCR9 antibody treatment on the number of CCR9+ CD4+ PBMC.

[0032] Figure 9: Comparison of binding to various ligands of the chimeric antibody AB1020243 and its humanized version.

[0033] Figure 9A: Human CCR9B; Figure 9B: Cynomolgus CCR9; Figure 9B: CXCR1; Figure 9D: CXCR2; Figure 9E: CCR5; Figure 9F: CCR8; Figure 9G: HEK cells not transfected with CCR9.

[0034] Figure 10: Comparison of antibody AB1020243 with its humanized version

[0035] Figure 10A: NK cell activation after binding to the Molt 4 cell line; Figure 10B: Kinetic properties of humanized and chimeric afuc AB1020243 antibodies.

[0036] Figure 11: The ability of anti-CCR9 antibodies to compete with CCL25 for binding to CCR9.

[0037] Figure 12: Binding of 243LO0326 and AB1020243 to ligands

[0038] Figure 12A: Human CCR9A; Figure 12B: Human CCR9B; Figure 12C: Cynomolgus CCR9A; Figure 12D: CCR5; Figure 12E: CCR8; Figure 12F: CXCR1; and Figure 12F: CXCR2.

[0039] Figure 13: The amount of AB1020243 antibody or 243LO0326 antibody bound to the cell surface over time

[0040] The amount of AB1020243 antibody or 243LO0326 antibody bound to the cell surface over time (Figure 13A) and the effect of antibody or CCL25 treatment on CCR9 receptor levels on the cell surface over time (Figure 13B).

[0041] Figure 14: The effects of anti-CCR9 antibodies on Molt 4 cell death

[0042] Figure 14A: PBMC and Jurkat cells (Figure 14B); and human or cynomolgus PBMC (Figure 14C).

[0043] Figure 15: Impact of fucosylation on potency

[0044] Figure 15B: Effect of afucosylation on ADDC. Figure 15A: ADCC activity of fucosylated vs. afucosylated species in a fucosylation addition study. PR: Relative potency.

[0045] Figure 16: Targeted in vivo depletion of CCR9+ T cells

[0046] Figure 16A: The effect of intravenous injection of 243LO0326 on the number of CCR9+ memory CD4+ T cells in the blood of cynomolgus monkeys. Figure 16B: High and low doses of 243LO0326 decrease CDR4+CCR9+ cells in the peripheral blood of cynomolgus monkeys over a 2-day period. Figure 16C: High and low doses of 243LO0326 decrease CDR4+CCR9+ cells in the ileum of cynomolgus monkeys, as measured on day 15 by FACS. Figure 16D: High and low doses of 243LO0326 decrease CDR4+CCR9+ cells in the intestinal mucosa of cynomolgus monkeys, as measured on day 15 by IHC.

[0047] Figure 17: Selective depletion of CCR9+ intestinal lymphocytes

[0048] Figure 18: The effects of 243LO0326 on IBD markers in human intestinal explants taken from IBD patients.

[0049] Figure 18A: CCR9 levels; Figure 18B: IL-6 levels; Figure 18C: GM-CSF levels; Figure 18D: IL-22 levels.

[0050] Figure 19: Blocking of CCR9 internalization

[0051] 243LO0326 inhibited CCL25-induced CCR9 internalization.

[0052] Figure 20: Alignment of CCR9A and CCR9B

5. DETAILED DESCRIPTION OF THE INVENTION

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which this disclosure belongs. Singleton, et al., "DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY", 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, "THE HARPER COLLINS DICTIONARY OF BIOLOGY", Harper Perennial, NY (1991) provide one skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0054] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein may be used in practicing or testing embodiments of this disclosure. Numerical ranges include the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written from left to right in the 5' to 3' orientation; amino acid sequences are written from left to right in the amino to carboxy orientation, respectively.

[0055] The headings provided herein are not limitations on the various aspects or embodiments of this disclosure.

[0056] Amino acids are referred to herein using the amino acid name, the three-letter abbreviation, or the single-letter abbreviation. The term "protein" as used herein includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein." In some instances, the term "amino acid sequence" is synonymous with the term "peptide." In some instances, the term "amino acid sequence" is synonymous with the term "enzyme." The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, both conventional one- and three-letter codes for amino acid residues may be used. The 3-letter code for amino acids as defined in accordance with the Joint Commission on Biochemical Nomenclature (JCBN) of the IUPACIUB. It is also understood that a polypeptide may be encoded by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0057] Other definitions of terms may appear throughout the specification. Before exemplary embodiments are described in more detail, it should be understood that this disclosure is not limited to the particular embodiments described, as such may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present disclosure will be defined solely by the appended claims.

[0058] Where a range of values is provided it is understood that each intermediate value, to within one tenth of a unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any indicated value or intermediate value in an indicated range and any other indicated or intermediate value in that indicated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range where either, neither or both of the limits are included in the smaller ranges is also encompassed within this disclosure, subject to any limit specifically excluded in the indicated range. Where the indicated range includes either or both of the limits, ranges excluding either or both of the included limits are also included in this disclosure.

[0059] It should be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a plurality of such agents, and reference to "the agent" includes reference to one or more agents and their equivalents known to those skilled in the art, and so on.

[0060] "About" may generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given value or range of values. Preferably, the term "about" should be understood herein as plus or minus (±) 5%, preferably ± 4%, ± 3%, ± 2%, ± 1%, ± 0.5%, ± 0.1%, of the numerical value of the number with which it is being used.

[0061] Embodiments described herein as "comprising" one or more features may also be considered to disclose corresponding embodiments "consisting of" and/or "consisting essentially of" such features.

[0062] The term "pharmaceutically acceptable" as used herein means approved by a regulatory agency of the federal government or a state government or listed in the United States Pharmacopoeia, European Pharmacopoeia or other generally recognized pharmacopoeia for use in animals and, more particularly, in humans.

[0063] Concentrations, quantities, volumes, percentages and other numerical values may be presented herein in a range format. It should also be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all individual numerical values or subranges encompassed within that range as if each numerical value and subrange were explicitly recited.

[0064] The term "epitope" refers to a region of the target protein (e.g., polypeptide) capable of binding to (e.g., being bound by) a binding molecule of the invention.

[0065] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between the members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind to antigen slowly and tend to dissociate readily, whereas high affinity antibodies generally bind to antigen more rapidly and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention.

[0066] The potency of a binding molecule can be expressed as an IC50 value. IC50 is the mean inhibitory concentration of a binding molecule. In functional assays, the IC50 is the concentration that reduces a biological response by 50% of its maximum. The IC50 can be calculated by a number of means known in the art.

[0067] The potency of a binding molecule can be expressed as an EC50 value. In functional assays, EC50 is the concentration that induces a median response between the baseline and the maximum after a specified exposure time. The EC50 can be calculated by a number of means known in the art.

5.1. Binding Molecules

[0068] The present invention relates to binding molecules that bind to CCR9. Chemokine receptor 9, CCR9 (also known as CDw199 and GPR-9-6) is a member of the beta chemokine receptor family. CCR9 is organized into 15 domains, corresponding to an N-terminal extracellular domain (Nt), seven transmembrane domains, three intracellular domains, three extracellular domains, and a C-terminal intracellular domain (Ct). In humans, CCR9 exists in two isoforms CCR9A and CCR9B. Isoform A has 12 additional N-terminal amino acids and has higher affinity for the ligand CCL25 compared to isoform B. Isoforms A and B are reported to be co-expressed in a 10:1 ratio. The previously described anti-CCR9 antibodies 91R and 92R only bind to the CCR9A isoform and do not bind to CCR9B [4].

[0069] Without wishing to be limited by theory, the present inventors' observation that a higher proportion of CCR9-positive CD4 T lymphocytes co-express the pro-inflammatory cytokines IFN-Y, IL-4, and IL-17 in the intestine compared to CCR9-negative CD4 T lymphocytes suggests that CCR9 may identify CD4 T lymphocytes that contribute to the pathogenesis of IBD.

[0070] The RNA, DNA, and amino acid sequences of CCR9 are known to those skilled in the art and can be found in many databases, for example, the National Center for Biotechnology Information (NCBI) and UniProt databases. Examples of such sequences found in UniProt are P51686-1 for human CCR9A and P51686-2 for human CCR9B; Q0H741 for cynomolgus CCR9; Q9WUT7 for mouse CCR9; and Q8CH33 for rat CCR9.

[0071] In some embodiments, the binding molecule of the invention binds to human CCR9. In some embodiments, the binding molecule binds to human CCR9A and/or human CCR9B. In some embodiments, the binding molecule binds to human CCR9A and binds to human CCR9B.

[0072] In some embodiments, the binding molecule binds to CCR9 from a non-human mammalian species, such as a non-human primate. In some embodiments, the binding molecule binds to cynomolgus CCR9. In some embodiments, the binding molecule binds to both human and cynomolgus CCR9, such as binding to both human CCR9A and human CCR9B and cynomolgus CCR9.

[0073] In some embodiments, the binding molecule does not bind to mouse and/or rat CCR9. In some embodiments, the binding molecule binds to human and cynomolgus CCR9, such as human CCR9A and human CCR9B and cynomolgus CCR9, but does not bind to mouse or rat CCR9.

[0074] In some embodiments, the binding molecule selectively binds to (also referred to interchangeably herein as specifically to) CCR9. By selective binding, it will be understood that the CDRs of a binding molecule together bind to CCR9, without significant cross-reactivity with any other molecule. In particular, a binding molecule that selectively binds to CCR9 may not have significant cross-reactivity with any other cytokine receptor. A binding molecule that selectively binds to CCR9 may not have significant cross-reactivity to any one, any two, any three, or all four of CCR5, CCR8, CXCR1, and CXCR2. For example, in some embodiments, the binding molecule does not bind to CCR5, CCR8, CXCR1, or CXCR2.

[0075] In some embodiments, a binding molecule that selectively binds to human CCR9, such as human CCR9A and/or human CCR9B, may not have significant cross-reactivity with CCR9 from another species, such as with mouse and/or rat CCR9. In some embodiments, a binding molecule that selectively binds to human CCR9 may have cross-reactivity with a closely related CCR9 molecule, such as cynomolgus CCR9, but may not have significant cross-reactivity with a more distantly related CCR9, such as mouse and/or rat CCR9.

[0076] Cross-reactivity may be assessed by any suitable method. For example, binding may be measured by testing the binding of the binding molecule to overexpressing cell lines expressing the receptor of interest. Binding may also be measured by a radioimmunoassay (RIA), BIACORE® (using recombinant CCR9 as the analyte and binding molecule as the ligand, or vice versa), KINEXA®, ForteBio Octet system, or other binding assays known in the art. By way of non-limiting example, cross-reactivity of the binding molecule with a molecule other than CCR9 may be considered significant if the binding molecule binds to the other molecule at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100% as strongly as it binds to CCR9. A binding molecule that selectively binds to CCR9 may bind to another molecule, such as a different cytokine receptor, such as any one, two, three, or all four of CCR5, CCR8, CXCR1, and CXCR2, at less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% the strength that it binds to CCR9. Preferably, the binding molecule binds to the other molecule at less than 20%, less than 15%, less than 10%, or less than 5%, less than 2%, or less than 1% the strength that it binds to CCR9. When used herein, CCR9 is preferably a naturally occurring CCR9, such as human CCR9, such as human CCR9A and/or human CCR9B.

[0077] In some embodiments, the binding molecule binds to a cell that expresses CCR9. The binding molecule may bind to a CCR9 receptor that is present on the cell membrane of a cell. The cell may be a cell that naturally expresses CCR9, or the cell may be engineered to express CCR9, such as by introducing a nucleic acid encoding CCR9 under conditions that allow expression of CCR9. The cell may be engineered to overexpress CCR9 such that the engineered cell line expresses higher levels of CCR9 than a corresponding unmodified cell. The cell may express human CCR9A or human CCR9B.

[0078] The cell may be a HEK cell line that has been engineered to express CCR9, such as human CCR9A and/or human CCR9B.

[0079] The Molt 4 cell line is a human acute lymphoblastic leukemia T lymphocyte cell line that naturally expresses CCR9 at high levels. The Molt 4 cell line can be obtained from ATCC, CRL-1582. In some embodiments, the binding molecule binds to a Molt 4 cell. In some embodiments, the binding molecule binds to a Molt 4 cell by binding to CCR9 on the surface of said Molt 4 cell or CCR9 present on the cell membrane of said Molt 4 cell.

[0080] In some embodiments, the binding molecule does not bind to a cell that does not express CCR9, such as a Jurkat cell. The Jurkat cell line can be obtained from ATCC as Jurkat, Clone E6.1. In some embodiments, the binding molecule binds to a CCR9 (e.g., a human CCR9) with a dissociation constant (KD) of <1 μM, <100 nM, <10 nM, <5 nM, <1 nM, <0.5 nM, <0.1 nM, or <10 pM. In some embodiments, the binding molecule binds to CCR9 (e.g., CCR9 present on a Molt 4 cell) with a KD between about 0.01 nM to about 0.45 nM, between about 0.025 nM to about 0.25 nM, or between about 0.05 nM to about 0.1 nM. In some embodiments, the binding molecule binds to CCR9 (e.g., CCR9 present on a Molt 4 cell) with a KD of about 0.09 nM.

[0081] In some embodiments, the binding molecule binds to CCR9 (e.g., CCR9 present on a Molt 4 cell) with a KD of between about 2 nM to about 5 nM, between about 2.5 nM to about 4 nM, or between about 3 nM to about 3.5 nM. In some embodiments, the binding molecule binds to CCR9 (e.g., CCR9 present on a Molt 4 cell) with a KD of about 3.4 nM.

[0082] KD (binding affinity) measurements may be performed by any suitable assay known in the art. Suitable assays include an affinity assay performable by a Ligand Tracer system, KinExA system (e.g., KinExA 3100, KinExA 3200, or KinExA 4000) (Sapidyne Instruments, Idaho), or ForteBio Octet system.

[0083] In some embodiments, the binding molecule is a CCR9 antagonist. For example, the binding molecule can prevent or inhibit the normal function of CCR9. For example, the binding molecule can prevent or inhibit the migration of a cell expressing CCR9 by antagonizing CCR9 present on the cell.

[0084] In some embodiments, the binding molecule inhibits the binding of a ligand to CCR9. For example, in some embodiments, the binding molecule binds to CCR9 and blocks or inhibits the binding of a ligand to the CCR9 molecule. This can be assessed by standard methods known in the art, including those described herein.

[0085] The binding molecule may be a competitive inhibitor of CCR9. For example, the binding molecule may compete with a CCR9 ligand for binding to CCR9. Competitive binding may be assessed by standard methods known in the art, including those described herein.

[0086] In some embodiments, the CCR9 ligand is CCL25. CCL25 (also known as TECK) is a cytokine belonging to the CC family of chemokines. The RNA, DNA, and amino acid sequences of CCL25 are known to those skilled in the art and can be found in many databases, e.g., the National Center for Biotechnology Information (NCBI) and UniProt databases. Examples of such sequences found in UniProt are at O15444 for human CCL25; O35903 for mouse CCL25; Q32PX4 for rat CCL25, and A0A2K5TSD9 and A0A2K5TSC0 for cynomolgus CCL25.

[0087] In some embodiments, the binding molecule blocks or inhibits the binding of CCL25 to CCR9. For example, CCL25 may have reduced binding to CCR9 in the presence of a binding molecule of the present invention. In some embodiments, the binding molecule is capable of binding to CCR9 present on the surface or membrane of a cell and inhibiting the binding of CCL25 to CCR9 on the cell.

[0088] In some embodiments, the binding molecule is capable of preventing or inhibiting the migration of a CCR9-expressing cell to which it binds. For example, a cell bound by a binding molecule of the invention may migrate less efficiently in response to a stimulus than a cell not bound by a binding molecule of the invention. In some embodiments, the binding molecule prevents or inhibits the migration of a CCR9-expressing cell in response to CCL25. In some embodiments, the binding molecule is capable of preventing or inhibiting the migration of a CCR9-expressing cell from the periphery to the intestine of a subject.

[0089] In some embodiments, the binding molecule of the invention is capable of inducing death of a CCR9-expressing cell to which it binds. For example, in some embodiments, the binding molecule is capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) against a CCR9-expressing cell to which it binds. In some embodiments, the binding molecule is capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) against a CCR9-expressing lymphocyte to which it binds. As used herein, “mediating antibody-dependent cell-mediated cytotoxicity” or “mediating ADCC” means that the binding molecule is capable of inducing ADCC.

[0090] ADCC is a cell-mediated immune defense mechanism whereby an immune effector cell is activated to lyse a target cell whose membrane surface antigens have been bound by binding molecules. Examples of such immune effector cells include natural killer cells (NK cells), macrophages, neutrophils, and eosinophils. Therefore, in some embodiments, the binding molecule is capable of inducing the activation of such an immune effector cell. For example, a binding molecule of the invention may be capable of inducing the activation of such an immune effector cell when the binding molecule is bound to a cell expressing CCR9.

[0091] In some embodiments, the binding molecule of the invention is capable of depleting CCR9-expressing cells in a cell population comprising CCR9-expressing cells and immune effector cells.

[0092] The ability of a binding molecule to induce killing, deplete, or mediate ADCC against a CCR9-expressing cell to which it binds can be determined using any of the methods described herein. For example, a binding molecule of the invention can be incubated with PBMCs and the percentage of CD4+CCR9+ cells remaining at the end of the incubation period is determined using a non-competitive CCR9 binding molecule. Varying concentrations of a binding molecule can be used to calculate a concentration-dependent killing effect of the binding molecule. From this, the EC50 of the binding molecule can be calculated. As used herein, the EC50 can be calculated based on the killing of CD4+CCR9+ cells by PBMCs following binding of a binding molecule of the invention to CD4+CCR9+ cells.

[0093] In some embodiments, the EC50 of a binding molecule of the invention (e.g., measured as described herein) is less than 100 nM, such as less than 50 nM, less than 25 nM, less than 10 nM, or less than 5 nM. The EC50 can be less than 3 nM, less than 2 nM, less than 1 nM, less than 500 pM, or less than 250 pM. The EC50 can be from about 2 pM to about 200 pM, such as from about 3.5 pM to about 200 pM. In some embodiments, the EC50 is from about 1 pM to about 5 nM, such as from about 4 pM to about 3 nM. In some embodiments, the EC50 is from about 2 nM to about 3 nM, such as from about 2.5 nM to about 3 nM, such as about 2.6 nM. In some embodiments, the EC50 is from about 0.25 nM to about 2.5 nM, such as from about 0.5 nM to about 1 nM, such as about 0.5 nM. In some embodiments, the EC50 is from about 50 pM to about 250 pM, such as about 150 pM to about 250 pM, such as about 200 pM. In some embodiments, the EC50 is from about 3.5 pM to about 50 pM, such as from about 3.5 pM to about 25 pM. In some embodiments, the EC50 is from about 3.5 pM to about 5 pM or from about 4 pM to about 10 pM. In some embodiments, the EC50 is from about 6 pM to about 60 pM, such as from about 25 pM to about 60 pM, preferably from about 25 pM to about 35 pM, such as about 30 pM.

[0094] In some embodiments, the EC50 when the binding molecule is bound to human CCR9 is from about 3.5 pM to about 10 pM, such as from about 4 pM to about 5 pM. In some embodiments, the EC50 where the binding molecule is bound to cynomolgus CCR9 is from about 6 pM to about 60 pM, such as from about 25 pM to about 60 pM, preferably from about 25 pM to about 35 pM, such as about 30 pM.

[0095] To initiate ADCC, the binding molecules can crosslink an immune effector cell to a cell expressing CCR9. Thus, in some embodiments, the binding molecules of the invention are capable of crosslinking an immune effector cell to a cell expressing CCR9. For example, binding molecules can be capable of crosslinking an immune effector cell to a cell expressing CCR9 by binding an Fc receptor on an immune effector cell. There are a number of Fc receptors that are specific to different classes of antibodies, including IgG (gamma receptors), IgE (eta receptors), IgA (alpha receptors), and IgM (mu receptors). Therefore, in some embodiments, the binding molecule is capable of binding to an Fc receptor. In some embodiments, the binding molecule is capable of binding to an FCYR, such as a receptor selected from FCYRI, FcYRIIa, FcYRIIb, FcYRIIIa, and FcyRIIIb. In some embodiments, the binding molecule is capable of binding to FcYRIIIa. In some embodiments, the binding molecule is capable of binding to an Fc receptor that is present on an immune effector cell.

[0096] Binding molecules can be bound by an Fc receptor on an immune effector cell via an Fc domain present on the binding molecule. Therefore, in some embodiments, a binding molecule of the invention comprises a region that is capable of binding to an Fc receptor, such as an Fc receptor as defined above. In some embodiments, the binding molecule comprises an immunoglobulin Fc domain, or a fragment thereof that retains the ability to bind to one or more Fc receptors, such as one or more Fc receptors as defined above. As used herein, the term "Fc domain" refers to the portion of a single immunoglobulin heavy chain starting at the hinge region and ending at the C-terminus of the antibody. Accordingly, a complete Fc domain can comprise at least a portion of a hinge domain (e.g., upper, middle, and/or lower hinge region), a CH2 domain, and a CH3 domain. In some embodiments, the binding molecule comprises a homodimer of an Fc domain. In some embodiments, the binding molecule comprises a region that binds to CCR9 and further comprises such an immunoglobulin Fc domain or fragment thereof. In some embodiments, the binding molecule is an immunoglobulin molecule or fragment thereof, which comprises an antigen-binding region that binds to CCR9 and an Fc-binding region that binds to one or more Fc receptors as defined above.

[0097] In some embodiments, the Fc-binding region, immunoglobulin Fc domain, or fragment thereof is an IgG Fc domain or fragment thereof, such as an IgG1, IgG2, IgG3, or IgG4 Fc domain or fragment thereof. In some embodiments, the Fc-binding region, immunoglobulin Fc domain, or fragment thereof is an IgG1 Fc domain or fragment thereof. In some embodiments, the Fc-binding region, immunoglobulin Fc domain, or fragment thereof is a human immunoglobulin Fc domain or fragment thereof, such as a human IgG1 Fc domain or fragment thereof. In some embodiments, the binding molecule comprises an immunoglobulin Fc domain having an amino acid sequence set forth in SEQ ID NO: 78. For example, in some embodiments, the Fc domain comprises amino acids 111-330 of SEQ ID NO: 78.

[0098] Binding of a binding molecule of the invention to an Fc receptor on an immune effector cell can crosslink the immune effector cell with a cell to which the antigen-binding portion of the binding molecule is attached. Thus, in some embodiments, the binding molecule of the invention is capable of inducing activation of immune effector cells. In some embodiments, crosslinking of an immune effector cell to a cell expressing CCR9 by the binding molecule activates ADCC by the effector cell.

[0099] Activation of immune effector cells can be assessed by the methods described herein, such as by an in vitro ADCC bioassay. Briefly, immune effector cells can be engineered to express a reporter, such as a luciferase reporter, that acts as a surrogate for effector function. Effector immune cells comprising such a reporter are added to target cells that have been preincubated with a binding molecule. Following incubation of the effector and target cells, reporter function (e.g., luminescence) is quantified. Such a method can therefore be used to identify a binding molecule that is capable of activating an immune effector cell. For example, if the assay is intended to assess the efficacy of a binding molecule to activate immune effector cells, the target cell can be a cell that is known to express CCR9. Such a method can be used to identify a target cell that expresses CCR9. For example, if the assay is intended to assess whether a target cell expresses CCR9, then the binding molecule may be a binding molecule known to bind to CCR9, such as a binding molecule of the invention.

[00100] In some embodiments, the binding molecule of the invention has an NK cell activation potency of <5 nM, such as <3 nM, <2 nM, <1 nM, or <0.5 nM as determined using a HEK cell line expressing CCR9A. In some embodiments, the binding molecule of the invention has an NK cell activation potency of 0.8 nM as determined using a Molt 4 cell line. In some embodiments, activation of antibody-dependent cell-mediated cytotoxicity by the effector cell causes lysis of the CCR9-expressing cell.

[00101] For a binding molecule to be considered capable of mediating ADCC against a CCR9-expressing cell to which it binds, the binding molecule must have an immune effector cell activation potency, such as an NK cell activation potency, of <20 nM as determined using a Molt 4 cell line, or <30 nM as determined using a CCR9A-expressing HEK cell line, and/or a PBMC killing EC50 of <3 nM.

[00102] In some embodiments, a binding molecule of the invention provides altered effector functions that in turn affect the biological profile of the binding molecule. For example, altering the glycosylation profile of a constant region domain of an immunoglobulin molecule can increase Fc receptor binding of the modified binding molecule. In other cases, constant region modifications consistent with this invention can moderate complement binding. Still other constant region modifications can be used to eliminate disulfide bonds or oligosaccharide chemical moieties that allow for improved localization due to antigen specificity or increased antibody flexibility. Similarly, modifications to the constant region according to the present invention can be readily made using well-known biochemical or molecular engineering techniques well within the purview of the skilled artisan.

[00103] Therefore, in some embodiments, a binding molecule of the invention may have enhanced effector functions that increase the ability of the binding molecule to interact with an immune effector cell, such as that which increases the ability of the binding molecule to crosslink an immune effector cell with a CCR9-expressing cell that is bound by the binding molecule, or that increases the ability of the binding molecule to induce ADCC. In some embodiments, the immunoglobulin Fc domain of a binding molecule provided herein is modified compared to the corresponding wild-type Fc domain, wherein said modification leads to an increased affinity for one or more Fc receptors, preferably one or more FCY receptors. In some embodiments, the immunoglobulin Fc domain is modified compared to the corresponding wild-type Fc domain, wherein said modification leads to an increased antibody-dependent cell-mediated cytotoxicity response.

[00104] A binding molecule may be made with an altered type of glycosylation, such as an afucosylated/hypofucosylated binding molecule with reduced amounts of fucosyl residues or a binding molecule with increased bisecting GlcNac structures. Such altered glycosylation profiles have been shown to increase the ADCC capability of binding molecules. Such modifications may be accomplished by any means known in the art, such as those described in the Examples section herein.

[00105] Therefore, in some embodiments, a binding molecule provided herein may be afucosylated. The binding molecule may not comprise fucose. The binding molecule may include at least one N-linked glycan that lacks a central fucose sugar unit.

[00106] In some embodiments, the linker molecule is afucosylated at position EU 297, also referred to herein as position N297 (Edelman et al., 1969, Proc Natl Acad Sci USA 63(1): 78-85). That is, the N-linked glycan at position N297 may be absent, may lack fucose, or may lack a central fucose sugar unit. The N-linked glycan at amino acid position N297 corresponds to position N180 in SEQ ID NO: 78. In some embodiments, the linker molecule comprises SEQ ID NO: 78 wherein N180 of SEQ ID NO: 78 is afucosylated. In some embodiments, the binding molecule comprises an Fc domain comprising amino acids 111-330 of SEQ ID NO: 78 wherein N180 of SEQ ID NO: 78 is afucosylated.

[00107] As described herein, the binding molecule may comprise an Fc domain or fragment thereof, such as an hIgG1 Fc domain or fragment thereof. In some embodiments, the binding molecule comprises an afucosylated Fc domain or fragment thereof, such as an afucosylated hIgG1 Fc domain or fragment thereof. Any of the Fc domains or Fc domain fragments as described herein may be present in an afucosylated form. The Fc domain or fragment may not comprise fucose. At least one N-linked glycan of the Fc domain or fragment may lack a central fucose sugar unit. The Fc domain or fragment can be afucosylated at position EU 297. In some embodiments, the binding molecule comprises an afucosylated Fc domain comprising amino acids 111-330 of SEQ ID NO: 78. For example, the binding molecule can comprise an afucosylated Fc domain comprising amino acids 111-330 of SEQ ID NO: 78 wherein N180 of SEQ ID NO: 78 is afucosylated. In some embodiments, the binding molecule comprises an afucosylated Fc domain and a non-fucosylated Fc domain. For example, the binding molecule can comprise an afucosylated Fc domain comprising amino acids 111-330 of SEQ ID NO: 78, wherein N180 of SEQ ID NO: 78 is afucosylated, and a non-afucosylated Fc domain comprising amino acids 111-330 of SEQ ID NO: 78. In some embodiments, the binding molecule is fully afucosylated. For example, all N-linked glycans of the binding molecule can be afucosylated. In some embodiments, the Fc domain of the binding molecule is fully afucosylated. For example, all N-linked glycans of the Fc domain can be afucosylated.

[00108] In some embodiments, the binding molecule is present in a composition comprising multiple copies of said binding molecule, wherein at least 50%, 75%, 90%, 95%, 98%, 99%, or 100% of the copies of the binding molecule in the composition are afucosylated, e.g. lacking fucose, lacking a central fucose sugar unit on at least one N-linked glycan, or being afucosylated at position EU 297, as described herein. In such a composition, the binding molecule has an amino acid sequence as disclosed herein, but the copies of the binding molecule in the composition may vary in their glycosylation pattern, e.g. some copies may contain fucose and some copies may lack fucose, e.g. lacking a central fucose sugar unit on at least one N-linked glycan, or being afucosylated at position EU 297, as described herein.

[00109] In any of the embodiments disclosed herein, the CCR9-expressing cell is a gut-resident cell. In any of the embodiments disclosed herein, the CCR9-expressing cell is a peripheral blood cell. In any of the embodiments disclosed herein, the CCR9-expressing cell expresses CD4, CD8, CD20, CD19, CD69, and/or CD103. In any of the embodiments disclosed herein, the CCR9-expressing cell expresses GM-CSF, IL-22, TNFα, IL-6, IFNY, IL-4, and/or IL-17. For example, the CCR9-expressing cell can express IFNY, IL-4, and/or IL-17. In any of the embodiments disclosed herein, the CCR9-expressing cell is a lymphocyte, for example a gut-resident lymphocyte or a peripheral blood lymphocyte. In some embodiments, the lymphocyte is a T lymphocyte, such as a CD4+ T lymphocyte. In some embodiments, the lymphocyte is a B lymphocyte.

[00110] In any of the embodiments disclosed herein, the immune effector cell is an NK cell, a macrophage, a neutrophil, an eosinophil, or a combination thereof. In any of the embodiments disclosed herein, the immune effector cell is an NK cell.

[00111] A binding molecule of the invention may have any one or more of the functional characteristics set forth above. Additionally or alternatively, it may have any one or more of the structural characteristics (e.g., sequence) set forth below.

[00112] Figure 3 and Figure 4 illustrate sequences of exemplified binding molecules of the invention. The binding molecules illustrated in Figure 3 and Figure 4 are antibodies, where the antibodies comprise a variable heavy domain (VH) and variable light domain (VL) region sequence as illustrated in these Figures. The binding molecule of the invention may comprise a VH and VL from any antibody illustrated in Figure 3 or Figure 4. The binding molecule of the invention may comprise a VH and VL from any antibody illustrated in Figure 4.

[00113] Table 3 provides the amino acid sequences of the VH and VL regions and Table 4 provides CDR sequences for exemplary binding molecules of the invention. The binding molecule of the invention may comprise the VH and VL of any binding molecule illustrated in Table 3. The binding molecule of the invention may comprise a set of six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) of any binding molecule illustrated in Table 4.

[00114] A binding molecule of the invention may comprise the CDR sequences of any antibody as shown in Figure 3 or Figure 4. For example, a binding molecule may comprise all six sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as indicated for any of the antibodies illustrated in Figure 3 or Figure 4. A binding molecule of the invention may comprise the CDR sequences of any antibody as shown in Figure 4. For example, a binding molecule may comprise all six sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as indicated for any of the antibodies illustrated in Figure 4. A binding molecule of the invention may comprise a set of six CDRs as illustrated in Table 4. For example, a binding molecule may comprise all six sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as indicated for any of the antibodies illustrated in Table 4.

[00115] A binding molecule of the invention may comprise a heavy chain variable region (VH) with a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable region (VL) with a set of CDRs LCDR1, LCDR2 and LCDR3. Thus, the binding molecule may comprise a heavy chain variable region (VH) comprising HCDR1, HCDR2, HCDR3 as indicated for any of the antibodies illustrated in Figure 3 or Figure 4, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 as indicated for the same antibody illustrated in Figure 3 or Figure 4. The binding molecule may comprise a heavy chain variable region (VH) comprising HCDR1, HCDR2, HCDR3 as indicated for any of the antibodies illustrated in Figure 4, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 as indicated for the same antibody illustrated in Figure 4. The binding molecule may comprise a heavy chain variable region (VH) comprising HCDR1, HCDR2, HCDR3 as indicated for any of the antibodies illustrated in Table 4, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 as indicated for the same antibody illustrated in Figure 4. LCDR3 as indicated for the same antibody illustrated in Table 4.

[00116] For example, in some embodiments, the binding molecule comprises: a) HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6 wherein XI and X2 of SEQ ID NO: 4 are any amino acid; b) HCDR1 of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, HCDR3 of SEQ ID NO: 9, LCDR1 of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO: 12; or c) HCDR1 of SEQ ID NO: 13, HCDR2 of SEQ ID NO: 14, HCDR3 of SEQ ID NO: 15, LCDR1 of SEQ ID NO: 16, LCDR2 of SEQ ID NO: 17 and LCDR3 of SEQ ID NO: 18.

[00117] In some embodiments, the binding molecule comprises: a) a VH region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3 and a VL region comprising LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6 wherein XI and X2 of SEQ ID NO: 4 are any amino acid; b) a VH region comprising HCDR1 of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, and HCDR3 of SEQ ID NO: 9 and a VL region comprising LCDR1 of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO: 12; or c) a VH region comprising HCDR1 of SEQ ID NO: 13, HCDR2 of SEQ ID NO: 14 and HCDR3 of SEQ ID NO: 15 and a VL region comprising LCDR1 of SEQ ID NO: 16, LCDR2 of SEQ ID NO: 17 and LCDR3 of SEQ ID NO: 18.

[00118] The binding molecule may comprise HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR2 of SEQ ID NO: 5, LCDR3 of SEQ ID NO: 6 and LCDR1having the amino acid sequence selected from: (a) SEQ ID NO: 4; (b) SEQ ID NO: 19; (c) SEQ ID NO: 20; (d) SEQ ID NO: 21; and (e) SEQ ID NO: 22 wherein X1 and X2 of SEQ ID NO: 4 are any amino acid, and wherein X1 of SEQ ID NO: 19 is P or S and X2 of SEQ ID NO: 19 is R, T, or G. Accordingly, in some embodiments, the binding molecule comprises: a) HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6 wherein X1 and X2 of SEQ ID NO: 4 are any amino acid; b) HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 19, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6 where Xi of SEQ ID NO: 19 is P or S and X2 of SEQ ID NO: 19 is R, T or G; c) HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 20, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6; d) HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 21, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6; or e) HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 22, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6.

[00119] In some embodiments, the binding molecule comprises: a) a VH region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and a VL region comprising LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6 wherein X1 and X2 of SEQ ID NO: 4 are any amino acid; b) a VH region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2 and HCDR3 of SEQ ID NO: 3, and a VL region comprising LCDR1 of SEQ ID NO: 19, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6 wherein X1 of SEQ ID NO: 19 is P or S and X2 of SEQ ID NO: 19 is R, T or G; c) a VH region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2 and HCDR3 of SEQ ID NO: 3, and a VL region comprising LCDR1 of SEQ ID NO: 20, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6; d) a VH region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2 and HCDR3 of SEQ ID NO: 3, and a VL region comprising LCDR1 of SEQ ID NO: 21, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6; or e) a VH region comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2 and HCDR3 of SEQ ID NO: 3, and a VL region comprising LCDR1 of SEQ ID NO: 22, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6.

[00120] The present invention encompasses the binding molecules defined herein having the recited CDR sequences (reference binding molecules), as well as functional variants thereof. A functional variant binds to the same target antigen as the reference binding molecule and preferably exhibits the same antigen cross-reactivity profile as the reference binding molecule. For example, the functional variant may bind to the same natural forms of CCR9 as the reference binding molecule, such as binding to the same subset of molecules from the following list: human CCR9A, human CCR9B, cynomolgus CCR9, rat CCR9, and mouse CCR9. Functional variants may have a different affinity for a target antigen when compared to the reference binding molecule, but substantially the same affinity is preferred.

[00121] In some embodiments, functional variants of a reference binding molecule show sequence variation in one or more CDRs when compared to corresponding reference CDR sequences. Thus, a functional binding molecule variant can comprise a functional variant of a CDR. When the term "functional variant" is used in the context of a CDR sequence, it means that the CDR has 1, 2, or 3 amino acid differences when compared to a corresponding reference CDR sequence, and when combined with the remaining 5 CDRs (or variants thereof) allows the variant binding molecule to bind to the same target antigen as the reference binding molecule, and preferably exhibit the same antigen cross-reactivity profile as the reference binding molecule.

[00122] In some embodiments, a binding molecule comprises: a light chain CDR1 with 1, 2, or 3 amino acid differences when compared to a corresponding reference CDR sequence; a light chain CDR2 with 1, 2, or 3 amino acid differences when compared to a corresponding reference CDR sequence; a light chain CDR3 with 1, 2, or 3 amino acid differences when compared to a corresponding reference CDR sequence; a heavy chain CDR1 with 1, 2, or 3 amino acid differences when compared to a corresponding reference CDR sequence; a heavy chain CDR2 with 1, 2, or 3 amino acid differences when compared to a corresponding reference CDR sequence; and a heavy chain CDR3 with 1, 2, or 3 amino acid differences when compared to a corresponding reference CDR sequence; wherein the binding molecule binds to the same target antigen as the reference binding molecule, and preferably exhibits the same antigen cross-reactivity as the reference binding molecule.

[00123] In some embodiments, a binding molecule comprises: a light chain CDR1 having at most 2 amino acid differences when compared to a corresponding reference CDR sequence; a light chain CDR2 having at most 2 amino acid differences when compared to a corresponding reference CDR sequence; a light chain CDR3 having at most 2 amino acid differences when compared to a corresponding reference CDR sequence; a heavy chain CDR1 having at most 2 amino acid differences when compared to a corresponding reference CDR sequence; a heavy chain CDR2 having at most 2 amino acid differences when compared to a corresponding reference CDR sequence; and a heavy chain CDR3 having at most 2 amino acid differences when compared to a corresponding reference CDR sequence; wherein the binding molecule binds to the same target antigen as the reference binding molecule and preferably exhibits the same antigen cross-reactivity profile as the reference binding molecule. In some embodiments, a binding molecule comprises a light chain CDR1 having at most 2 amino acid differences when compared to a corresponding reference CDR sequence.

[00124] A binding molecule may comprise: a light chain CDR1 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; a light chain CDR2 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; a light chain CDR3 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; a heavy chain CDR1 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; a heavy chain CDR2 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; and a heavy chain CDR3 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; wherein the binding molecule binds to the same target antigen as the reference binding molecule and preferably exhibits the same antigen cross-reactivity profile as the reference binding molecule.

[00125] In some embodiments, a variant binding molecule may have at most 5, 4, or 3 total amino acid differences in its CDRs when compared to a corresponding reference binding molecule, with the proviso that there are at most 3, at most 2, or at most 1 amino acid differences per CDR. Preferably, a variant binding molecule has at most 2 (more preferably at most 1) total amino acid differences in its CDRs when compared to a corresponding reference binding molecule, with the proviso that there are at most 2 amino acid differences per CDR. More preferably, a variant binding molecule has at most 2 (more preferably at most 1) total amino acid differences in its CDRs when compared to a corresponding reference binding molecule, with the proviso that there is at most 1 amino acid difference per CDR.

[00126] For example, such a binding molecule may comprise all six HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of a reference binding molecule (such as any of the antibodies illustrated in Figure 3 or Figure 4 or Table 3), wherein any one or more of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences comprises 1, 2, or 3 amino acid changes compared to the respective CDR sequences of the reference binding molecule. A binding molecule may comprise all six HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of a reference binding molecule (such as any of the antibodies illustrated in Figure 3 or Figure 4), except that it has 1, 2, or 3 amino acid changes, e.g., substitutions, in any of said CDR sequences. The binding molecule may comprise 1, 2 or 3 amino acid changes, e.g. substitutions, in any one or more of said light chain CDRs and/or any one or more of said heavy chain CDRs. The binding molecule may comprise 1, 2 or 3 amino acid changes, e.g. substitutions, in any one or more of HCDR1, HCDR2, LCDR1, LCDR2 and LCDR3. The binding molecule may comprise 1, 2 or 3 amino acid changes, e.g. substitutions, in LCDR1.

[00127] The foregoing may be applied to any of the antibodies illustrated in Figure 3 or Figure 4 or Table 4, or to any of the binding molecules described herein, wherein the amino acid differences are defined relative to the CDR sequences thereof, and wherein the variant binding molecule binds to the same target antigen as said binding molecules and preferably exhibits the same antigen cross-reactivity. Thus, the reference binding molecule in any of the embodiments herein may be an antibody having VH and VL sequences of any of the antibodies illustrated in Figure 3 or Figure 4 or binding molecules of Table 3, or an antibody having the set of six CDRs of any of the antibodies illustrated in Figure 3 or Figure 4 or Table 4.

[00128] The amino acid difference may be an amino acid substitution, insertion, or deletion. In some embodiments, the amino acid difference is a conservative amino acid substitution as described herein.

[00129] In some embodiments, the binding molecule comprises all six sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as indicated for any of the antibodies illustrated in Figure 3 or Figure 4 or Table 4 or as indicated above, or a functional variant of any of these. For example, such a binding molecule may comprise all six sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as indicated for any of the antibodies illustrated in Figure 3 or Figure 4 or Table 4 or as indicated above, wherein any one or more of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences comprise 1, 2, or 3 amino acid changes, e.g. substitutions, as compared to the respective sequences as recited in Figure 3 or Figure 4 or Table 4 or above. A binding molecule may comprise all six sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as indicated for any of the antibodies illustrated in Figure 3 or Figure 4 or binding molecules of Table 3 or as indicated above, except for having 1, 2, or 3 amino acid changes, e.g., substitutions, in any of said CDR sequences. The binding molecule may comprise 1, 2, or 3 amino acid changes, e.g., substitutions, in any one or more of said light chain CDRs and/or any one or more of said heavy chain CDRs. The binding molecule may comprise 1, 2, or 3 amino acid changes, e.g., substitutions, in any one or more of HCDR1, HCDR2, LCDR1, LCDR2, and LCDR3. The binding molecule may comprise 1, 2, or 3 amino acid changes, e.g., substitutions, in LCDR1.

[00130] A binding molecule of the invention may comprise the heavy chain variable region (VH) and the light chain variable region (VL) of any antibody as shown in Figure 3 or Figure 4 or the binding molecule of Table 3.

[00131] For example, in some embodiments, the binding molecule comprises: a) a VH region amino acid sequence comprising SEQ ID NO: 51 and a VL region amino acid sequence comprising SEQ ID NO: 52; b) a VH region amino acid sequence comprising SEQ ID NO: 51 and a VL region amino acid sequence comprising SEQ ID NO: 53; c) a VH region amino acid sequence comprising SEQ ID NO: 51 and a VL region amino acid sequence comprising SEQ ID NO: 54; d) a VH region amino acid sequence comprising SEQ ID NO: 51 and a VL region amino acid sequence comprising SEQ ID NO: 55 (DVVMTQTPLSLPVSLGDQASISCRSSQSLVHX1NX2NTYLHWYLQKP GQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVFF CSQSTHVPWTFGGGTKLEIK wherein (a) XI and X2 are each any amino acid (SEQ ID NO: 76); or (b) X1 is P or S and X2 is R, T or G (SEQ ID NO: 77)); or e) a VH region amino acid sequence comprising SEQ ID NO: 56 and a VL region amino acid sequence comprising SEQ ID NO: 57.

[00132] In some embodiments, the binding molecule comprises: a) a VH region amino acid sequence comprising SEQ ID NO: 58 and a VL region amino acid sequence comprising SEQ ID NO: 59; or b) a VH region amino acid sequence comprising SEQ ID NO: 60 and a VL region amino acid sequence comprising SEQ ID NO: 61.

[00133] The present invention encompasses binding molecules as described herein comprising the sequences of the recited VH and VL regions (reference binding molecules), as well as functional variants thereof. A functional variant binds to the same target antigen as the reference binding molecule and preferably exhibits the same antigen cross-reactivity as the reference binding molecule. Functional variants may have a different affinity for the target antigen when compared to the reference binding molecule, but substantially the same affinity is preferred.

[00134] The VH region sequence of a binding molecule may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the corresponding VH region sequence of a reference binding molecule. The VL region sequence of a binding molecule may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the corresponding VL region sequence of a reference binding molecule.

[00135] In some embodiments, the CDRs of such a binding molecule are identical to the CDRs of the reference binding molecule and all differences are outside of these CDRs, for example the differences between the binding molecule and the reference binding molecule are in the framework regions of the VH and/or VL sequences.

[00136] In some embodiments, the CDRs of such a binding molecule are functional variants as described above. In some embodiments, such a binding molecule comprises amino acid differences in one or more CDRs and amino acid differences in one or more framework regions as compared to the VH and/or VL sequences of the corresponding reference binding molecule.

[00137] In some embodiments, a binding molecule has the same framework sequences as the reference binding molecules. In another embodiment, the binding molecule comprises a framework region with at most 2, preferably at most 1, amino acid differences (when compared to a corresponding reference framework sequence). Thus, each framework region can have at most 2, preferably at most 1, amino acid differences (when compared to a corresponding reference framework sequence).

[00138] In some embodiments, a binding molecule may have at most 5, 4, or 3 total amino acid differences in the framework regions thereof when compared to a corresponding reference binding molecule, with the proviso that there are at most 2 (preferably at most 1) amino acid differences per framework region. Such a binding molecule may have at most 2 (more preferably at most 1) total amino acid differences in the framework regions thereof when compared to a corresponding reference binding molecule, with the proviso that there are at most 2 amino acid differences per framework region. Such a binding molecule may have at most 2 (more preferably at most 1) total amino acid differences in the framework regions thereof when compared to a corresponding reference binding molecule, with the proviso that there is at most 1 amino acid difference per framework region.

[00139] Thus, a binding molecule may comprise a VH region and a VL region as described herein, wherein: the VH region has at most 14 amino acid differences (at most 2 amino acid differences in each CDR and at most 2 amino acid differences in each framework region) when compared to the VH region of a reference binding molecule herein; and the VL region has at most 14 amino acid differences (at most 2 amino acid differences in each CDR and at most 2 amino acid differences in each framework region) when compared to the VL region of a reference binding molecule herein; wherein the variant binding molecule binds to the same target antigen as the reference binding molecule and preferably exhibits the same antigen cross-reactivity as the reference binding molecule.

[00140] Said variant VH or VL regions may be referred to as "functional equivalents" of the reference VH or VL regions.

[00141] In some embodiments, a binding molecule can comprise a VH region and a VL region as described herein, wherein: the VH region has at most 7 amino acid differences (e.g., at most 1 amino acid difference in each CDR and at most 1 amino acid difference in each framework region) when compared to a VH region of a reference binding molecule herein; and the VL region has at most 7 amino acid differences (e.g., at most 1 amino acid difference in each CDR and at most 1 amino acid difference in each framework region) when compared to a VL region of a reference binding molecule herein; wherein the variant binding molecule binds to the same target antigen as the reference binding molecule and preferably exhibits the same antigen cross-reactivity as the reference binding molecule.

[00142] In some embodiments, the binding molecule comprises: a) a VH region comprising the amino acid sequence of SEQ ID NO: 51, or a sequence that has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto; and b) a VL region comprising the amino acid sequence of SEQ ID NO: 55 or a sequence that has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto, wherein (i) XI and X2 of SEQ ID NO: 55 are each any amino acid (SEQ ID NO: 76); or (ii) X1 of SEQ ID NO: 55 is P or S and X2 of SEQ ID NO: 55 is R, T, or G (SEQ ID NO: 77).

[00143] For example, in some embodiments, the binding molecule comprises: a) a VH region comprising the amino acid sequence of SEQ ID NO: 51, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto; and b) a VL region comprising the amino acid sequence of SEQ ID NO: 76, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto.

[00144] For example, in some embodiments, the binding molecule comprises: a) a VH region comprising the amino acid sequence of SEQ ID NO: 51, or a sequence that has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto; and b) a VL region comprising the amino acid sequence of SEQ ID NO: 77, or a sequence that has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto. c) In some embodiments, the binding molecule comprises: d) a VH region comprising the amino acid sequence of SEQ ID NO: 51, or a sequence that has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto; and e) a VL region comprising the amino acid sequence of SEQ ID NO: 52, or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto.

[00145] In some embodiments, the binding molecule comprises: a) a VH region comprising the amino acid sequence of SEQ ID NO: 51, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto; and b) a VL region comprising the amino acid sequence of SEQ ID NO: 53, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto.

[00146] In some embodiments, the binding molecule comprises: a) a VH region comprising the amino acid sequence of SEQ ID NO: 51, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto; and b) a VL region comprising the amino acid sequence of SEQ ID NO: 54, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto.

[00147] In some embodiments, the binding molecule comprises: a) a VH region comprising the amino acid sequence of SEQ ID NO: 56, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto; and b) a VL region comprising the amino acid sequence of SEQ ID NO: 57, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto.

[00148] In some embodiments, the binding molecule comprises: a) a VH region comprising the amino acid sequence of SEQ ID NO: 58, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto; and b) a VL region comprising the amino acid sequence of SEQ ID NO: 59, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto.

[00149] In some embodiments, the binding molecule comprises: a) a VH region comprising the amino acid sequence of SEQ ID NO: 60, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto; and b) a VL region comprising the amino acid sequence of SEQ ID NO: 61, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto.

[00150] In some embodiment, the binding molecule comprises: a) a VH region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to a reference amino acid sequence of SEQ ID NO: 51, 56, 58, 60, or a functional variant thereof; and c) a VL region comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity to a reference amino acid sequence of SEQ ID NO: 52, 53, 54, 55, 57, 59, 61, or a functional variant thereof, wherein (i) X1 and X2 of SEQ ID NO: 55 are each any amino acid (SEQ ID NO: 76); or (ii) X1 of SEQ ID NO: 55 is P or S and X2 of SEQ ID NO: 55 is R, T or G (SEQ ID NO: 77).

[00151] The foregoing may be applied to any of the antibodies illustrated in Figure 3 or Figure 4 or binding molecules of Table 3, or to any of the binding molecules described herein, wherein the amino acid differences are defined relative to the VH and/or VL sequences thereof, and wherein the binding molecule binds to the same target antigen as said binding molecules and preferably exhibits the same antigen cross-reactivity. Thus, the reference binding molecule in any of the embodiments herein may be an antibody having a VH and VL sequence of any of the antibodies illustrated in Figure 3 or Figure 4 or binding molecules of Table 3.

[00152] The amino acid difference may be an amino acid substitution, insertion, or deletion. In some embodiments, the amino acid difference is a conservative amino acid substitution as described herein.

[00153] The binding molecule of the invention may be provided in isolated form.

[00154] In some embodiments, the binding molecule of the invention is an immunoglobulin molecule. In some embodiments, the binding molecule is an antibody or an antigen-binding fragment thereof.

[00155] In particular, an antibody is a protein including at least one or two heavy chain (H) variable regions (abbreviated herein as VH) and at least one or two light chain (L) variable regions (abbreviated herein as VL). The VH and VL regions may be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDRs"), interspersed with regions that are more conserved, termed "framework regions" (FW). The extent of the framework region and CDRs has been precisely defined (see Kabat, E.A., et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991, and Chothia, C. et al., J. Mol. Biol. 196:901-917, 1987). Preferably, each VH and VL is composed of three CDRs and four FWs, arranged from the amino terminus to the carboxy terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4.

[00156] The heavy or light chain of the antibody can additionally include all or part of a heavy or light chain constant region. In some embodiments, the binding molecule comprises all or part of a heavy chain constant region of SEQ ID NO: 78 and all or part of a light chain constant region of SEQ ID NO: 79. In some embodiments, the binding molecule comprises a heavy chain constant region and a light chain constant region as shown in Table 7. The binding molecule can comprise a functional fragment of a heavy chain constant region and/or a light chain constant region as shown in Table 7, such as a fragment wherein the binding molecule retains the ability to bind to one or more Fc receptors as described herein. In some embodiments, the binding molecule can comprise a heavy chain constant region and a light chain constant region as shown in Table 7, and can further comprise a VH region and a VL region as disclosed herein, for example the VH and VL sequences of any of the antibodies illustrated in Figure 3 or Figure 4 or binding molecules of Table 1. In some embodiments, the binding molecule is an antibody that is a tetramer of two immunoglobulin heavy chains and two immunoglobulin light chains, wherein the immunoglobulin heavy and light chains are interconnected by, for example, disulfide bonds. The heavy chain constant region includes three domains, CH1, CH2, and CH3. In some embodiments, the binding molecule has a heavy chain constant region of SEQ ID NO: 78. The light chain constant region is comprised of one domain, CL. In some embodiments, the binding molecule has a light chain constant region of SEQ ID NO: 79. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen.

[00157] The "Kabat numbering system" is generally used when referring to a residue in the variable region (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., "Sequences of Immunological Interest", 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[00158] Amino acid position numbering as in Kabat refers to the numbering system used for heavy chain variable domains or light chain variable domains of the antibody assembly in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or more amino acids corresponding to a shortening of, or insertion into, a FW or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after residue 82 of the heavy chain FW.

[00159] Kabat residue numbering can be determined for a given antibody by aligning regions of antibody sequence homology with a "standard" Kabat numbered sequence. In contrast, Chothia refers to the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The table below lists the positions of the amino acids comprising the variable regions of antibodies in each system. Table 1: Positions of amino acids comprising the variable regions of antibodies 1Numbering of Kabat 2Numbering of Chothia

[00160] ImMunoGeneTics (IMGT) also provides a numbering system for immunoglobulin variable regions, including CDRs. See, e.g., Lefranc, M. P. et al., Dev. Comp Immunol. 27: 55-77 (2003). The IMGT numbering system is based on an alignment of more than 5,000 sequences, structural data, and characterization of hypervariable loops and allows easy comparison of variable regions and CDRs for all species. According to the IMGT numbering schemes, HCDR1 is at positions 26-35, HCDR2 is at positions 51-57, HCDR3 is at positions 93-102, LCDR1 is at positions 27-32, LCDR2 is at positions 50-52, and LCDR3 is at positions 89-97.

[00161] The term "antibody" includes intact immunoglobulins of the types IgA, IgG, IgE, IgD, IgM (as well as their subtypes), in which the immunoglobulin light chains may be of the kappa or lambda types. The binding molecule of the invention may be, or may comprise, a full-length antibody, or may be or comprise an antigen-binding fragment thereof. The term antigen-binding fragment, as used herein, refers to a portion of an antibody that binds to CCR9, for example, a molecule in which one or more immunoglobulin chains are not full-length, but which bind to CCR9. In some embodiments, the antigen-binding fragment is one or more selected from an Fv fragment, a Fab fragment, an F(ab')2 fragment, a Fab' fragment, a dsFv fragment, an scFv fragment, an sc(Fv)2 fragment, a dAb fragment, a single chain antibody, or a combination thereof. For example, in some embodiments, the antigen-binding fragment is a Fab fragment. In some embodiments, the binding molecule comprises a Fab domain.

[00162] The antibodies of the invention or antigen-binding fragments thereof may have any antibody format. In some embodiments, the antibody has the "conventional" format described above. Alternatively, the antibody may be, in some embodiments, a Fab fragment. The antibody or antigen-binding fragment according to the invention may also be a Fab', an Fv, a scFv, a Fd, a V domain NAR, an IgNAR, an intrabody, an IgG CH2, a minibody, a single domain antibody, an Fcab, a scFv-Fc, F(ab‘)2, a di-scFv, a bispecific T-cell engager (BiTE®), an F(ab')3, a tetrabody, a triabody, a diabody, a DVD-Ig, a (scFv)2, or a mAb2.

[00163] In some embodiments, the binding molecule is a monoclonal antibody (mAb). In some embodiments, the binding molecule is a polyclonal antibody.

[00164] A "monoclonal antibody" (mAb) refers to a homogeneous population of antibodies involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies which typically include different antibodies directed against different antigenic determinants. The term "monoclonal antibody" encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain mutants (scFv), fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, "monoclonal antibody" refers to such antibodies prepared in any number of ways including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

[00165] In some embodiments, the antibody or antigen-binding fragment is one or more selected from a murine antibody, a humanized antibody, a chimeric antibody, a fully human antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a multispecific antibody, or a combination thereof. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody. In some embodiments, an antibody of the invention does not induce an anti-human antibody response, e.g., a murine anti-human antibody response.

[00166] The antibodies of the invention and antigen-binding fragments thereof may be derived from any species by recombinant means. For example, the antibodies or antigen-binding fragments may be mouse, rat, goat, horse, swine, bovine, chicken, rabbit, camelid, donkey, human, or chimeric versions thereof. For use in administration to humans, the non-human-derived antibodies or antigen-binding fragments may be genetically or structurally altered to be less antigenic upon administration to the human patient.

[00167] Especially preferred are human or humanized antibodies, especially as recombinant human or humanized antibodies. The term "humanized antibody" refers to an antibody derived from a non-human (e.g., murine) immunoglobulin, which has been engineered to contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which complementarity determining region (CDR) residues are replaced with CDR residues from a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and capacity (Jones et al., 1986, Nature, 321: 522-525; Riechmann et al., 1988, Nature, 332: 323-327; Verhoeyen et al., 1988, Science, 239: 1534-1536). In some cases, residues in the Fv framework region of a human immunoglobulin are replaced by the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capacity.

[00168] Humanized antibodies may be further modified by substituting additional residues in the Fv framework region and/or within the substituted non-human residues to refine and optimize the specificity, affinity, and/or capacity of the antibodies. In general, humanized antibodies will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin while all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin (Fc) constant region or domain, typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. Nos. 5,225,539 or 5,639,641.

[00169] In some embodiments, an antibody of the invention is a human antibody. The term "human antibody" means an antibody produced in a human or an antibody having an amino acid sequence corresponding to an antibody produced in a human prepared using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides.

[00170] In some embodiments, an antibody of the invention is a chimeric antibody. The term "chimeric antibodies" refers to antibodies in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both the light and heavy chains corresponds to the variable region of antibodies derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capacity while the constant regions are homologous to the sequences in antibodies derived from another (typically human) to avoid eliciting an immune response in that species.

[00171] There are five major classes of heavy chain constant region, classified as IgA, IgG, IgD, IgE and IgM, each with characteristic effector functions designated by isotype. For example, IgG is separated into four subclasses known as IgG1, IgG2, IgG3 and IgG4. Ig molecules interact with several classes of cellular receptors. For example, IgG molecules interact with three classes of FCY receptors (FCYR) specific to the IgG antibody class, namely FCYRI, FCYRII and FCYRIII. The sequences important for binding of IgG to FCYR receptors have been reported to be located in the CH2 and CH3 domains. The antibodies of the invention or antigen-binding fragments thereof may be of any isotype, i.e. IgA, IgD, IgE, IgG and IgM, and synthetic multimers of the four immunoglobulin (Ig) chain structure. In preferred embodiments, the antibodies or antigen-binding fragments thereof are of the IgG isotype. The antibodies or antigen-binding fragments may be of any subclass of IgG, e.g., IgG1, IgG2, IgG3, or IgG4 isotype. In preferred embodiments, the antibodies or antigen-binding fragments thereof are of an IgG1 or IgG2 isotype.

[00172] In some embodiments, the antibodies comprise a heavy chain constant region that is of the IgG isotype. In some embodiments, the antibodies comprise a portion of a heavy chain constant region that is of the IgG isotype. In some embodiments, the IgG constant region or portion thereof is an IgG1, IgG2, IgG3, or IgG4 constant region. Preferably, the IgG constant region or portion thereof is an IgG1 or IgG2 constant region. Antibody molecules can also have other formats, for example lgG1 with YTE (Dall'Acqua et al., (2002) J. Immunology, 169: 5171- 5180; Dall'Acqua et al., (2006) J. Biol. Chem. 281 (33):23514-24) and/or TM (Oganesyan et al., (2008) Acta Cryst D64:700-4) mutations in the Fc region.

[00173] Antibodies of the invention or antigen-binding fragments thereof may comprise a lambda light chain or a kappa light chain. Engineered antibodies and antigen-binding fragments thereof include those in which modifications have been made to framework residues within the VH region and/or VL region. Such modifications may improve the properties of the antibody, for example, to decrease the immunogenicity of the antibody and/or to improve the production and purification of the antibody.

[00174] Binding molecules of the invention include both intact and modified forms of the binding molecules disclosed herein. For example, a binding molecule of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as a pharmaceutical agent, a detection agent, and/or a protein or peptide that can mediate the association of a binding molecule disclosed herein with another molecule (e.g., a streptavidin core region or a polyhistidine tail). Non-limiting examples of detection agents include: enzymes, such as alkaline phosphatase, glucose-6-phosphate dehydrogenase (“G6PDH”), alpha-D-galactosidase, glucose oxidase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, and peroxidase, e.g., horseradish peroxidase; dyes; fluorescent markers or fluorescence such as fluorescein and its derivatives, fluorochrome, rhodamine compounds and derivatives, GFP (GFP for green fluorescent protein), dansyl, umbelliferone, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine; fluorophores such as cryptates and lanthanide chelates, e.g. Europium etc. (Perkin Elmer and Cis Biointernational); chemiluminescent or chemiluminescent markers such as isoluminol, luminol and the dioxetanes; bioluminescent markers such as luciferase and luciferin; sensitizers; coenzymes; enzyme substrates; radiolabels including, but not limited to, bromine77, carbon14, cobalt57, fluorine8, gallium67, gallium68, hydrogen3 (tritium), indium111, indium113m, iodine123m, iodine125, iodine126, iodine131, iodine133, mercury107, mercury203, phosphorus32, rhenium99m, rhenium101, rhenium105, ruthenium95, ruthenium97, ruthenium103, ruthenium105, scandium47, selenium75, sulfur35, technetium99, technetium99m, tellurium121m, tellurium122m, tellurium125m, thulium165, thulium167, thulium-168 and yttrium-199; particles, such as latex or carbon particles, metal sol, crystallite, liposomes, cells, etc., which may be additionally labeled with a dye, catalyst or other detectable group; molecules such as biotin, digoxigenin or 5-bromodeoxyuridine; toxin fractions, such as, for example, a toxin fraction selected from a Pseudomonas exotoxin group (PE or a cytotoxic fragment or mutant thereof), diphtheria toxin or a cytotoxic fragment or mutant thereof, botulinum toxin A, B, C, D, E or F, ricin or a cytotoxic fragment thereof, for example ricin A, abrin or a cytotoxic fragment thereof, saporin or a cytotoxic fragment thereof, blueberry antiviral toxin or a cytotoxic fragment thereof and bryodin 1 or a cytotoxic fragment thereof.

[00175] The binding molecules of the invention also include derivatives that are modified (e.g., by covalently attaching any type of molecule to the binding molecule) such that the covalent bond does not prevent the binding of the binding molecule to its target (e.g., epitope), or otherwise impair the biological activity of the binding molecule. Suitable derivatives can be produced by methods including, but not limited to, fucosylation, glycosylation, acetylation, PEGylation, phosphorylation, and amidation.

[00176] Additional embodiments are multispecific (bispecific, trispecific, etc.) binding molecules and other conjugates, for example with small cytotoxic molecules. The binding molecules of the present invention, including antibodies of the present invention, can be obtained using conventional techniques known to those skilled in the art and their utility confirmed by conventional binding studies - exemplary methods are described in Examples 3 and 4. By way of example, a simple binding assay is to incubate the cell expressing an antigen with the antibody. If the antibody is labeled with a fluorophore, binding of the antibody to the antigen can be detected by FACS analysis.

[00177] The antibodies of the present invention can be produced in a variety of animals, including mice, rats, rabbits, goats, sheep, monkeys or horses. The antibodies can be produced following immunization with individual capsular polysaccharides or with a plurality of capsular polysaccharides. Blood isolated from these animals contains polyclonal antibodies - multiple antibodies that bind to the same antigen. The antigens can also be injected into chickens to generate polyclonal antibodies in the egg yolk. To obtain a monoclonal antibody that is specific for a single epitope of an antigen, antibody-secreting lymphocytes are isolated from an animal and immortalized by fusing them with a cancer cell line. The fused cells are called hybridomas and will grow continuously and secrete antibodies in culture. Individual hybridoma cells are isolated by dilution cloning to generate clones of cells that all produce the same antibody; these antibodies are called monoclonal antibodies. Methods for producing monoclonal antibodies are conventional techniques known to those skilled in the art (see, for example, "Making and Using Antibodies: A Practical Handbook." G. C. Howard. CRC Books. 2006. ISBN 0849335280). Polyclonal and monoclonal antibodies are often purified using Protein A/G or antigen affinity chromatography.

[00178] The antibody or antigen-binding fragment thereof of the invention may be prepared as an anti-CCR9 monoclonal antibody, which may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature 256:495 (1975). Using the hybridoma method, a mouse, hamster, or other suitable host animal is immunized as described above to elicit production by lymphocytes of antibodies that will specifically bind to an immunizing antigen. The lymphocytes may also be immunized in vitro. After immunization, the lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol, to form hybridoma cells that may then be selected and separated from unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies specifically directed against a chosen antigen as determined by immunoprecipitation, immunoblotting, or an in vitro binding assay, e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), can then be propagated in vitro culture using standard methods (Goding, "Monoclonal Antibodies: Principles and Practice", Academic Press, 1986) or in vivo as ascites tumors in an animal. The monoclonal antibodies can then be purified from the culture medium or ascites fluid using known methods.

[00179] Alternatively, the binding molecule (e.g., a monoclonal antibody) can also be produced using recombinant DNA methods as described in U.S. Patent No. 4,816,567. Polynucleotides encoding a monoclonal antibody are isolated from mature B cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using standard procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors which, when transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that otherwise do not produce immunoglobulin protein, monoclonal antibodies are generated by the host cells. Likewise, recombinant monoclonal antibodies or antigen-binding fragments thereof of the desired species can be isolated from phage display libraries expressing CDRs of the desired species as described in McCafferty et al., Nature 348:552-554 (1990); Clackson et al., Nature, 352:624-628 (1991); and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[00180] The polynucleotide(s) encoding a binding molecule of the invention may be further modified in a number of different ways using recombinant DNA technology to generate alternative binding molecules. In some embodiments, the constant domains of the light and heavy chains of, e.g., a mouse monoclonal antibody may be replaced (1) with those regions of, e.g., a human antibody to generate a chimeric antibody or (2) with a different immunoglobulin polypeptide to generate a fusion antibody. In some embodiments, the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody. Site-directed or high-density mutagenesis of the variable region may be used to optimize the specificity, affinity, etc. of a monoclonal antibody.

[00181] In some embodiments, the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof. Human antibodies can be directly prepared using various techniques known in the art. Immortalized human B lymphocytes immunized in vitro or isolated from an immunized individual that produce an antibody directed against a target antigen can be generated. See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J. Immunol. 147 (1): 86-95 (1991); U.S. Patent 5,750,373.

[00182] In some embodiments, the antibody or antigen-binding fragment thereof can be selected from a phage library, where such phage library expresses human antibodies, as described, for example, in Vaughan et al., Nat. Biotech. 14:309-314 (1996); Sheets et al., Proc. Natl. Acad. Sci. USA, 95:6157-6162 (1998); Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); and Marks et al., J. Mol. Biol. 222:581 (1991). Techniques for generating and using antibody phage libraries are also described in U.S. Patent Nos. 5,969,108; 6,172,197; 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe et al., J. Molec. Biol. 376:1182-1200 (2008), each of which is incorporated by reference in its entirety.

[00183] Affinity maturation strategies and chain shuffling strategies are known in the art and can be employed to generate high affinity human antibodies or antigen-binding fragments thereof. See Marks et al., BioTechnology 10:779-783 (1992), incorporated by reference in its entirety.

[00184] In some embodiments, the antibody or antigen-binding fragment thereof (e.g., a monoclonal antibody) can be a humanized antibody. Methods for engineering, humanizing, or surface-coating non-human or human antibodies can also be used and are well known in the art. A humanized, surface-coated, or similarly engineered antibody can have one or more amino acid residues from a non-human source, for example, but not limited to, mouse, rat, rabbit, non-human primate, or other mammal. These non-human amino acid residues are replaced with residues that are often referred to as "imported" residues, which are typically taken from a variable, constant, or other "imported" domain of a known human sequence. Such imported sequences can be used to reduce immunogenicity or to reduce, enhance, or modify binding, affinity, association rate, dissociation rate, avidity, specificity, half-life, or any other suitable characteristic, as known in the art. Suitably, the CDR residues may be directly and most substantially involved in influencing binding to CCR9. Accordingly, part or all of the non-human or human CDR sequences are preferably retained while the non-human sequences of the variable and constant regions may be replaced by human or other amino acids.

[00185] The antibodies may also optionally be humanized, surface-coated, engineered or human-engineered antibodies with retention of high affinity for the CCR9 antigen and other favorable biological properties. To achieve this goal, humanized (or human) or engineered anti-CCR9 antibodies and surface-coated antibodies may optionally be prepared by a process of analyzing the original sequences and various humanized and engineered conceptual products using three-dimensional models of the original, engineered and humanized sequences. Three-dimensional models of immunoglobulins are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display likely three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the likely role of residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen, such as CCR9. In this way, FW residues can be selected and combined from the consensus sequences and imported such that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.

[00186] Humanization, surface coating or engineering of antibodies or anti-CCR9 antigen-binding fragments thereof of the present invention may be carried out using any known method, such as those but not limited to those described in Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988); Sims et al., J. Immunol. 151:2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993); U.S. Pat. Nos. 5,639,641; 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; 4,816,567, 7,557,189; 7,538,195; and 7,342,110; International Application Nos. PCT/US98/16280; PCT/US96/18978; PCT/US91/09630; PCT/US91/05939; PCT/US94/01234; PCT/GB89/01334; PCT/GB91/01134; PCT/GB92/01755; International Patent Application Publication Nos. WO90/14443; WO90/14424; WO90/14430; and European Patent Publication No. EP 229246; each of which is incorporated in its entirety herein by reference, including the references cited therein.

[00187] Humanized anti-CCR9 antibodies and antigen-binding fragments thereof can also be prepared in transgenic mice containing human immunoglobulin loci that are capable upon immunization of producing the full human antibody repertoire in the absence of endogenous immunoglobulin production. This approach is described in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

[00188] In some embodiments, a fragment (e.g., antibody fragment) of the antibody (e.g., anti-CCR9 antibody) is provided. Various techniques are known for producing antibody fragments. Traditionally, such fragments are derived by proteolytic digestion of intact antibodies, as described, for example, by Morimoto et al., J. Biochem. Biophys. Meth. 24:107-117 (1993) and Brennan et al., Science 229:81 (1985). In some embodiments, the anti-CCR9 antibody fragments are produced recombinantly. The Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli or other host cells, thereby allowing for the production of large quantities of such fragments. Such anti-CCR9 antibody fragments can also be isolated from the antibody phage libraries discussed above. Anti-CCR9 antibody fragments may also be linear antibodies as described in U.S. Patent No. 5,641,870. Other techniques for producing antibody fragments will be apparent to the skilled practitioner.

[00189] Techniques for producing CCR9-specific single chain antibodies may be adapted in accordance with the present invention. See, e.g., U.S. Patent No. 4,946,778). Additionally, methods for constructing Fab expression libraries may be adapted to allow rapid and efficient identification of monoclonal Fab fragments having the desired specificity for CCR9 or their derivatives, fragments, analogs or homologs. See, e.g., Huse et al., Science 246:1275-1281 (1989). Antibody fragments may be produced by techniques known in the art including, but not limited to: F(ab')2 fragment produced by pepsin digestion of an antibody molecule; Fab fragment generated by reduction of the disulfide bonds of an F(ab')2 fragment; Fab fragment generated by treatment of the antibody molecule with papain and a reducing agent; or Fv fragments.

[00190] A modified binding molecule (e.g., antibody or antigen-binding fragment thereof as provided herein) may comprise any type of binding region that provides for the association of the binding molecule with CCR9. In this sense, the binding region may be a variable region, which may comprise or be derived from any type of mammal that can be induced to mount a humoral response and generate immunoglobulins against the desired antigen. As such, the variable region of an anti-CCR9 antibody or antigen-binding fragment thereof may be of, for example, human, murine, non-human primate (e.g., cynomolgus monkeys, macaques, etc.), or lupine origin. In some embodiments, both the variable and constant regions of the modified antibody or antigen-binding fragment thereof are human. In some embodiments, the variable regions of a compatible antibody (usually derived from a non-human source) may be engineered or specifically customized to improve the binding properties or reduce the immunogenicity of the molecule. In this regard, the variable regions useful in the present invention may be humanized or otherwise altered by the inclusion of imported amino acid sequences.

[00191] In some embodiments, the variable domains in both the heavy and light chains of an antibody or antigen-binding fragment thereof are altered by at least partial replacement of one or more CDRs and/or by partial replacement of the framework region and sequence changes. Although the CDRs may be derived from an antibody of the same class or even subclass of the antibody from which the framework regions are derived, it is conceived that the CDRs will be derived from an antibody of a different class and in certain embodiments from an antibody of a different species. It is not necessary to replace all of the CDRs with the complete CDRs of the donor variable region to transfer antigen-binding capability from one variable domain to another. Rather, it is only necessary to transfer those residues that are necessary to maintain antigen-binding site activity. Given the explanations set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762 it will be well within the competence of those skilled in the art to carry out routine experimentation to obtain a functional antibody with reduced immunogenicity.

[00192] Notwithstanding the alterations in the variable region, those skilled in the art will appreciate that a modified antibody or antigen-binding fragment thereof of this invention can comprise an antibody (e.g., full-length antibody or antigen-binding fragment thereof) in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics, such as increased effector function, including improved ability to induce ADCC when compared to an antibody of approximately the same immunogenicity comprising a native or unaltered constant region. In some embodiments, the constant region of the modified antibody will comprise a human constant region. Modifications to the constant region compatible with this invention comprise additions, deletions, or substitutions of one or more amino acids in one or more domains. That is, a modified antibody disclosed herein can comprise alterations or modifications in one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and/or in the light chain constant domain (CL). In some embodiments, a modified constant region is contemplated in which one or more domains are partially or entirely deleted. In some embodiments, the modified antibody will comprise domain deletion constructs or variants in which the entire CH2 domain has been removed (ΔCH2 constructs). In some embodiments, the omitted constant region domain can be replaced with a short amino acid spacer (e.g., 10 residues) that provides some of the molecular flexibility typically conferred by the absent constant region.

[00193] Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods, and hybrid methods, such as, for example, segment approach methods. Protocols for determining percent identity are routine procedures within the scope of one of skill in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by summing the scores of individual residue pairs and imposing gap penalties. Non-limiting methods include, for example, CLUSTAL W, see, for example, Julie D. Thompson et al., "CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice", 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, e.g., Osamu Gotoh, "Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments", 264(4) J. MoI. Biol. 823–838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all input sequences. Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, "Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences", 8(5) CABIOS 501–509 (1992); Gibbs sampling, see, for example, C. E. Lawrence et al., "Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment," 262(5131) Science 208-214 (1993); Align-M, see, for example, Ivo Van WaIIe et al., "Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences", 20(9) Bioinformatics:1428-1435 (2004).

[00194] Thus, the percentage of sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize alignment scores using a gap-opening penalty of 10, a gap-extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by standard single-letter codes).

[00195] The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, % identity can be calculated as the number of identical nucleotides/amino acids divided by the total number of nucleotides/amino acids, multiplied by 100. Calculations of % sequence identity can also take into account the number of gaps and the length of each gap that needs to be introduced to optimize the alignment of two or more sequences. Sequence comparisons and the determination of the percent identity between two or more sequences can be carried out using specific mathematical algorithms, such as BLAST, which will be familiar to a skilled artisan.

[00196] The present invention further encompasses variants and equivalents that are substantially homologous to a binding molecule of the invention (e.g., an antibody, such as a murine, chimeric, humanized or human antibody, or antigen-binding fragments thereof). These may contain, for example, conservative substitution mutations, i.e., the replacement of one or more amino acids with similar amino acids. For example, conservative substitution refers to the replacement of one amino acid with another within the same general class such as, for example, an acidic amino acid with another acidic amino acid, a basic amino acid with another basic amino acid, or a neutral amino acid with another neutral amino acid. It is well known in the art what is meant by a conservative amino acid substitution.

[00197] Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions, or additions. These changes are preferably of a minimal nature, i.e., conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag. Table 2: Conservative Amino Acid Substitutions

[00198] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline and α-methyl serine) may be substituted for amino acid residues of the binding molecules of the present invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code and unnatural amino acids may be substituted for amino acid residues of polypeptides. The binding molecules of the present invention may also comprise non-naturally occurring amino acid residues.

[00199] Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methane-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethyl-homo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Various methods are known in the art for incorporating non-natural amino acid residues into proteins. For example, an in vitro system can be employed in which nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90: 10145-9, 1993. In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271: 19991-8, 1996). In a third method, E. coli cells are grown in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired unnatural amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The unnaturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See Koide et al., Biochem. 33: 7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by chemical modification in vitro. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2: 395-403, 1993).

[00200] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues of binding molecules of the present invention.

[00201] The essential amino acids in the binding molecules of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244:1081-5, 1989). The sites of biological interaction can also be determined by physical analysis of the structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with amino acid mutation of putative contact sites. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309: 59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g., translocation or protease components) of the binding molecules of the present invention.

[00202] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:21526, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of permissible substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

5.2. Compositions

[00203] Also provided herein are compositions comprising a binding molecule of the invention. The composition may comprise any binding molecule as described herein. The composition may comprise more than one binding molecule as described herein.

[00204] In another aspect, a composition comprising a binding molecule that binds to CCR9 is provided, wherein said binding molecule is capable of (i) inhibiting the binding of CCL25 to CCR9 and (ii) mediating antibody-dependent cell-mediated cytotoxicity against a CCR9-expressing cell to which it binds.

[00205] A composition as described herein may be a pharmaceutical composition, such as a composition comprising a binding molecule of the present invention and further comprising at least one pharmaceutically acceptable carrier or diluent. The term "pharmaceutical composition" refers to a preparation that is in such a form as to permit the biological activity of the active ingredient to be effective and that does not contain additional components that are unacceptably toxic to a subject to which the composition would be administered. Such a composition may be sterile and may comprise a pharmaceutically acceptable carrier, such as physiological saline. Suitable pharmaceutical compositions may comprise one or more of a buffer (e.g., acetate, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), a stabilizing agent (e.g., human albumin), a preservative (e.g., benzyl alcohol), and absorption enhancer to enhance bioavailability, and/or other conventional solubilizing or dispersing agents.

[00206] In some embodiments, a pharmaceutical composition of the invention can comprise a sterile, non-toxic, pharmaceutically acceptable carrier such as physiological saline, non-toxic buffers, preservatives, and the like. Suitable formulations for use in the therapeutic methods disclosed herein are described in "Remington's Pharmaceutical Sciences", 22nd ed., Ed. Lloyd V. Allen, Jr. (2012).

[00207] In some embodiments, a pharmaceutical composition of the invention may be comprised within one or more formulations selected from a capsule, a tablet, an aqueous suspension, a solution, a nasal spray, or a combination thereof.

[00208] In some embodiments, the pharmaceutical composition comprises more than one type of binding molecule of the invention. For example, a pharmaceutical composition can comprise two or more selected from an antibody, an antigen-binding fragment, or a combination thereof.

[00209] The term "a pharmaceutically effective amount" of a binding molecule means an amount sufficient to achieve effective binding to a target and to achieve a benefit, for example, to improve the symptoms of a disease or condition or to detect a substance or a cell.

[00210] In some embodiments, a pharmaceutical composition can comprise a buffer (e.g., acetate, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizing agent (e.g., human albumin), etc.

[00211] Suitably, the binding molecule of the invention binds to the CCR9 molecule with sufficient affinity that the binding molecule is useful as a therapeutic agent or a diagnostic reagent in targeting CCR9.

5.3. Therapeutic methods

[00212] The invention also relates to the use of binding molecules and compositions of the invention in therapeutic methods. The invention provides a binding molecule of the invention for use in such therapeutic methods, for example a binding molecule of the invention for use in a method of treating the human or animal body by therapy. Where therapeutic methods and uses are described herein with reference to the binding molecule of the invention, the same methods and uses are also included where the binding molecule is present in a composition, such as a pharmaceutical composition, such as a pharmaceutical composition as described herein.

[00213] Thus, the invention encompasses the binding molecule defined above and the pharmaceutical composition defined above for use in a method of treating a CCR9-mediated disease or condition.

[00214] In one aspect, there is provided a binding molecule of the invention for use in the treatment of a CCR9-mediated disease or condition. Also provided is a composition of the invention, e.g., a pharmaceutical composition of the invention, for use in the treatment of a CCR9-mediated disease or condition. Further, there is provided the use of a binding molecule of the invention in the manufacture of a medicament for the treatment of a CCR9-mediated disease or condition.

[00215] In another aspect, a method of treating a CCR9-mediated disease or condition in a subject is provided, the method comprising administering to the subject an effective amount of a binding molecule of the invention.

[00216] In one aspect, a binding molecule is provided for use in treating a CCR9-mediated disease or condition in a subject wherein said binding molecule is capable of (i) inhibiting the binding of CCL25 to CCR9 and (ii) mediating antibody-dependent cell-mediated cytotoxicity against a CCR9-expressing cell to which it binds.

[00217] In one aspect, a method of treating a CCR9-mediated disease or condition in a subject is provided, the method comprising administering to the subject an effective amount of a binding molecule that binds to CCR9, wherein said binding molecule is capable of (i) inhibiting the binding of CCL25 to CCR9, and (ii) mediating antibody-dependent cell-mediated cytotoxicity against a CCR9-expressing cell to which it binds.

[00218] In some embodiments of any of the treatment aspects, the CCR9-mediated disease or condition that is treated is an inflammatory bowel disease.

[00219] In some embodiments, the binding molecule is capable of depleting CCR9-expressing cells in the subject, e.g., in the intestine of the subject. Without being limited by theory, this may be due to the ability of the binding molecule to prevent migration of a CCR9-expressing cell from the periphery into the intestine of a subject and/or due to the ability of the binding molecule to induce death of a CCR9-expressing cell to which it binds.

[00220] Thus, in one aspect, there is provided a method of depleting cells expressing CCR9 in a subject in need thereof, comprising administering to the subject an effective amount of a binding molecule of the invention.

[00221] In some embodiments, the CCR9 polypeptide is comprised within a CCR9 polypeptide sequence or a fragment thereof.

[00222] A "CCR9 polypeptide" can comprise the full-length CCR9 polypeptide sequence or can comprise a CCR9 fragment of any length of the full-length CCR9 polypeptide sequence (e.g., comprising a polypeptide sequence of 5%, 15%, 25%, 35%, 45%, 55%, 65%, 75%, 85%, or 95% of the full-length CCR9 polypeptide sequence) that comprises an epitope that can bind (e.g., be bound by) a binding molecule of the invention.

[00223] The binding molecule may be advantageously used in methods for detecting a CCR9 epitope and associated diagnostic methods.

[00224] The term "treat" or "treating" as used herein encompasses therapeutic measures that cure, slow, alleviate symptoms of, and/or halt the progression of a diagnosed pathological condition or disorder. Thus, those in need of treatment include those already having the disorder. Preferably, the term "treat" or "treating" as used herein means corrective treatment. The term "treat" or "treatment" encompasses the treatment of inflammatory bowel diseases and the treatment of their symptoms and diseases/disorders associated therewith. In some embodiments, the term "treat" or "treatment" refers to a symptom of IBD, such as inflammation in the intestine, abdominal pain, diarrhea, blood in the stool. In some embodiments, a subject is successfully "treated" for a CCR9-mediated disease or disorder (e.g., inflammatory bowel disease) according to the methods provided herein if the patient experiences, for example, complete, partial, or transient relief or elimination of symptoms associated with the CCR9-mediated disease or disorder (e.g., inflammatory bowel disease). "Treating" or "treatment" also includes prophylactic treatment, for example to prevent the onset of the disease, such as to prevent the onset of IBD.

[00225] “Prevent” refers to prophylactic or preventative measures that prevent and/or slow the development of a targeted pathological condition or disorder. Thus, those in need of prevention include those prone to or susceptible to the disorder. In some embodiments, a disease or disorder (e.g., inflammatory bowel disease) is successfully prevented according to the methods provided herein if the patient develops, transiently or permanently, e.g., fewer or less severe symptoms associated with the disease or disorder, or a later onset of symptoms associated with the disease or disorder, than a patient who was not subjected to the methods of the invention.

[00226] The terms "subject", "individual" and "patient" are used interchangeably herein to refer to a mammalian subject. In some embodiments, the "subject" is a human, domestic animals, farm animals, sport animals and zoo animals, e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, etc. In some embodiments, the subject is a cynomolgus monkey (Macaca fascicularis). In preferred embodiments, the subject is a human. In the methods of the invention, the subject may not have been previously diagnosed as having inflammatory bowel disease. Alternatively, the subject may have been previously diagnosed as having inflammatory bowel disease. The subject may also be one who has risk factors for the disease or one who is asymptomatic for inflammatory bowel disease. The subject may also be one who has or is at risk for developing inflammatory bowel disease. Thus, in some embodiments, a method of the invention can be used to confirm the presence of inflammatory bowel disease in a subject. For example, the subject may have previously been diagnosed with inflammatory bowel disease by alternative means. In some embodiments, the subject has previously received therapy for inflammatory bowel disease.

[00227] In some embodiments, the treatment methods of the invention comprise one or more administration steps selected from oral, intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal, inhalation, topical, or a combination thereof.

[00228] In some embodiments, the binding molecule is delivered directly to the site of the adverse cell population (e.g., thereby increasing exposure of the diseased tissue to the therapeutic agent). In some embodiments, administration is directly to the airway, e.g., by inhalation or intranasal administration.

[00229] In some embodiments, the inflammatory bowel disease is Crohn's disease, such as ileal or ileocolonic Crohn's disease. In some embodiments, the inflammatory bowel disease is ulcerative colitis.

5.4. Detection Methods

[00230] In another aspect, methods are provided for detecting the presence or absence of a CCR9 polypeptide. A binding molecule of the invention can be used in such methods.

[00231] In some embodiments, a method of detecting the presence or absence of a CCR9 polypeptide comprises: a) contacting a sample with a binding molecule of the invention to provide a binding molecule-antigen complex; b) detecting the presence or absence of said binding molecule-antigen complex; c) wherein the presence of the binding molecule-antigen complex confirms the presence of a CCR9 polypeptide.

[00232] In some embodiments, any of steps (i)-(iii) above are performed ex vivo or in vitro. In some embodiments, all of steps (i)-(iii) above are performed ex vivo or in vitro. In some embodiments, any of steps (i)-(iii) above are performed in vivo.

[00233] The binding molecule may be any binding molecule described herein, for example a binding molecule comprising a set of six CDRs as illustrated for any antibody in Figure 3 or Figure 4 or Table 4, or a binding molecule comprising a VH and VL as illustrated for any antibody in Figure 3 or Figure 4 or binding molecules of Table 3.

[00234] In some embodiments, the detection methods described herein are performed in vitro. In some embodiments, the detection methods described herein are performed ex vivo. In some embodiments, the detection methods described herein are performed on a sample from a subject, such as a sample that has been previously obtained from the subject. The detection methods may or may not include a step of obtaining the sample from the subject. Suitably, a “sample” is a sample obtained from a subject (e.g., biopsy), cell line, tissue culture, or other source of cells that potentially express CCR9. In some embodiments, a sample is a biopsy from a subject. Said biopsy may be from the intestine of a subject suffering from an inflammatory bowel disease or from the intestine of a subject at risk for suffering from an inflammatory bowel disease. The sample may be an isolated sample from a subject.

[00235] The invention encompasses a corresponding use of the binding molecule of the invention to detect a CCR9 polypeptide (e.g., epitope of the CCR9 polypeptide).

[00236] In some embodiments, the presence of the binding molecule-antigen complex is indicative of the presence of an inflammatory bowel disease and the absence of the binding molecule-antigen complex is indicative of the absence of an inflammatory bowel disease.

[00237] In some embodiments, the inflammatory disease is an inflammatory bowel disease comprising a cell that expresses a CCR9 polypeptide (e.g., CCR9 polypeptide epitope).

[00238] Thus, the present invention encompasses the use of the binding molecule of the invention in methods of diagnosing a subject with an inflammatory bowel disease, preferably wherein said inflammatory bowel disease comprises a cell expressing CCR9.

[00239] In some embodiments, a detection method or diagnostic method may comprise measuring the level of CCR9 expression in a sample from a subject and comparing the measured CCR9 expression level to a reference level of CCR9, wherein an increase in the measured CCR9 expression level compared to the CCR9 reference level is indicative of the presence of inflammatory bowel disease. In some embodiments, said reference level of CCR9 is the level of CCR9 expression in a non-IBD (e.g., normal) sample of the same type, such as from the same tissue type.

[00240] An "antigen-binding molecule complex" means a complex (e.g., macromolecular complex) comprising an antigen that is bound to its respective binding molecule, for example CCR9 that is bound to a binding molecule of the invention. The term "antigen-binding molecule complex" may be used synonymously with the terms "bound CCR9 binding molecule complex" and "bound CCR9 binding molecule".

[00241] A binding molecule-antigen complex may be detected by any means known to one of skill in the art. In some embodiments, the binding molecule is labeled with a detectable marker. Said marker may be an epifluorescent label.

[00242] In some embodiments, a binding molecule-antigen complex is detected via a secondary (e.g., detection) antibody that binds to the binding molecule and/or the binding molecule-antigen complex.

[00243] Suitably, said secondary antibody comprises a detection means, such as a marker/label to aid in detection. Said detection means is preferably conjugated to the secondary antibody. Examples of suitable labels include detectable labels such as radiolabels or fluorescent or colored molecules, enzymatic labels or chromogenic labels - e.g., dyes that provide a visible color change upon binding of the detection antibody to an antigen. By way of example, the label may be fluorescein isothiocyanate (FITC), R-phycoerythrin, Alexa 532, CY3 or digoxigenin. The label may be a reporter molecule, which is detected directly, such as by detection of its fluorescent signal, or by exposure of the label to photographic or X-ray film. Alternatively, the label is not directly detectable, but may be detected, for example, in a two-phase system. An example of indirect label detection is binding of an antibody to the label.

[00244] In some embodiments, said secondary antibody comprises a fluorescent label and a binding molecule-antigen complex is detected by the fluorescence emitted from a binding molecule-antigen-secondary antibody complex. A "binding molecule-antigen-secondary antibody complex" means a complex comprising an antigen (e.g., CCR9) that has bound to a binding molecule, wherein said complex has further bound to a secondary antibody that binds to said binding molecule and/or binding molecule-antigen complex.

[00245] Suitably, a binding molecule-antigen complex is detected when the signal (e.g., fluorescence) emitted from the detection marker is greater than the signal detected in a control comprising no binding molecule (e.g., no binding molecule that binds to a CCR9). Said control may alternatively comprise a CCR9, but the sample is not applied to said control.

[00246] In another aspect, a second binding molecule is provided that binds to CCR9 wherein said second binding molecule does not compete for binding to CCR9 with a first binding molecule.

[00247] The first binding molecule may be any binding molecule as disclosed herein that binds to CCR9. A suitable first binding molecule may be a binding molecule as illustrated in Figure 4. A suitable first binding molecule may be a binding molecule comprising VH and VL from any binding molecule illustrated in Table 3 or comprising a set of six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) from any binding molecule illustrated in Table 4. A suitable first binding molecule may be a binding molecule that: a) comprises a heavy chain variable region (VH) having a set of CDRs HCDR1, HCDR2, and HCDR3 and a light chain variable region (VL) having a set of CDRs LCDR1, LCDR2, and LCDR3, from an antibody as shown in Figure 4; b) comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody shown in Figure 4; c) comprises a heavy chain variable region (VH) sequence of SEQ ID NO: 51 and a light chain variable region (VL) sequence of SEQ ID NO: 55, wherein (i) XI and X2 of SEQ ID NO: 55 are each any amino acid (SEQ ID NO: 76); or (ii) X1 of SEQ ID NO: 55 is P or S and X2 of SEQ ID NO: 55 is R, T or G (SEQ ID NO: 77); or d) comprises a heavy chain variable region (VH) sequence of SEQ ID NO: 51 and a light chain variable region (VL) sequence of SEQ ID NO: 52, 53 or 54; or e) comprises a heavy chain variable region (VH) sequence of SEQ ID NO: 56 and a light chain variable region (VL) sequence of SEQ ID NO: 57.

[00248] Advantageously, the invention encompasses the use of the second binding molecule (which does not compete for binding with the first binding molecule) to determine the abundance of cells expressing CCR9 in a sample that has been contacted with the first binding molecule.

[00249] Thus, a method is provided for assessing the depletion of CCR9-expressing cells by a first binding molecule, the method comprising: a) contacting said first binding molecule with a population of cells, wherein the population of cells comprises CCR9-expressing cells and immune effector cells, under conditions suitable to permit antibody-dependent cell-mediated cytotoxicity by the effector cells; b) contacting said population of cells with a second binding molecule that binds to CCR9 and that does not compete for binding to CCR9 with the first binding molecule of step (i); c) detecting CCR9-expressing cells in the population of cells that are bound by the second binding molecule of (ii); d) comparing the amount of CCR9-expressing cells detected in step (iii) with the amount of CCR9-expressing cells in the original population of cells used in step (i), and thereby determining the amount of CCR9-expressing cells that were depleted in step (i).

[00250] In some embodiments, the second binding molecule comprises the VH and VL sequences illustrated in Table 6 or the set of six CDRs illustrated in Table 6. For example, the first binding molecule can comprise the VH and VL of any binding molecule illustrated in Table 3 or comprise a set of six CDRs of any binding molecule illustrated in Table 4, and the second binding molecule can comprise the VH and VL sequences illustrated in Table 6 or the set of six CDRs illustrated in Table 6.

[00251] In some embodiments, the second binding molecule is a binding molecule as described herein that binds to CCR9 and is functional; an LCDR2 of SEQ ID NO: 5 or a functional variant thereof; and e) an LCDR3 of SEQ ID NO: 48, or a functional variant thereof, wherein a functional variant is as defined herein.

[00252] In some embodiments, the second binding molecule is a binding molecule that binds to CCR9 and that comprises a heavy chain variable region (VH) having a set of CDRs HCDR1, HCDR2, and HCDR3 and a light chain variable region (VL) having a set of CDRs LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 44, HCDR2 of SEQ ID NO: 45, and HCDR3 of SEQ ID NO: 46, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 47, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 48.

[00253] In some embodiments, the second binding molecule is a binding molecule that binds to CCR9 and that comprises: a) a VH region comprising the amino acid sequence of SEQ ID NO: 72, or a sequence that has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto; and b) a VL region comprising the amino acid sequence of SEQ ID NO: 73, or a sequence that has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity thereto. 5.5 Polynucleotides, Vectors, and Host Cells

[00254] In another aspect, a polynucleotide comprising a nucleic acid sequence encoding a binding molecule of the invention is provided.

[00255] In some embodiments, the polynucleotide is an isolated polynucleotide.

[00256] In some embodiments, where the binding molecule comprises more than one polypeptide chain (e.g., where the binding molecule is an immunoglobulin molecule or fragment thereof, comprising at least one heavy chain and at least one light chain), the polynucleotide encodes one, some, or all of the polypeptide chains of the binding molecule. For example, the polynucleotide can encode both the heavy chain polypeptide and the light chain polypeptide of a binding molecule. In some embodiments, the polynucleotide encodes a VH region of a binding molecule. In some embodiments, a polynucleotide of the invention encodes a VL region of a binding molecule. In some embodiments, the polynucleotide encodes both a VH region and a VL region of a binding molecule.

[00257] In some embodiments, the polynucleotide additionally encodes a leader sequence (e.g., that functions as a secretory sequence to control transport of a polypeptide from the cell).

[00258] Variants of a polynucleotide described above are encompassed by the invention. Polynucleotide variants may contain alterations in the coding regions, non-coding regions, or both. In some embodiments, a polynucleotide variant comprises an alteration that produces silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced by a silent substitution due to degeneracy of the genetic code. A polynucleotide variant may be produced for a variety of reasons, for example, to optimize expression codons for a particular host (changing codons in human mRNA to those preferred by a bacterial host such as E. coli).

[00259] In another aspect there is provided a vector comprising the polynucleotide of the invention operatively associated with a promoter; or comprising a polynucleotide encoding the VH region of a binding molecule of the invention and a polynucleotide encoding the VL region of a binding molecule of the invention wherein said polynucleotides are operatively associated with one or more promoter(s). As used herein, the term "promoter" means any nucleic acid sequence that regulates the expression of a polynucleotide sequence by directing transcription of the polynucleotide sequence. As used herein, the terms "operably associated" and "operably linked" mean that the promoter is in a correct functional location and/or orientation relative to a polynucleotide sequence that it regulates to control initiation of transcription and/or expression of that sequence. In some embodiments, the nucleic acid sequences encoding the VH region and the VL region are operatively associated with the same promoter. In some embodiments, the nucleic acid sequences encoding the VH region and the VL region are each operably associated with a separate promoter. In some embodiments, the separate promoters are promoters of the same type. In some embodiments, the separate promoters are promoters of different types.

[00260] Examples of vectors include viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, liposome-encapsulated DNA or RNA expression vectors, and certain eukaryotic cells, such as producer cells. Once assembled into the vector, the polynucleotide of the invention can be operably linked to an appropriate promoter for expression of the polypeptide in a desired host.

[00261] A vector may comprise additional nucleic acid sequence(s) that control expression of the polynucleotide. For example, a vector may comprise one or more enhancer and repressor sequences. As used herein, the term "enhancer" means a nucleic acid sequence that binds to one or more proteins to increase transcriptional activation of a polynucleotide. As used herein, the term "repressor" means a nucleic acid sequence that binds to one or more proteins to decrease transcriptional activation of a polynucleotide.

[00262] Another aspect provided herein is a host cell comprising a polynucleotide of the invention or a polynucleotide as disclosed herein, or a host cell comprising a vector of the invention or a vector as disclosed herein, wherein the host cell is capable of producing a polypeptide encoded by the polynucleotide or vector.

[00263] Another aspect provided herein is a method of producing a binding molecule of the invention comprising expressing a polynucleotide or vector of the invention in a host cell. For example, in some embodiments, the method comprises culturing a host cell under conditions suitable to produce a binding molecule of the invention encoded by a polynucleotide or vector present in the host cell.

[00264] Suitable host cells for expression of a binding molecule of the invention include prokaryotic, yeast, insect or higher eukaryotic cells (preferably in which the polynucleotide is under the control of appropriate promoters). Prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin as described herein. Cell-free translation systems may also be employed. In some embodiments, the host cell is a cell that does not fucosylate the Fc region of the binding molecule. In some embodiments, the host cell is a cell that expresses an enzyme that bypasses the fucose pathway to produce GDP-D-rhamnose. 5.6 Fucosylation

[00265] The effector function of the antibody can be modified by generating antibodies with altered glycosylation patterns. For example, an antibody can be made that has an altered type of glycosylation, such as an afucosylated/hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC capability of antibodies. Such carbohydrate modifications can be achieved by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. For example, EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. PCT Publication WO 03/035835 describes a variant of the CHO cell line, Lec13 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also [5]). PCT Publication WO 99/54342 describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures that result in increased ADCC activity of the antibodies (see also [6]). WO 2011/035884 A1 describes processes for producing molecules lacking fucose in their glyco moieties. WO 2011/035884 A1 particularly describes cells for producing molecules, wherein the cells comprise at least one enzyme that uses GDP-6-deoxy-D-yloxy-4-hexulose as a substrate, wherein the enzyme converts the substrate to GDP-D-rhamnose, GDP-D-perosamine, GDP-deoxy-D-tallose, GDP-6-deoxy-Daltrose or GDP-4-keto-3,6-dideoxy-D-mannose. 5.7 Additional Kits and Assays

[00266] In one aspect, a kit comprising a binding molecule of the invention is provided. Further encompassed is the use of said kit in the methods of the present invention.

[00267] In some embodiments, a kit further comprises an isolated (e.g., purified) antigen or a cell expressing an antigen. For example, the kit can further comprise an isolated (e.g., purified) CCR9 or a cell expressing CCR9. In some embodiments, the kit comprises one or more containers. In some embodiments, the kit comprises all components necessary and/or sufficient to perform a detection assay as described herein, including all controls, instructions for performing assays, and any software necessary for analysis and presentation of results.

[00268] A binding molecule of the invention may be used in assays for immunospecific binding by any method known in the art. Immunoassays that may be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blotting, RIA, ELISA, ELISPOT, sandwich immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays.

[00269] A binding molecule of the invention may be employed histologically, such as in immunofluorescence, immunoelectron microscopy, or non-immunological assays, for example, for in situ detection of CCR9 or its conserved variants or peptide fragments. In situ detection may be accomplished by removing a histological specimen from a patient and applying thereto a labeled binding molecule of the invention, for example, applied by overlaying the labeled binding molecule on a biological sample. By using such a procedure it is possible to determine not only the presence of CCR9, or conserved variants or peptide fragments, but also its distribution in the tissue examined. Using the present invention, those skilled in the art will readily appreciate that any of a wide variety of histological methods (such as staining procedures) may be modified so as to achieve such in situ detection. Table 3: Amino acid sequence of the VH and VL regions of exemplary binding molecules of the invention. Table 4: Amino acid sequence of the CDRs of exemplary binding molecules 5.8 EXAMPLE 1 - CCR9 expression in various cells and tissues

[00270] Cells were isolated from various human tissues and cynomolgus monkeys. The proportion of cells in a given tissue and/or a given cell type expressing CCR9 was determined by flow cytometry.

[00271] As illustrated in Figure 1A, cells expressing CCR9 and CD4 were found in the colon, ileum, and thymus of human and cynomolgus tissue, and also found in the mesenteric lymph node in cynomolgus tissue. In contrast, CCR9+ CD4+ cells were present only at a low level in the blood. CCR9 mRNA was also found to be co-expressed with CD3+ cells from cynomolgus ileum (data not shown).

[00272] CCR9 expression was evaluated in human tissue. The highest proportion of CCR9-expressing cells was found in thymic CD4+ T cells (76% of cells express CCR9), with high levels also observed in CD4+ T cells (47% of cells express CCR9) and CD8+ T cells (37% of cells express CCR9) from the ileum of IBD patients. Some CCR9 expression was also observed in colon cells from IBD patients that were either CD4+ T cells (9% of cells express CCR9) or CD8+ T cells (12% of cells express CCR9), with only low numbers of CCR9-expressing cells in blood CD4+ and CD8+ T cells (<5%) from IBD patients. CCR9 expression was observed in 28% of B cells in blood, with a lower level (11% and 16%, respectively) in B cells in the colon and ileum. Only very low numbers of monocytes, dendritic cells (DCs) and plasmacytoid dendritic cells (pDCs) expressed CCR9 (<5%).

[00273] Figure 1B shows the percentage of B cells expressing CCR9 in the ileum (left graph) and colon (right graph) of healthy and IBD patients. For each B cell type (naïve B cell, memory B cell, plasma cell), results from healthy subjects are provided on the left and results for IBD patients are provided on the right. It was found that there was a decrease in the number of B cells expressing CCR9 in IBD, with the largest decrease observed in memory B cells. It is hypothesized that this may be due to elevated expression of the CCR9 ligand leading to receptor internalization in IBD.

[00274] Figure 1C shows the percentage of effector T cells and Tregs from the colon or ileum of healthy subjects or patients with IBD that express CCR9. For each indicated tissue type, the % of effector T cells expressing CCR9 is shown on the left, and the % of Tregs expressing CCR9 is shown on the right. There was no apparent difference in the effector T/Treg ratio in any tissue type between healthy and IBD patients. 5.9 EXAMPLE 2 - Association of CCR9 with Proinflammatory Cytokine Expression

[00275] Coexpression of the pro-inflammatory cytokines IFN-Y, IL-4, and IL-17 was assessed in human CD4+CCR9+ and CD4+CCR9- cells using flow cytometry. Cells were isolated from human colon or ileum and were treated with PMA/ionomycin and Brefeldin A (a protein transport inhibitor) for 4 hours to stimulate cytokine production. CD4+ T cells were identified by gating based on single live cells that were CD45+, CD3+, and CD8-. CD4+ CCR9+ cells were found to express higher levels of pro-inflammatory cytokines than CD4+ CCR9- cells (Figure 2A). The difference in levels between CD4+CCR9- and CD4+CCR9+ cells was greatest for IL-17 expression (in the range of two- to six-fold increase), although the mean difference was still around two- to three-fold for IL-4 and IFN-Y (Figure 2B). 5.10 EXAMPLE 3 - Generation of anti-CCR9 antibodies

[00276] Anti-CCR9 antibodies were generated using hybridoma technology after immunization of CD1 mice with alternating HEK cell lines overexpressing human CCR9 and mouse CCR9. Three groups of mice were used. For Group 1, mice were immunized with alternating CHO and HEK cells overexpressing hCCR9; mice in Group 2 were immunized with alternating HEK cells overexpressing hCCR9 and HEK cells overexpressing mCCR9; mice in Group 3 were immunized with HEK cells overexpressing mCCR9. The recombinant cell lines were administered at 1e7 cells/100 μL diluted in PBS, emulsified with equal volumes of complete Freund's adjuvant, and injected into the mice at two sites, 100 μL per site. For the three subsequent injections, cells were emulsified in incomplete Freund's adjuvant and injections were performed as above. The final boost was performed on day 24 by injecting 200 μL of cells in PBS intraperitoneally.

[00277] Tail vein bleeds were obtained from mice prior to immunization, on day 13 after the first immunization, and on day 20 after the second immunization. IgG titers to human CCR9, mouse CCR9, and parental HEK and CHO cells were determined by serum ELISA. Animals with the highest antigen-specific titers were forwarded for hybridoma generation. 5.10.1 Assessment of the immune response of mice to hCCR9 and mCCR9

[00278] Individual mouse serum IgG titers to hCCR9 and mCCR9 were determined by ELISA. The HEK and CHO hCCR9 and mCCR9 cell lines described above, along with the parental HEK and CHO cells, were plated onto 96-well Poly-D-Lysine microtiter plates at 40,000 cells/well and cultured overnight at 37 °C in a CO2 incubator. After overnight incubation, the supernatant was removed and the cells fixed for 5 min in 3.7% formaldehyde/PBS solution at RT. All subsequent incubations were carried out at room temperature. After removal of the formaldehyde solution, the wells were then blocked by the addition of 3% marvel blocking buffer/PBS. After 1 h, the blocking buffer was removed, the wells were washed again with PBS, and serum samples were added in a dilution series (50 μL per well from a 1:200 dilution). After incubation for 1 h, the wells were washed three times with PBS supplemented with 0.05% (v/v) Tween 20. A 1:500 europium-labeled anti-mouse IgG in Delfia assay buffer (Perkin Elmer) was then added to the wells at 50 μL per well. After a further 1 h incubation and five washes as above, Enhancement solution (Perkin Elmer) was added to the wells at 50 μL per well, the plates were incubated at room temperature for 10 min on an orbital shaker. The plates were then read using a PerkinElmer EnVision 2103 multilabel plate reader.

[00279] Serum titration curves for hCCR9, mCCR9 as well as parental HEK and CHO cells were plotted and the respective area under the curves (AUC) calculated. 5.10.2 Isolation of monoclonal mouse IgG

[00280] Four days after the final boost, lymph nodes were aseptically harvested and cells were isolated by mechanical disruption and counted. These cells were mixed with SP2/0 myeloma cells and fused using an electrofusion device. The resulting fusions were mixed with a methylcellulose-based semi-solid medium and plated on OmniTray plates. The semi-solid medium comprised CloneMatrix and DMEM supplemented with 20% FCS, 10% BM Condimed H1, 1 mM sodium pyruvate and OPI medium supplement, 2% hypoxanthine azerine and FITC-conjugated goat anti-mouse IgG. Cells in semi-solid medium were cultured for 13 days at 37°C in a 5% CO2 incubator. During this incubation period, clonal colonies are formed from a single parent hybridoma cell. These colonies secrete IgG that is trapped in the vicinity of the colony by FITC-conjugated anti-IgG present in the semisolid medium. The resulting immune complex formation can be seen around the cell as a fluorescent "halo" when visualized by a ClonePix FL colony picker (Molecular Devices). These haloed colonies are then picked into 96-well microtiter plates.

[00281] After 3-5 days of culture, supernatants from picked colonies were harvested and tested for CCR9 binding. 5.10.3 DNA sequencing and purification of mouse IgGs

[00282] Messenger RNA (mRNA) was extracted from hybridoma cells using magnetic oligo (dT) particles and reverse transcribed into cDNA. PCR amplification was performed using poly-C and constant region VH or VL primers specific for all mouse IgG subclasses. 5.10.4 Mouse IgG Purifications

[00283] Cells were seeded in 24-well plates and overcultured in serum-free HL-1 medium supplemented with HyperZero and glutamine. After 10 days, supernatants were transferred to 96-well master blocks and mouse IgGs of all subclasses (IgG1, IgG2a, IgG2b, and IgG3) were purified from culture supernatants of overcultured cells on ProPlus resin (Phynexus) using Perkin Elmer Minitrack. Captured mouse IgGs were eluted with 75 μL of 100 mM HEPES, 140 mM NaCl pH 3.0, then neutralized with an equal volume of 200 mM HEPES pH 8.0. Purified IgGs were quantified using absorbance reading at 280 nm in a UV-Star 384-well plate. 5.10.5 Reformatting of mouse IgGs

[00284] Mouse hybridoma IgG clones were molecularly reformatted to generate constructs expressing the mouse VH and VL domains and the relevant mouse IgG constant domains for each hybridoma essentially as described by Persic et al 1997 [7] with the following modifications. An OriP fragment was included in the expression vectors to facilitate use with transient CHO cells and to allow episomal replication. The VH domain was cloned into the relevant vector containing the mouse heavy chain constant domains and regulatory elements to express the entire IgG1 heavy chain in mammalian cells. Similarly, the VL domain was cloned into a vector for the expression of the appropriate mouse light chain constant domains (lambda or kappa) and regulatory elements to express the entire IgG light chain in mammalian cells. To obtain IgGs, mammalian suspension CHO cells were transiently transfected with the heavy and light chain IgG vectors. IgGs were expressed and secreted into the medium. Collections were filtered prior to purification, then IgG was purified using Protein A chromatography. Culture supernatants were loaded onto an appropriately sized Ceramic Protein A column (Pall 20078-036) and washed with 50 mM Tris-HCl pH 8.0, 250 mM NaCl. Bound IgG was eluted from the column using 0.1 M Sodium Citrate (pH 3.0) and neutralized by the addition of Tris-HCl (pH 9.0). The eluted material was buffer exchanged to PBS using Nap10 columns (GE Lifesciences 17-0854-02) and the IgG concentration was determined spectrophotometrically using an extinction coefficient based on the amino acid sequence of the IgG [8]. Purified IgG was analyzed for purity using SDS-PAGE. Purified IgGs were analyzed for binding to human CCR9 on HEK cells.

[00285] Figure 3 shows the amino acid sequences of the variable heavy chain (Figure 3A) and variable light chain (Figure 3B) sequences for nine unique hits identified in this analysis. The cDNA for AB1020243, for example, was prepared from clone ZY10OI-A02 and the sequence for the VH and VL domains determined. FW = framework region. The locations of the CDRs are underlined as indicated by the Kabat assignment (Kabat et al., "Sequences of Immunological Interest", 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The VH and VL sequences of humanized AB1020243 (AB1020243-fgl) are illustrated in Figure 4, where they are aligned with the original chimeric sequences.

[00286] To further improve the potency and efficacy of AB1020243, site-directed scanning mutagenesis of H&L CDRs was performed. CDRs were mutated by site-directed mutagenesis of mammalian expression vectors with low-redundancy nucleotide codon (NNC) mixtures designed to maximize the abundance of alternative amino acids (aa) while eliminating undesirable amino acids (Met, Trp) from the library. A total of 566 CDR variants were produced and CDR-optimized versions of humanized AB1020243 were generated. The screening library was expressed in CHO cells in 24-well plates prior to protein A purification of the variants. After identification of improved variants, intra- and inter-CDR recombination was performed by site-directed mutagenesis to combine beneficial variants into the final lead antibody candidates such as a-fuc huIgG1. The VH and VL sequences of the CDR-optimized antibodies AB243LO0331 and AB243LO0326 are illustrated in Figure 4, where they are aligned with the parent sequences of AB1020243 and with the humanized form of AB1020243. LCDR1 residues that were modified are shown in bold underlined text. 5.11 EXAMPLE 4 - Assessment of CCR9 binding and potency 5.11.1 Cellular binding

[00287] Antibody binding to CCR9-expressing cells was assessed using HEK cells overexpressing human CCR9A or using Molt4 cells. The Molt4 cell line is derived from human T cells and is known to express CCR9 [9]. Test antibodies were diluted to a concentration of 10 μg/mL in PBS supplemented with 1% fetal bovine serum. Antibodies were titrated as a single point using 8-point three-fold titrations in a 96-well plate and cells were stained at 4 °C for 20 min. Cells were washed and stained with a secondary goat anti-mouse or anti-human IgG PE at 2.5 μg/mL in sterile PBS + 1% FBS at 4 °C for 20 min.

[00288] Figure 5A shows that at least AB1020243, AB1020229, and AB1020011 bound to Molt4 cells. Binding to Molt4 cells was compared to binding to Jurkat cells, which do not express human CCR9. As illustrated in Figure 5B, binding to Molt4 cells was similar to that observed in Figure 5A, while no significant binding to Jurkat cells was detected.

[00289] The AB1020243 antibody (hybridoma mIgG) was also tested for binding to different forms of CCR9. Briefly, the test antibody was diluted to a concentration of 60 μg/mL in assay buffer containing Hanks' balanced salt solution (Sigma HB264) plus 0.1% bovine serum albumin (Sigma A9576). The antibody was titrated in duplicate using single relative titrations at 24 spots in a clear, non-clumping, black bottom 384 microplate (Corning® NBS™ 3655) such that the final volume of test antibody was 10 uL per well. Alexa Fluor 647-labeled human IgG (H+L) secondary detection antibody (Jackson Immuno-Research; 209-665-082) was prepared according to the manufacturer's instructions and diluted 1:1000 in assay buffer as described above. Secondary detection antibody was dispensed into the assay plate containing test antibodies using a Multidrop dispenser (Thermo Scientific) in a volume of 10 μL per well.

[00290] Transfected CCR9 overexpressing HEK cell lines or untransfected parental control cells were suspended in assay buffer at a concentration of 1x106 cells per mL and 10 μL of cells were dispensed into the assay plate containing test antibody and secondary antibody using a Multidrop dispenser (Thermo Scientific). The assay plates were incubated overnight at 4 °C and were read on a mirrorball® (laser scanning fluorescence cytometer; TTP-Labtech) using excitation at 488 nm and emission using the FL4 channel (667-685 nm). Data were graphed in GraphPad Prism version 8.0.0 for Windows (GraphPad Software, San Diego, CA, USA) and expressed as Log (mg/mL antibody) v's Fluorescence (FL4) counts. As illustrated in Figure 6, AB1020243 showed good binding to both human forms of CCR9 (CCR9A and CCR9B) and to cynomolgus CCR9. It showed little or no binding to mouse or rat CCR9. 5.11.2 In vitro ADCC bioassay

[00291] The potency and effector function of the antibodies were assessed using an NK cell activation assay as follows: All potency assays were performed at an Effector (NK-92 MI CD16a NFAT cells) to Target (Molt4; HEK cells overexpressing CCR9 or Jurkat cells) ratio of 5:1. The Effector NK-92 cell line does not naturally express the FcYRIIIa (CD16) receptor. A modified form of NK-92 that overexpresses the high-affinity (ha) CD16 V158 FcYRIIIa receptor, which has increased binding to Fc domains, was used (Boissel et al., Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 April 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract 2302). This receptor allows NK cells to cross-link to target cells via the antibody that is bound to the target cells. The NK-92 cells used also express an NFAT luciferase reporter, which can be used as a surrogate for effector function. Binding of the target-bound antibody to the NK cell induces activation of the NFAT transcription factor, which drives a luciferase reporter gene. The luminescence signal is proportional to ADCC activity.

[00292] Both target and effector cells were prepared in assay medium containing Advanced RPMI (Gibco™; 12633) with 10% Ultra Low IgG FBS (Gibco™; 16250-078). Target cells were resuspended at a concentration of 8x105 cells per mL and effector cells were resuspended at a concentration of 8x106 cells per mL. Test antibodies were diluted to a concentration of 60 μg/mL and 4-fold duplicate titrations were performed at 12 points in a 384-well polypropylene microplate (Greiner Bio-one International; 781280).

[00293] Target cells were distributed into 384-well clear bottom white tissue culture treated plates (Corning® 3560) using a Multidrop dispenser (Thermo Scientific) in a volume of 12.5 μL per well. Test antibodies (6.25 μL per well) were then added to the plates containing target cells using a Bravo Automated Liquid Handling Platform (Agilent). Target cells and antibodies were pre-incubated for 30 minutes at 37°C in an O2/CO2 incubator. Effector cells were then added using a Multidrop dispenser (Thermo Scientific) in a volume of 6.25 μL per well. Assay plates containing target cells, effector cells, and antibodies were incubated for an additional 5 hours at 37°C in an O2/CO2 incubator.

[00294] At the end of the incubation period, luciferase detection reagent was prepared according to the manufacturer's instructions (Steady-Glo® Luciferase Assay System; Promega E2520) and 25 μL was dispensed into each well using manual pipetting. Assay plates were covered with aluminum foil and lysed for 20 min at room temperature on the plate shaker. Assay plates were read on an EnVision® 2105 multimode plate reader (Perkin Elmer) using the ultrasensitive luminescence mode. Data were analyzed in GraphPad Prism version 8.0.0 for Windows (GraphPad Software, San Diego, CA, USA) using a four-parameter logistic log (agonist) versus response equation. Data were expressed as Log antibody concentration (Molar) versus response (Counts per second).

[00295] Figure 7 shows the activation of NK cells by (fucosylated) antibodies when bound to MOLT4/HEK cells. The non-fucosylated forms of the antibodies were produced from a dual expression cassette, in which the expression of IgG heavy chain is co-expressed with the RMD enzyme to produce afucosylated protein in CHO cells. The potency of the AB1020243 afuc antibody when bound to HEK cells expressing human CCR9A was measured as 0.5 nM. The potency of the humanized AB1020243 afuc antibody when bound to Molt4 cells was measured as 0.8 nM. 5.11.3 PBMC Killing Assay

[00296] Antibodies were serially titrated (10-fold) with a starting concentration of 10 μg/mL at 8 points. Antibodies were added to 1x106 human peripheral blood mononuclear cells (PBMCs) in a 96-well U-bottom plate and incubated overnight. The next day, the percentage of remaining CCR9-expressing CD4 T cells was measured by flow cytometry using a non-competitive CCR9 antibody. The VH/VL and CDR sequences of the non-competitive antibody are presented in Table 5 and Table 6. As illustrated in Figure 8, all antibodies AB1020243, AB1020229, and AB1020011 showed killing of CCR9+ PBMCs. The EC50 (n=4) for AB1020243 afuc was measured as 2.6 nM. Table 5: Amino acid sequences of the VH and VL regions of the noncompetitive CCR9 antibody. Table 6: Amino acid sequence of the CDRs of the non-competitive CCR9 antibody. Table 7: Heavy and light chain constant region amino acid sequences 5.12 EXAMPLE 5 - Properties of humanized AB1020243

[00297] Humanized AB1020243 was produced as in Example 3. In all experiments described below, humanized AB1020243 was in an afucosylated form produced from a dual expression cassette, in which expression of IgG heavy chain is co-expressed with GDP-6-deoxy-D-yl-4-hexulose reductase (RMD) to produce afucosylated protein in CHO cells. 5.12.1 CCR9 Cross-Reactivity of Humanized AB1020243

[00298] The binding of the parental (chimeric) AB1020243 antibody was compared to that of the humanized version based on the methods described in Example 4.

[00299] As illustrated in Figure 9, the humanized (“human”) antibody retained similar binding to the parental (“chimeric”) AB1020243 against CCR9 and cynomolgus HEK cells expressing human CCR9B (Figure 9A and Figure 9B). No significant binding was observed against the parental HEK JI cell line, which does not express CCR9 (Figure 9G). Even at higher concentrations, humanized AB1020243 showed no significant off-target binding to CXCR1 (Figure 9C), CXCR2 (Figure 9D), CCR5 (Figure 9E), or CCR8 (Figure 9F). 5.12.2 Humanized AB1020243 Retains High Potency

[00300] Humanization of AB1020243 surprisingly did not lead to any significant change in potency in the NK cell activation assay (Figure 10). Both chimeric and humanized forms of AB1020243 afuc produced the same degree of activation of the NK-92 cell line described above using MOLT4 cells. This is in contrast to a reference afucosylated mouse IgG1 anti-CCR9 antibody (3C3, ATCC HB-12653) for which a large reduction in potency was observed following humanization. Similarly, humanization did not alter the potency of AB1020243 afuc bound to HEK cells overexpressing cynomolgus CCR9A.

[00301] The kinetic properties of the AB1020243 afuc antibody were also found to be not significantly altered by humanization. CCR9+ live cell measurements were made using fluorescently labeled antibodies using Ligand Tracer technology. Briefly, cell affinities were measured on a LigandTracer instrument (Ridgeview Instruments) using a fluorescence detector at 633 nm at room temperature (~22°C). Human IgG1 format IgGs were specifically labeled with the fluorophore DyLight 650 Maleimide at an engineered cysteine residue in the CH2 region of the IgG. The labeled IgGs were added to the Nunclon D circular plate with pre-adhered discrete spots of parental CHO K1 (control) and CCR9-expressing CHO K1 cells. The IgG binding and dissociation fluorescence profiles were measured on the instrument and then analyzed with a bivalent (1:2) fitting model in TraceDrawer software (Ridgeview Instruments). The dissociation rate (kd) of AB1020243 afuc was measured as 3.85 x 105 s-1 and remained similar after humanization, being measured as 4.3 x 10-5 s1. This is consistent with the fact that there was no reduction in potency observed after humanization (Figure 10B). 5.12.3 Ability to compete with CCL25

[00302] Humanized AB1020243 was tested for its ability to compete with CCL25 in an HTRF IP-One Gq assay (Cis-Bio; 62IPAPEB). The IP-One assay is a competitive immunoassay using terbium cryptate-labeled anti-IP1 Mab and d2-labeled IP1. IP1 is a downstream metabolite of the IP cascade and accumulates in cells upon Gq receptor activation and is stable in the presence of LiCl. LiCl is added to the cell stimulation buffer, causing IP1 to accumulate upon receptor activation.

[00303] Test antibodies were exchanged into IP-one buffer (Krebs Ringer solution + LiCl) and were serially diluted neat in IP-one buffer using a one plus one dilution series in 16 points (in triplicate) in a 384-well polypropylene microplate (Greiner Bio-one International; 781280) and 3.5 mL of each antibody was added to white 384 shallow well plates (Costar; 4513) using a Bravo Automated Liquid Handling Platform (Agilent). MOLT 4 cells were harvested and resuspended in IP-One assay buffer to 5.4x106 cells/mL and 7.5 μL of cells were dispensed to the antibody-containing plates using a Multidrop dispenser (Thermo Scientific) so that the final cell concentration was 40,000 cells/well. Antibodies were preincubated with cells for 30 minutes. Recombinant Human CCL25/TECK Protein (R&D systems; 9046-TK) was reconstituted to 200 nM according to the manufacturer's instructions and 3.5 μL was dispensed onto the plates containing antibody and cells using a Multidrop dispenser (Thermo Scientific) such that the final assay concentration was 50 nM. Assay plates were incubated at 37 °C for 90 minutes before being processed according to the IP-One HTRF protocol using the reagents provided with the kit. Figure 11 shows that humanized AB1020243 and the CDR-optimized antibodies 234LO0331 and 243LO0326 retain similar abilities to compete with CCL25 as the parental chimeric AB1020243 antibody. The antibodies of the present invention therefore contrast with the previously described mouse CCR9 antibodies 92R and 91R which, for example, do not inhibit CCL25-induced migration of CCR9+ MOLT-4 cells and only partially compete with CCL25 for binding to MOLT-4 cells [4]. 5.13 EXAMPLE 6 - Properties of CDR-optimized antibodies

[00304] As set forth in Example 3, CDR-optimized forms of humanized AB1020243 were produced. In all experiments described herein, the CDR-optimized antibodies were in an afucosylated form. The CDR-optimized antibodies were formatted to reduce the risk of Asn deamidation in LCDR1. 5.13.1 CCR9 Binding

[00305] CDR-optimized AB1020243 was tested for its ability to bind to different forms of CCR9 and to different cell types, based on the methods described in Example 4. As illustrated in Figure 12, 243LO0326 and the parent antibody AB1020243 showed similar binding to both human isotypes CCR9A and CCR9B and to cynomolgus CCR9A. Both 243LO0326 and 243LO0331 showed no significant binding to mouse or rat CCR9, even at high concentrations (data not shown). 243LO0326 and the parent antibody AB1020243 also failed to show any off-target receptor binding to CCR5, CCR8, CXCR1, or CXCR2. 5.13.2 Affinity

[00306] As shown in Figure 13A, 243LO0326 remained membrane-bound for an extended period of time, with most of the antibody remaining on the cell surface after 5 hours at 37°C. In comparison, the amount of the parental antibody AB1020243 bound to the cell surface dropped significantly within the first hour. Affinity (KD) was measured using Ligand Tracer as 0.09 nM for 243LO0326 and 3.4 nM for AB1020243. Briefly, affinities in cells were measured on a LigandTracer instrument (Ridgeview Instruments) using a fluorescence detector at 633 nm, at room temperature (~22°C). IgGs in the human IgG1 format were specifically labeled with the fluorophore DyLight 650 Maleimide at an engineered cysteine residue in the CH2 region of the IgG. Labeled IgG were added to the Nunclon D circular plate, with pre-adhered discrete spots of parental CHO K1 (control) and CCR9-expressing CHO K1 cells. IgG binding and dissociation fluorescence profiles were measured on the instrument and then analyzed with a bivalent (1:2) fitting model in TraceDrawer software (Ridgeview Instruments).

[00307] As shown in Figure 13B, only a slight reduction in the level of CCR9 receptor was observed on the membrane with antibody treatment. For comparison, CCL25 treatment induced a large decrease in CCR9 receptor on the membrane. It was further demonstrated that anti-CCR9 antibodies were able to prevent CCL25-induced internalization (Figure 19). 5.13.3 Potency

[00308] CDR-optimized AB1020243 was tested for its ability to induce NK cell activation and PBMC killing as described in Example 4. Figure 14A shows the effects of the antibodies in the NK cell activation assay. 243LO0326 was found to be more potent than the parent antibody AB1020243. Figure 14B shows the killing of PBMC by 243LO0326 and the parent antibody AB1020243. 243LO0326 showed improved potency, with an EC50 of 4 pM, compared to an EC50 of 200 pM for AB1020243. Figure 14C shows that 243LO0346 has a similar ability to induce death of CD4+CCR9+ human and cynomolgus PBMC, with an EC50 measured at 4–5 pM using human PBMC and an EC50 of 30 pM using cynomolgus PBMC. 5.13.4 Afucosylation

[00309] Fucosylation of anti-CCR9 antibodies enhances cell killing, as shown in the NK cell ADCC activation assay (Figure 15A). The impact of fucosylation level on antibody (243LO0326) potency was assessed in a fucosylation addition study using the in vitro ADCC bioassay (see section 5.11.2). ADCC potency of the antibody was maintained compared to 100% non-fucosylated molecules for up to approximately 10% fucosylation. Acceptable potency was maintained for fucosylation up to 50% (Figure 15B). Table 8: ADCC activity of fucosylated vs. afucosylated species – addition study 1 Approximate fucosylation levels, Tox fuc levels = 0.8% 2 %RP (relative potency) is a combined value from two independent assay runs 3 NR = not reportable - sample does not meet adequacy criteria due to severe loss of potency 5.14 EXAMPLE 7 - In vitro and in vivo effects 5.14.1 Antibody depletion of CCR9+ cells in cynomolgus monkeys

[00310] Monkeys were injected intravenously with a single dose of isotype control antibody and 10 mg/kg 243LO0326. The percentage of CCR9+ memory CD4+ T cells was assessed in the blood at multiple time points. Figure 16A shows that there was a decrease in CCR9+ cells present in the blood by day 15 with 10 mg/kg 243LO0326 completely abolishing CCR9+ cells at this time point. Particularly at both high and low doses (10 mg/kg), 243LO0326 depleted CCR9+ T cells in peripheral blood (Figure 16B), ileum (Figure 16C), and mucosa (Figure 16D). The depletion of CCR9+ T cells was tissue and cell specific (Figure 17). 243LO0326 selectively depleted CCR9+ intestinal lymphocytes with relative preservation of extraintestinal populations, without impact on thymic T cells. 5.14.2 Intestinal explant model

[00311] 6 mm punch biopsies of the intestinal mucosal layer were taken from healthy human subjects and human subjects with inflammatory bowel disease as described in Vadstrup et al. [10]. Explant tissue was cultured in the presence or absence of 243LO0326 for 12-84 hours. The percentage of CCR9+ cells was assessed along with the key mediators of inflammation: IL-6, GM-CSF and IL-22. Figure 18 shows that antibody treatment depleted CCR9+ CD4+ cells (Figure 18A) and reduced inflammatory mediators (Figure 18B to Figure 18D) in each of the explants tested. Explant tissue from one of the healthy subjects also showed high levels of IL-6, GM-CSF and IL-22 that were decreased by anti-CCR9 treatment. 5.15 EXAMPLE 7 - Anti-CCR9 antibodies block CCL25-induced CCR9 internalization

[00312] IHC data surprisingly suggest that there is quantitatively lower expression of CCR9 in inflamed Crohn's ileum. Ex vivo, CCR9 internalizes upon activation by CCL25, which is expressed at higher levels in inflamed Crohn's ileum. This suggests that in the inflamed intestine, CCR9 on pathogenic T cells is internalized upon activation by CCL25. CCR9-expressing cells in Crohn's ileum, however, show pro-inflammatory and resident memory phenotypes.

[00313] In an assay to detect CCR9 internalization, the CDR-optimized afucosylated antibody 243LO0326 was able to prevent CCL25-induced internalization at a concentration of 2 nM (Figure 19). CCL25-induced internalization was assessed by FACS. Advantageously, blocking CCL25-induced internalization prevents receptor recycling. The antibodies of the invention thus advantageously induce ADCC, block CCL25 binding to the CCR9 receptor, and prevent CCL25-induced CCR9 receptor recycling in CCR9-expressing T cells. 5.16 Conclusion

[00314] Taken together, these data show that anti-CCR9 treatment is a viable treatment option for patients with IBD. CCR9+ T cells expressing pro-inflammatory cytokines are expressed in the intestine and blood of patients with inflammatory bowel disease (Figure 1 and Figure 2). The anti-CCR9 antibodies described above bind human CCR9 on expressing T cells (Figure 5 and Figure 6). Afucosylated versions of these antibodies activate NK cells to promote ADCC (Figure 7 and Figure 15), leading to targeted cell killing (Figure 8). Humanization of one of the antibody clones surprisingly did not reduce the affinity of the antibody for human CCR9 compared to the chimeric parental antibody (Figure 9), maintained the ability to activate NK cells (Figure 10A), and retained the ability to compete with CCL25 (Figure 11).

[00315] Further CDR optimization of one of the humanized antibodies was performed. These CDR-optimized afucosylated antibodies (243LO0326 and 243LO0331) bound to the CCR9 membrane for an extended period of time compared to the parental antibody and had an affinity (KD) of 0.09 to 3.4 nM (Figure 13). The CDR-optimized antibodies were further able to induce NK cell activation and PBMC killing. 243LO0326 was more potent than AB1020243 (Figure 14). 243LO0326 is further able to inhibit CCL25-induced CCR9 internalization (Figure 19). Treatment with 243LO0326 also decreases CCR9 T cells in the blood (Figure 16). 243LO0326 treatment of intestinal explant tissue from IBD patients depletes CCR9+ CD4+ cells (Figure 16A) and reduces inflammatory mediators (Figure 16B to Figure 16D). 243LO0326 is therefore highly potent and specific for CCR9. Cell depletion occurs through ADCC; however, in the absence of ADCC effector cells such as NK cells, 243LO0326 also blocks the binding between CCR9 and its ligand CCL25.

[00316] Given that the CCR9 antibodies disclosed herein are capable of binding to both hCCR9 isoforms, CCR9A and CCRB, and compete with CCL25 for binding to CCR9, the epitope is expected to be located within the extracellular N-terminus of CCR9B, i.e., amino acid positions 1-36 of CCR9B: MADDYGSESTSSMEDYVNFNFTDFYCEKNNVRQFAS (SEQ ID NO: 80), corresponding to amino acid positions 13-48 of CCR9A (Table 9, Table 10, Figure 20). Table 9: Antibody epitope sequence Table 10: Sequence of hCCR9 isoforms

[00317] The CCR9-targeted antibodies disclosed herein are capable of blocking the binding of CCL25 to CCR9 and depleting CCR9+ T cells. In autoimmune inflammatory bowel disease, the antibodies disclosed herein are expected to deplete pathogenic CCR9+ T cells in blood and intestinal tissue and block the binding of CCL25 to CCR9 where pathogenic T cells are not fully depleted. This dual mechanism of action is expected to provide a sustained treatment effect. Specific depletion of topical intestinal CCR9+ effector memory T cells will induce and maintain durable remission and disease modification. REFERENCES [1] M. F. Neurath, Nat Rev Immunol 14, 329 (2014). [2] M. Gajendran et al., Dis Mon 64, 20 (2018). [3] J. Torres et al., J Crohns Colitis 14, 4 (2020). [4] B. Somovilla-Crespo et al., Frontiers in Immunology 9, (2018). [5] R. L. Shields et al., J Biol Chem 277, 26733 (2002). [6] P. Umana et al., Nat Biotechnol 17, 176 (1999). [7] L. Persic et al., Gene 187, 9 (1997). [8] C. N. Pace et al., Protein Sci 4, 2411 (1995). [9] J. Minowada, T. Ohnuma, and G. E. Moore, JNCI: Journal of the National Cancer Institute 49, 891 (1972). [10] K. Vadstrup et al., PLOS ONE 11, e0155335 (2016).

Claims (33)

1. A binding molecule that binds to CCR9 and comprises a heavy chain variable region (VH) having a set of CDRs HCDR1, HCDR2, and HCDR3 and a light chain variable region (VL) having a set of CDRs LCDR1, LCDR2, and LCDR3, characterized in that (a) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; (b) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, and HCDR3 of SEQ ID NO: 9, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO: 12; or (c) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 13, HCDR2 of SEQ ID NO: 14, and HCDR3 of SEQ ID NO: 15, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 16, LCDR2 of SEQ ID NO: 17, and LCDR3 of SEQ ID NO: 18; and wherein any one or more of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprises 1, 2 or 3 amino acid substitutions compared to said sequences. 2. The binding molecule of claim 1, wherein the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and wherein the amino acid sequence of the VL region comprises LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6, and LCDR1 having the amino acid sequence selected from: (a) SEQ ID NO: 4; (b) SEQ ID NO: 19; (c) SEQ ID NO: 20; (d) SEQ ID NO: 21; and (e) SEQ ID NO: 22. 3. The binding molecule of claim 1(a) or 2, wherein: (i) the amino acid sequence of the VH region comprises SEQ ID NO: 51, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto, and; the amino acid sequence of the VL region comprises SEQ ID NO: 52 or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; (ii) the amino acid sequence of the VH region comprises SEQ ID NO: 51, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto, and; the amino acid sequence of the VL region comprises SEQ ID NO: 53 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; (iii) the amino acid sequence of the VH region comprises SEQ ID NO: 51, or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and; the amino acid sequence of the VL region comprises SEQ ID NO: 54 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; (iv) the amino acid sequence of the VH region comprises SEQ ID NO: 51, or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and the amino acid sequence of the VL region comprises SEQ ID NO: 55 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; or (v) the amino acid sequence of the VL region comprises SEQ ID NO: 56, or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto and; the amino acid sequence of the VL region comprises SEQ ID NO: 57 or a sequence that has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto. 4. The binding molecule of claim 1, wherein: (i) the binding molecule comprises a VH region and a VL region as defined in claim 1(b), wherein the amino acid sequence of the VH region comprises SEQ ID NO: 58, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and wherein the amino acid sequence of the VL region comprises SEQ ID NO: 59, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; or (ii) the binding molecule comprises a VH region and a VL region as defined in claim 1(c), wherein the amino acid sequence of the VH region comprises SEQ ID NO: SEQ ID NO: 60, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto; and the amino acid sequence of the VL region comprises SEQ ID NO: 61, or a sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto. 5. Binding molecule according to any one of claims 1 to 3, characterized in that the binding molecule inhibits the binding of CCL25 to CCR9. 6. The binding molecule of any one of claims 1 to 5, wherein the binding molecule is an anti-CCR9 antibody or an antigen-binding fragment thereof; optionally wherein the anti-CCR9 antibody or antigen-binding fragment thereof is humanized, chimeric or fully human. 7. The binding molecule of claim 6, which is: (a) an IgG immunoglobulin or a fragment thereof; or (b) an IgG1 immunoglobulin or a fragment thereof. 8. The binding molecule of any one of claims 1 to 7, wherein said binding molecule comprises an immunoglobulin Fc domain or fragment thereof that retains the ability to bind to one or more Fc receptors; optionally wherein the immunoglobulin Fc domain or fragment: (a) is an IgG Fc domain or fragment thereof; (b) is a human IgG Fc domain or fragment thereof; (c) is a human IgG1 Fc domain or fragment thereof; (d) is modified compared to the corresponding wild-type Fc domain, wherein said modification leads to an increased affinity for one or more FCY receptors; (e) is modified compared to the corresponding wild-type Fc domain, wherein said modification leads to an increased antibody-dependent cell-mediated cytotoxicity response; and/or (f) comprises an afucosylated N-linked glycan at amino acid position 297. 9. The binding molecule of any one of claims 1 to 8, wherein the binding molecule is: (a) afucosylated; (b) afucosylated at amino acid position 297; or (c) present in a composition comprising multiple copies of said binding molecule, wherein at least 50%, 75%, 80%, 90%, 95%, 98%, 99%, or 100% of the copies of the binding molecule in the composition are afucosylated; and/or wherein the binding molecule: (a) binds to human CCR9; (b) binds to human CCR9A and human CCR9B; (c) binds to cynomolgus CCR9; and/or (e) does not bind to CCR5, CCR8, CXCR1, or CXCR2; and/or wherein the binding molecule: (a) is capable of mediating antibody-dependent cell-mediated cytotoxicity against a CCR9-expressing cell to which it binds; (b) is capable of mediating antibody-dependent cell-mediated cytotoxicity against a CCR9-expressing lymphocyte to which it binds; (c) can be bound by an FCYR; (d) can be bound by FcYRIIIa; (e) is capable of cross-linking an immune effector cell to a CCR9-expressing cell; (f) is capable of cross-linking an immune effector cell to a CCR9-expressing cell and activating antibody-dependent cell-mediated cytotoxicity by the effector cell; (g) is capable of inhibiting CCL25-induced CCR9 receptor internalization; (h) is capable of inhibiting CCL25-mediated migration of CCR9-expressing T cells to the intestine, (i) is capable of cross-linking an immune effector cell to a CCR9-expressing cell and activating antibody-dependent cell-mediated cytotoxicity by the effector cell, thereby causing lysis of the CCR9-expressing cell; and/or (j) is capable of depleting CCR9-expressing cells in a cell population comprising CCR9-expressing cells and immune effector cells. 10. Binding molecule according to any one of the preceding claims, characterized in that the binding molecule is capable of binding to human CCR9 with an affinity (KD) of about 0.1 nM, optionally 0.09 nM. 11. A binding molecule that binds to CCR9, wherein the binding molecule competes for binding to CCR9 with the binding molecule as defined in any one of claims 1 to 10. 12. The binding molecule of claim 11, wherein the binding molecule is: (a) afucosylated; (b) afucosylated at amino acid position 297; or (c) present in a composition comprising multiple copies of said binding molecule, wherein at least 50%, 75%, 80%, 90%, 95%, 98%, 99%, or 100% of the copies of the binding molecule in the composition are afucosylated, or wherein the immunoglobulin Fc domain or fragment: (d) is an IgG Fc domain or fragment thereof; (e) is a human IgG Fc domain or fragment thereof; (f) is a human IgG1 Fc domain or fragment thereof; (g) is modified compared to the corresponding wild-type Fc domain, wherein said modification leads to an increased affinity for one or more FCY receptors; (h) is modified compared to the corresponding wild-type Fc domain, wherein said modification leads to an enhanced antibody-dependent cell-mediated cytotoxicity response; and/or (i) comprises an afucosylated N-linked glycan at amino acid position 297. 13. A binding molecule that binds to CCR9, wherein the binding molecule does not compete for binding to CCR9 with the binding molecule according to any one of claims 1 to 10. 14. A therapeutic agent comprising an anti-CCR9 antibody as an active ingredient, wherein the anti-CCR9 antibody competes with CCL25 for binding to human CCR9. 15. The therapeutic agent of claim 14, wherein the anti-CCR9 antibody inhibits CCL25-mediated CCR9 internalization; optionally wherein the anti-CCR9 antibody competes with CCL25 for binding to human CCR9A and CCR9B. 16. The therapeutic agent of any one of claims 14 to 15, wherein the anti-CCR9 antibody specifically binds to an epitope on CCR9, wherein the epitope is located within the sequence comprising SEQ ID NO: 80; and/or wherein the anti-CCR9 antibody is: (a) afucosylated; (b) afucosylated at amino acid position 297; or (c) present in a composition comprising multiple copies of said binding molecule, wherein at least 50%, 75%, 80%, 90%, 95%, 98%, 99%, or 100% of the copies of the binding molecule in the composition are afucosylated. 17. The therapeutic agent of any one of claims 14 to 15, wherein the immunoglobulin Fc domain or fragment: (a) is an IgG Fc domain or fragment thereof; (b) is a human IgG Fc domain or fragment thereof; (c) is a human IgG1 Fc domain or fragment thereof; (d) is modified compared to the corresponding wild-type Fc domain, wherein said modification leads to an increased affinity for one or more FCY receptors; (e) is modified compared to the corresponding wild-type Fc domain, wherein said modification leads to an increased antibody-dependent cell-mediated cytotoxicity response; and/or (f) comprises an afucosylated N-linked glycan at amino acid position 297. 18. Use of the binding molecule as defined in claim 13, characterized in that it is to determine the abundance of CCR9+ cells in a sample that has been contacted with a binding molecule of any one of claims 1 to 10. 19. A method for assessing depletion of cells expressing CCR9 by a binding molecule that binds to CCR9 and comprises a heavy chain variable region (VH) having a set of CDRs HCDR1, HCDR2, and HCDR3 and a light chain variable region (VL) having a set of CDRs LCDR1, LCDR2, and LCDR3, wherein (a) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; (b) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8 and HCDR3 of SEQ ID NO: 9, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11 and LCDR3 of SEQ ID NO: 12; or (c) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 13, HCDR2 of SEQ ID NO: 14 and HCDR3 of SEQ ID NO: 15, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 16, LCDR2 of SEQ ID NO: 17 and LCDR3 of SEQ ID NO: 18, or as defined in any one of claims 1 to 12, characterized in that the method comprises: (i) contacting said binding molecule with a population of cells, wherein the population of cells comprises cells expressing CCR9 and immune effector cells, under conditions suitable to allow antibody-dependent cell-mediated cytotoxicity by the effector cells; (ii) contacting said population of cells with a binding molecule that binds to CCR9 and that does not compete for binding to CCR9 with the binding molecule of step (i); (iii) detecting CCR9-expressing cells in the cell population that are bound by the binding molecule of (ii); (iv) comparing the amount of CCR9-expressing cells detected in step (iii) with the amount of CCR9-expressing cells in the original cell population used in step (i) and thereby determining the amount of CCR9-expressing cells that were depleted in step (i). 20. The binding molecule of claim 13, the use of claim 17, or the method of claim 19, wherein said binding molecule does not compete for binding to CCR9 with the binding molecule comprising a heavy chain variable region (VH) having a set of CDRs HCDR1, HCDR2, and HCDR3 and a light chain variable region (VL) having a set of CDRs LCDR1, LCDR2, and LCDR3, wherein (a) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; (b) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8 and HCDR3 of SEQ ID NO: 9, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11 and LCDR3 of SEQ ID NO: 12; or (c) the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 13, HCDR2 of SEQ ID NO: 14 and HCDR3 of SEQ ID NO: 15, and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 16, LCDR2 of SEQ ID NO: 17 and LCDR3 of SEQ ID NO: 18, or according to any one of claims 1 to 12, comprises a VH region amino acid sequence comprising HCDR1 of SEQ ID NO: 44, HCDR2 of SEQ ID NO: 45 and HCDR3 of SEQ ID NO: 46, and a VL region amino acid sequence comprising LCDR1 of SEQ ID NO: 47, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 48. 21. An isolated polynucleotide encoding the binding molecule that binds to CCR9 and comprising a heavy chain variable region (VH) having a set of CDRs HCDR1, HCDR2, and HCDR3 and a light chain variable region (VL) having a set of CDRs LCDR1, LCDR2, and LCDR3, wherein (a) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; (b) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8 and HCDR3 of SEQ ID NO: 9, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11 and LCDR3 of SEQ ID NO: 12; or (c) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 13, HCDR2 of SEQ ID NO: 14 and HCDR3 of SEQ ID NO: 15, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 16, LCDR2 of SEQ ID NO: 17 and LCDR3 of SEQ ID NO: 18, or as defined in any one of claims 1 to 13. 22. Vector characterized by the fact that it comprises: (a) the polynucleotide, as defined in claim 21, operationally associated with a promoter; or (b) a polynucleotide encoding the VH region, as defined in any one of claims 1 to 3, and a polynucleotide encoding the VL region, as defined in any one of claims 1 to 3, characterized by the fact that said polynucleotides are operationally associated with one or more promoters. 23. A host cell comprising the polynucleotide as defined in claim 21 or the vector as defined in claim 22. 24. A method of producing a binding molecule that binds to CCR9 and comprises a heavy chain variable region (VH) having a set of CDRs HCDR1, HCDR2, and HCDR3 and a light chain variable region (VL) having a set of CDRs LCDR1, LCDR2, and LCDR3, wherein (a) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; (b) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8 and HCDR3 of SEQ ID NO: 9, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11 and LCDR3 of SEQ ID NO: 12; or (c) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 13, HCDR2 of SEQ ID NO: 14, and HCDR3 of SEQ ID NO: 15, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 16, LCDR2 of SEQ ID NO: 17, and LCDR3 of SEQ ID NO: 18, or as defined in any one of claims 1 to 13, characterized in that it comprises expressing a polynucleotide as defined in claim 21, or a vector as defined in claim 22, in a host cell. 25. Pharmaceutical composition characterized by the fact that it comprises a binding molecule that binds to CCR9 and comprises a heavy chain variable region (VH) with a set of CDRs HCDR1, HCDR2 and HCDR3 and a light chain variable region (VL) with a set of CDRs LCDR1, LCDR2 and LCDR3, in which (a) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2 and HCDR3 of SEQ ID NO: 3, and in which the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5 and LCDR3 of SEQ ID NO: 6; (b) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8 and HCDR3 of SEQ ID NO: 9, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11 and LCDR3 of SEQ ID NO: 12; or (c) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 13, HCDR2 of SEQ ID NO: 14 and HCDR3 of SEQ ID NO: 15, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 16, LCDR2 of SEQ ID NO: 17 and LCDR3 of SEQ ID NO: 18, or as defined in any one of claims 1 to 12, and a pharmaceutically acceptable carrier or diluent. 26. Pharmaceutical composition, according to claim 25, characterized by the fact that it is for use as a medicine. 27. Use of the binding molecule that binds to CCR9 and comprises a heavy chain variable region (VH) with a set of CDRs HCDR1, HCDR2, and HCDR3 and a light chain variable region (VL) with a set of CDRs LCDR1, LCDR2, and LCDR3, wherein (a) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; (b) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8 and HCDR3 of SEQ ID NO: 9, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11 and LCDR3 of SEQ ID NO: 12; or (c) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 13, HCDR2 of SEQ ID NO: 14, and HCDR3 of SEQ ID NO: 15, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 16, LCDR2 of SEQ ID NO: 17, and LCDR3 of SEQ ID NO: 18, or as defined in any one of claims 1 to 13, the therapeutic agent as defined in any one of claims 14 to 16, or the composition as defined in claim 25, characterized in that it is for the manufacture of a medicament for treating a disease or condition in a subject. 28. Use of a binding molecule that binds to CCR9, characterized by the fact that it is for the manufacture of a medicament for treating a disease or condition in a subject, and wherein: (a) said binding molecule is capable of mediating antibody-dependent cell-mediated cytotoxicity against a CCR9-expressing cell to which it binds (b) binding of the binding molecule to CCR9 does not induce internalization of CCR9 and/or (c) the binding molecule competes with the binding of CC25 to CCR9, optionally human CCR9, optionally human CCR9A and CCR9B. 29. Use according to either of claims 27 or 28, characterized in that the disease is a CCR9-mediated disease or in which the disease is mediated by cells expressing CCR9; optionally in which the disease is an inflammatory bowel disease. 30. Use according to claim 27, characterized in that the disease is selected from the group consisting of Crohn's disease, ileal/ileocolonic Crohn's disease, and ulcerative colitis; or in which the disease is selected from the group consisting of T-cell acute lymphoblastic leukemia, prostate cancer, breast cancer, melanoma, circulating cells of a solid tumor, liver fibrosis, and acute inflammation of the liver. 31. A method for detecting the presence of a CCR9 polypeptide in a sample, comprising: contacting a sample with a binding molecule that binds to CCR9 and comprises a heavy chain variable region (VH) having a set of CDRs HCDR1, HCDR2, and HCDR3 and a light chain variable region (VL) having a set of CDRs LCDR1, LCDR2, and LCDR3, wherein (a) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; (b) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8 and HCDR3 of SEQ ID NO: 9, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11 and LCDR3 of SEQ ID NO: 12; or (c) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 13, HCDR2 of SEQ ID NO: 14 and HCDR3 of SEQ ID NO: 15, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 16, LCDR2 of SEQ ID NO: 17 and LCDR3 of SEQ ID NO: 18, according to any one of claims 1 to 13, to provide an antigen-binding molecule complex; detect the presence or absence of said antigen-binding molecule complex; wherein the presence of the antigen-binding molecule complex confirms the presence of a CCR9 polypeptide. 32. A kit comprising one or more containers comprising the binding molecule that binds to CCR9 and comprising a heavy chain variable region (VH) having a set of CDRs HCDR1, HCDR2, and HCDR3 and a light chain variable region (VL) having a set of CDRs LCDR1, LCDR2, and LCDR3, wherein (a) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; (b) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8 and HCDR3 of SEQ ID NO: 9, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11 and LCDR3 of SEQ ID NO: 12; or (c) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 13, HCDR2 of SEQ ID NO: 14 and HCDR3 of SEQ ID NO: 15, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 16, LCDR2 of SEQ ID NO: 17 and LCDR3 of SEQ ID NO: 18 or as defined in any one of claims 1 to 13, the therapeutic agent as defined in any one of claims 14 to 16 or the composition as defined in any one of claims 25. 33. Use of the binding molecule that binds to CCR9 and comprises a heavy chain variable region (VH) with a set of CDRs HCDR1, HCDR2, and HCDR3 and a light chain variable region (VL) with a set of CDRs LCDR1, LCDR2, and LCDR3, wherein (a) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; (b) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8 and HCDR3 of SEQ ID NO: 9, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11 and LCDR3 of SEQ ID NO: 12; or (c) the amino acid sequence of the VH region comprises HCDR1 of SEQ ID NO: 13, HCDR2 of SEQ ID NO: 14 and HCDR3 of SEQ ID NO: 15, and wherein the amino acid sequence of the VL region comprises LCDR1 of SEQ ID NO: 16, LCDR2 of SEQ ID NO: 17 and LCDR3 of SEQ ID NO: 18 or as defined in any one of claims 1 to 13, the therapeutic agent as defined in any one of claims 14 to 16 or the composition as defined in any one of claims 25, characterized in that it is for the manufacture of a medicament for use in therapy.