Methods of treating inflammation

Abstract

To provide depleting antigen binding sites which bind to CXCR3, and methods and compositions for treating or preventing autoimmune and inflammatory diseases, in particular, fatty liver disease, the accumulation of lipid deposits in the liver, steatohepatitis, graft rejection and diseases associated with effector and / or memory T cells.SOLUTION: The present invention provides a method for treating a disease associated with effector and / or memory T cells in a subject, the method comprising, consisting essentially of or consisting of administering to the subject a depleting antigen binding site that binds to CXCR3, thereby treating a disease associated with effector and / or memory T cells in the subject.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to methods and compositions for treating or preventing diseases associated with CXCR3 expression, in particular non-alcoholic steatohepatitis (NASH).

[0002] Related Applications This application claims priority to Australian provisional patent application AU 2018903814, the contents and disclosure of which are incorporated herein by reference in their entirety. [Background technology]

[0003] 2. Background of the Invention Chemokines, or chemoattractant cytokines, comprise a family of small molecular weight (approximately 8-10 KDa) inducible secreted molecules that typically function as activators and chemoattractants for leukocytes and can regulate angiogenesis, wound healing, and tumorigenesis. Most of the knowledge about chemokine function has come from studies of the immune system because of their involvement in immunoregulation and control of inflammation. Their roles in many other biological systems remain less understood.

[0004] Chemokines are typically classified into four subfamilies according to the number of conserved cysteine ​​residues at their amino termini. Most chemokines belong to two main subfamilies with four cysteine ​​residues. These subfamilies are typically classified according to the presence or absence of an amino acid between the two amino-terminal cysteine ​​residues, and are thereby called CC and CXC chemokines. CXC chemokines (typically restricted to higher vertebrates) are further classified according to the presence or absence of a glutamic acid-lysine-arginine (ELR) motif at their amino termini, usually adjacent to the first cysteine ​​residue.

[0005] Chemokine receptors such as CXCR3 are typically G protein-coupled receptors (GPCRs) linked to pertussis toxin (PTX)-sensitive Gi proteins. The N-terminal domain of CXCR is thought to be important for determining ligand binding specificity.

[0006] CXCR3, also known as G protein-coupled receptor 9 (GPR9), CD182, CD183, CKR-L2, CMKAR3, GPR9, IP10-R, Mig-R, MigR, and CXC motif chemokine receptor 3, is a 38 kDa seven-transmembrane protein. CXCR3 is expressed primarily on activated T lymphocytes and NK cells, and on some epithelial cells. CXCR3 is preferentially expressed on Th1 cells (whereas Th2 cells favor the expression of CCR3 and CCR4). CXCR3 ligands that attract Th1 cells may concomitantly block the migration of Th2 cells in response to CCR3 ligands, thereby promoting the polarization of effector T cell recruitment. CXCR3 can control leukocyte trafficking. Binding of chemokines to CXCR3 induces a variety of cellular responses, most notably integrin activation, cytoskeletal changes and chemotactic migration. CXCR3-ligand interactions attract Th1 cells and promote Th1 cell maturation.

[0007] CXCR3 is expressed on effector / memory T cells cultured in vitro and on T cells present in many types of inflamed tissue. In addition, CXCL9, CXCL10, and CXCL11 are commonly produced by local cells in inflammatory lesions and are involved in the expression of CXCR3. suggest that CXCR3 and its chemokines are involved in the recruitment of inflammatory cells. CXCR3 has been implicated in cancer, wound healing, responses to infection and tissue transplantation, and autoimmune diseases including atherosclerosis, multiple sclerosis, pulmonary fibrosis, type 1 diabetes, autoimmune myasthenia gravis, nephrotoxic nephritis, acute cardiac allograft rejection, and celiac disease.

[0008] There is a need for new and / or improved methods for treating diseases associated with CXCR3 activation.

[0009] The reference to any prior art in this specification is not an admission or suggestion that this prior art forms part of common general knowledge in any jurisdiction, or that this prior art would be reasonably expected to be understood, considered relevant, and / or could be combined with other pieces of prior art by a person of ordinary skill in the art. Summary of the Invention [Means for solving the problem]

[0010] Summary of the Invention In one aspect, the present invention provides a method for treating a disease associated with effector and / or memory T cells in a subject, the method comprising administering to a subject a depleting T cell that binds to CXCR3. The present invention provides a method comprising, consisting essentially of, or consisting of administering an antigen binding site to a subject, thereby treating a disease associated with effector and / or memory T cells in the subject.

[0011] In another aspect, the present invention also provides a method for treating fatty liver disease in a subject, comprising, consisting essentially of, or consisting of administering to the subject an exhaustable antigen binding site that binds CXCR3, thereby treating fatty liver disease in the subject.

[0012] In a further aspect, the present invention provides a method for preventing or delaying the onset of fatty liver disease in a subject, comprising, consisting essentially of, or consisting of administering to the subject an exhaustable antigen binding site that binds CXCR3, thereby preventing or delaying the onset of fatty liver disease in the subject.

[0013] Fatty liver disease can be associated with, caused by, or the result of alcoholism.Alternatively, fatty liver disease can be associated with, caused by, or the result of dietary causes other than alcohol (non-alcoholic fatty liver disease, NAFLD).NAFLD can be simple fatty liver, or simple fatty liver that includes one or more signs of inflammation and fibrosis.

[0014] Fatty liver disease can be characterized by hepatic steatosis, elevated circulating or hepatic triglyceride levels and / or elevated circulating or hepatic cholesterol levels.

[0015] In a further aspect, the present invention provides a method for treating non-alcoholic steatohepatitis (NASH) in a subject, the method comprising, consisting essentially of, or consisting of an exhaustible antigen binding site that binds to CXCR3, thereby treating NASH in the subject.

[0016] In another aspect, the present invention also provides a method for preventing or delaying the onset of non-alcoholic steatohepatitis (NASH) in a subject, comprising or comprising administering to a subject an exhaustible antigen binding site that binds to CXCR3, thereby preventing or delaying the onset of NASH. The present invention provides a method comprising:

[0017] In another aspect, the present invention also provides a method of reducing or treating inflammation in a subject at risk for or suffering from an autoimmune disease, comprising, consisting essentially of, or consisting of a depletable antigen binding site that binds to CXCR3, thereby reducing or treating inflammation in the subject.

[0018] Reducing or treating inflammation can include decreasing the proportion of one or more pro-inflammatory cytokines in an individual. Additionally, reducing or treating inflammation can include increasing the proportion of one or more anti-inflammatory cytokines in a subject.

[0019] In one aspect, the invention provides a method for inhibiting recruitment or migration of CXCR3+ cells to a site of inflammation in a subject, the method comprising administering to the subject an effective amount of a depletable antigen binding site that binds CXCR3, thereby inhibiting recruitment or migration of CXCR3+ cells to a site of inflammation in the subject.

[0020] In any embodiment of the present invention, the antigen-binding moiety that binds to CXCR3 is a depleting antibody. The depleting antibody has the ability to cause or mediate a decrease in the activity or viability of cells expressing CXCR3. Typically, the depleting antibody has the ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC).

[0021] The antigen binding site may be modified to provide the ability to deplete cells, or may be modified to enhance the existing ability of the antigen binding site to deplete cells.

[0022] In certain embodiments, the cells are CXCR3+ / CD4+ T cells, CXCR3+ / CD8+ T cells and / or CXCR3+ / CD19+ B cells. In further embodiments, the subject has received, is receiving or will receive a graft or transplant.

[0023] In another aspect, the invention provides a method of treating or preventing accumulation of lipid deposits in the liver of a subject, comprising administering to the subject an exhaustable antigen binding site that binds to CXCR3, thereby treating or preventing accumulation of lipid deposits in the liver of the subject.

[0024] In another aspect, the present invention provides a method for treating or preventing fatty liver disease in a subject, comprising administering to the subject an exhaustible antigen binding site that binds to CXCR3, thereby treating or preventing fatty liver disease in the subject. Fatty liver disease may be non-alcoholic fatty liver disease (NAFLD) or may be caused by or result from alcoholism.

[0025] In another aspect, the present invention provides a method for treating or preventing steatohepatitis in a subject, comprising administering to the subject a depletable antigen binding site that binds to CXCR3, thereby treating or preventing steatohepatitis. The steatohepatitis can be alcoholic steatohepatitis (ASH) or non-alcoholic steatohepatitis (NASH). Preferably, the steatohepatitis is NASH.

[0026] In another aspect, the invention provides a method for inhibiting graft or transplant rejection in a subject who has received, is receiving, or will receive a graft or transplant, comprising administering to the subject an effective amount of a depletable antigen binding site that binds CXCR3, thereby inhibiting graft or transplant rejection in the subject.

[0027] In certain embodiments of the methods of the present invention, the graft is an allograft or a xenograft. Alternatively, the transplant is an allograft or a xenograft. In certain instances, the graft or transplant is selected from among heart, kidney, lung, liver, pancreas, pancreatic islets, brain tissue, stomach, large intestine, small intestine, cornea, skin, trachea, bone, bone marrow, muscle, and bladder grafts or transplants. In some instances, the antibody or antigen-binding fragment is administered to the subject before, simultaneously with, and / or after the subject receives the graft or transplant.

[0028] In one embodiment of the method of the present invention, the antibody or antigen-binding fragment is administered to the subject more than once. In some examples, the method further comprises administering to the subject one or more other therapeutic agents, such as a cytokine (e.g., interleukin 2), a chemokine, or an antibody. In certain embodiments, the therapeutic agent is an immunosuppressant, such as a glucocorticoid, cyclosporine, rapamycin, voclosporin, sirolimus, everolimus, tacrolimus, mycophenolate, mofetil, mycophenolic acid, mizoribine, S1P-R agonist, malononitrile amide, an anti-CD3 antibody, an anti-CD25 antibody, an anti-CD52 antibody, an anti-CD20 antibody, or an anti-tumor necrosis factor antibody.

[0029] Also provided is the use of a depletable antigen-binding site that binds CXCR3 for the preparation of a medicament for depleting CXC3+ cells; inhibiting recruitment or migration of CXCR3+ cells to sites of inflammation; and / or inhibiting graft rejection in a subject that has received, has received, or is to receive a graft.

[0030] In another aspect, the invention provides a method of treating fibrosis in a subject, comprising administering to the subject an antigen binding site described herein, thereby treating the fibrosis.

[0031] In any embodiment of the invention, the antigen binding site binds or specifically binds to CXCR3 and inhibits CXCR3 activity.

[0032] Preferably, the antigen-binding site comprises an antigen-binding domain of an antibody, which binds or specifically binds to CXCR3 and inhibits CXCR3 activity.

[0033] CXCR3 activities that may be inhibited by any of the antigen binding sites of the present invention include binding of a ligand to CXCR3; ligand-induced conformational changes in CXCR3; CXCR3 activation; G protein activation; CXCR3-mediated cell signaling; CXCR3-mediated cell migration, inflammation, tumor growth, angiogenesis or metastasis responses in vitro or in vivo; CXCR3-mediated tumor cell proliferation; and / or CXCR3-mediated recruitment of inflammatory cells, leukocyte (e.g. neutrophils, eosinophils, mast cells or T cells) migration, integrin activation, chemotactic migration and Th1 cell maturation.

[0034] Preferably, the antigen-binding site described herein binds or specifically binds to human CXCR3. Preferably, the antigen-binding site binds or specifically binds to a human CXCR3 molecule comprising, consisting essentially of or consisting of the amino acid sequence set out in SEQ ID NO:7.

[0035] Preferably, the antigen-binding site inhibits or reduces CXCR3 activity induced by one or more ligands selected from the group consisting of CXCL9 (MIG), CXCL10 (IP10) and CXCL11 (I-TAC). For example, the antigen-binding site may inhibit migration of cells, preferably immune cells, stimulated by a CXCR3 ligand. Reduction or inhibition of CXCR3 activity may be determined by any of the methods described herein, in particular in Example 4.

[0036] The antigen-binding sites described herein may bind to CXCR3 and may not detectably or substantially bind to CXCR1, CXCR2, and / or C5aR. Binding of the antigen-binding sites to CXCR1, CXCR2, and / or C5aR may be determined by any of the methods described herein, in particular by flow cytometry as described in Example 2.

[0037] The antigen-binding site described herein may bind to CXCR3 and have an EC of less than 2 nM, less than 1 nM, or less than 0.5 nM. 50 Preferably, the EC of the antigen-binding site 50 is about 0.2 nM or less. 50 is determined using flow cytometry or ELISA assays as described herein, particularly in Example 2.

[0038] The antigen-binding sites described herein have an IC of about 20, 15, 12, 10, 8, 6, 5, 4, 3, 2, or 1 nM in competitive binding assays with MIG, ITAC, and / or IP10. 50 It can be shown that

[0039] The antigen-binding sites described herein can inhibit migration of immune cells expressing CXCR3 at concentrations of 10 μg / ml, 1 μg / ml or less. Preferably, the migration assay is performed by any of the methods described herein, particularly Example 4.

[0040] The antigen binding sites described herein may bind to the first and / or second N-terminal loop in CXCR3. For example, the antigen binding site may bind to CXCR3 within residues 1-23 (numbering according to human CXCR3) of the CXCR3 protein. Preferably, the antigen binding site of the invention binds to a peptide comprising the sequence: MVLEVSDHQVLNDAEVAALLENF (SEQ ID NO: 8), or a fragment thereof. Preferably, the antigen binding site of the invention does not bind to a sequence of CXCR3 that is C-terminal to residue 23. Alternatively, the antigen binding site may bind to CXCR3 within residues 23-44 (numbering according to human CXCR3) of the CXCR3 protein. Preferably, the antigen binding site of the invention binds to a peptide comprising the sequence: FSSSYDYGENESDSCCTSPPCP (SEQ ID NO: 10), or a fragment thereof.

[0041] The invention also provides an antigen-binding site that binds to the N-terminal region of CXCR3 and inhibits binding of CXCL9(MIG), CXCL10(IP10) and / or CXCL11(I-TAC) to CXCR3 or a CXCL9(MIG), CXCL10(IP10) and / or CXCL11(I-TAC)-mediated activity of CXCR3. Preferably, the N-terminal region comprises, consists essentially of or consists of residues 1-23 (numbering according to human CXCR3). Preferably, the CXCL9(MIG), CXCL10(IP10) and / or CXCL11(I-TAC)-mediated activity is CXCL9(MIG), CXCL10(IP10) and / or CXCL11(I-TAC)-mediated chemotaxis of immune cells (e.g. neutrophils).

[0042] As described herein, an antigen-binding site comprises: (i) single chain Fv fragment (scFv); (ii) dimeric scFv (di-scFv); (iii) one of (i) or (ii) linked to a constant region, Fc or heavy chain constant domain (CH)2 and / or CH3, of an antibody; or (iv) one of (i) or (ii) linked to a protein that binds to an immune effector cell. The compound may be in the form:

[0043] Further, as described herein, an antigen-binding site may comprise: (i) Diabody; (ii) triabody; (iii) tetrabodies; (iv) Fab; (v) F(ab')2; (vi) Fv; (vii) one of (i)-(vi) linked to a constant region, Fc or heavy chain constant domain (CH)2 and / or CH3, of an antibody; or (viii) one of (i) to (vi) linked to a protein that binds to an immune effector cell; The compound may be in the form:

[0044] The antigen-binding site is sometimes called the antigen-binding domain of an antibody.

[0045] Preferably, the antigen-binding site described herein is an antibody or an antigen-binding fragment thereof. Typically, the antigen-binding site is an antibody, such as a monoclonal antibody.

[0046] As used herein, an antigen binding site may be a variable domain.

[0047] In any aspect or embodiment, the antibody is a naked antibody, specifically, the antibody is in unconjugated form and has not been configured to form a conjugate.

[0048] Reference herein to a protein or antibody that "binds to" CXCR3 gives literal support to a protein or antibody that "specifically binds to" or "specifically binds to" CXCR3.

[0049] The present invention also provides an antigen-binding domain or antigen-binding fragment of the above antibody.

[0050] The present invention also provides a fusion protein comprising an antigen binding site, an immunoglobulin variable domain, an antibody, a dab (single domain antibody), a di-scFv, an scFv, a Fab, a Fab', an F(ab')2, an Fv fragment, a diabody, a triabody, a tetrabody, a linear antibody, a single chain antibody molecule, or a multispecific antibody as described herein.

[0051] The invention also provides conjugates in the form of antigen binding sites, immunoglobulin variable domains, antibodies, dabs, di-scFvs, scFvs, Fabs, Fab's, F(ab')2, Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single chain antibody molecules, or multispecific antibodies or fusion proteins as described herein conjugated to a label or a cytotoxic agent.

[0052] The invention also provides antibodies for binding to an antigen binding site, immunoglobulin variable domain, antibody, dab, di-scFv, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, tetrabody, linear antibody, single chain antibody molecule, or multispecific antibody, fusion protein, or conjugate described herein.

[0053] The invention also provides nucleic acids encoding an antigen binding site, immunoglobulin variable domain, antibody, dab, di-scFv, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, tetrabody, linear antibody, single chain antibody molecule, or multispecific antibody, fusion protein or conjugate as described herein.

[0054] In one example, such a nucleic acid is included in an expression construct in which the nucleic acid is operably linked to a promoter. Such an expression construct can be a vector, for example, a plasmid.

[0055] In the example of the invention relating to a single polypeptide chain antigen binding site, the expression construct may include a promoter linked to a nucleic acid encoding the polypeptide chain.

[0056] In examples involving multiple polypeptide chains forming an antigen binding site, the expression construct includes a nucleic acid encoding a polypeptide comprising, e.g., a VH, operably linked to a promoter, and a nucleic acid encoding a polypeptide comprising, e.g., a VL, operably linked to a promoter.

[0057] In another example, the expression construct may comprise, for example, the following components operably linked in 5'-3' order: (i) Promoter (ii) a nucleic acid encoding a first polypeptide; (iii) an internal ribosome entry site; and (iv) a nucleic acid encoding a second polypeptide. is a bicistronic expression construct comprising wherein the first polypeptide comprises a VH and the second polypeptide comprises a VL, or vice versa.

[0058] The present invention also contemplates separate expression constructs, one encoding a first polypeptide comprising a VH and the other encoding a second polypeptide comprising a VL. For example, the present invention also contemplates (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH, operably linked to a promoter; and (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL, operably linked to a promoter; A composition comprising:

[0059] The invention provides a cell comprising a vector or nucleic acid described herein. Preferably, the cell is isolated, substantially purified or recombinant. In one example, the cell comprises an expression construct of the invention or: (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH, operably linked to a promoter; and (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL, operably linked to a promoter; Including, Here, the first and second polypeptides associate to form an antigen-binding site as described herein.

[0060] Examples of cells of the invention include bacterial cells, yeast cells, insect cells or mammalian cells.

[0061] The present invention also provides a pharmaceutical composition comprising an antigen binding site as described herein or a CDR and / or FR sequence, or an immunoglobulin variable domain as described herein, an antibody, dab (single domain antibody), di-scFv, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, tetrabody, linear antibody, single chain antibody molecule, or multispecific antibody, fusion protein, or conjugate, together with a pharma- ceutically acceptable carrier, diluent or excipient.

[0062] The present invention also relates to an antigen-binding site comprising the antigen-binding sites described herein or comprising the CDRs and / or F Diagnostic compositions are provided comprising an R sequence, or an antigen binding site as described herein, an immunoglobulin variable domain, an antibody, a dab, a di-scFv, a scFv, a Fab, a Fab', a F(ab')2, an Fv fragment, a diabody, a triabody, a tetrabody, a linear antibody, a single chain antibody molecule, or a multispecific antibody, a fusion protein or conjugate, a diluent and optionally a label.

[0063] The invention also provides kits or articles of manufacture comprising the antigen binding site described herein or comprising the CDR and / or FR sequences or immunoglobulin variable domains described herein, antibodies, dabs, di-scFvs, scFvs, Fabs, Fab's, F(ab')2, Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single chain antibody molecules, or multispecific antibodies, fusion proteins or conjugates.

[0064] The antigen-binding sites, proteins or antibodies described herein may comprise a human constant region, e.g., an IgG constant region, such as IgG1, IgG2, or IgG3, or a mixture thereof. In the case of an antibody or protein comprising a VH and a VL, the VH may be linked to a heavy chain constant region and the VL may be linked to a light chain constant region.

[0065] In any embodiment of the invention, the antigen binding site comprises an Fc region that has been modified to have an enhanced ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Preferably, the enhanced ability to induce ADCC is conferred by mutation, deletion or modification of amino acids in the Fc region that interact with the Fc receptor. Preferably, the mutated, deleted or modified amino acids are at positions 239, 330 and / or 332 or equivalent positions 239, 330 and / or 332 according to SEQ ID NO: 1 (wherein alanine is at position 118). Preferably, the amino acids are mutated to S239D, A330L and I332E. Typically, the Fc comprises, consists essentially of or consists of the amino acid sequence shown in SEQ ID NO:3.

[0066] In any embodiment of the invention, the antigen binding site comprises an Fc region that has not been modified to have a reduced ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC), preferably wherein the amino acids at or equivalent to positions 234, 235 and / or 331 according to SEQ ID NO:1 (wherein alanine is at position 118) are not F, E and / or S, respectively. In other words, the amino acid at position 234 is not F, the amino acid at position 235 is not E and / or the amino acid at position 331 is not S.

[0067] In any embodiment of the invention, the antigen binding site does not include an Fc region comprising, consisting essentially of or consisting of the amino acid sequence set forth in SEQ ID NO:2.

[0068] It will be understood that the functional properties of the antigen-binding sites described herein apply mutatis mutandis to the antibodies of the invention.

[0069] The antigen binding sites described herein may be purified, substantially purified, isolated and / or recombinant.

[0070] The antigen binding sites described herein can be part of the supernatant harvested from the culture in which a hybridoma expressing the antigen binding sites described herein was grown.

[0071] In another aspect, the present invention also provides a method for depleting CXCR3+ cells in a subject, the method comprising administering to the subject an effective amount of an antigen-binding site described herein.

[0072] The present invention also provides a cell comprising a vector or a nucleic acid molecule described herein.

[0073] The present invention also provides animals or tissues derived from the cells described herein.

[0074] As used herein, unless the context requires otherwise, the term "comprise" and conjugations of this term (e.g., "comprising," "comprises," and "comprised") do not exclude additional additives, components, integers, or steps.

[0075] Further aspects of the invention and further embodiments of the aspects described in the previous paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0076] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]Long-lasting effect of CXCR3 depletion after a single intravenous (IV) injection. Mice were injected with depleting anti-CXCR3 antibodies. CD4+, CD8+ and B cells were quantified 24 and 36 days after injection. The results show that cell depletion using depleting anti-CXCR3 antibodies is long-lasting. [Diagram 2] Anti-CXCR3 depletion does not significantly affect Treg (CD4+; FoxP3+) numbers. Depleting anti-CXCR3 mAb mIgG1 (5 mg / kg) or mIgG1 isotype control (5 mg / kg) were injected intravenously (IV) into FoxP3-GFP reporter mice. Four days later, CD4+ splenic lymphocyte populations were analyzed using anti-mCXCR3 from Biolegend. These data confirm that only the CXCRr3+ Treg subset was depleted, whereas the ratio CD4+ FoxP3- / CD4+ FoxP3+ was not altered. [Diagram 3] Skin graft model. Anti-CXCR3 depleting antibody Balb / c to C57BL / 6 skin transplantation experiments. Mice receiving anti-CXCR3 depleting antibody show evidence of tolerated allograft skin 200 days after transplantation. [Figure 4] Targeting CXCR3+ cells in NASH ameliorates steatohepatitis and fibrosis. A depleting anti-CXCR3 mAb was shown to prevent NASH development and reduce collagen deposition (fibrosis) in mice. [Diagram 5] Depletion of CXCR3+ cells prevents the development of NASH. To induce NASH, mice were fed an MCD diet. Mice were also given either an isotype control; a depleting anti-CXCR3 mAb or a non-depleting anti-CXCR3 mAb. Only mice given the depleting antibody were prevented from developing NASH. [Figure 6] Anti-CXCR3 mAb depletes NKT cells and activated intrahepatic CD8 T cells. MCD diet increases the frequency of CD8+ CXCR3+ T cells; MCD diet increases the frequency of NKT cells; NKT cells are CXCR3+ and are depleted after treatment with Fc-modified anti-CXCR3 mAB. [Figure 7]Anti-CXCR3 treatment reduces the release of ALT in the serum and the infiltration of monocytes in the liver. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0077] Detailed Description of the Embodiments It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features described or apparent from the text or drawings, all of which different combinations constitute various alternative aspects of the invention.

[0078] Further aspects of the invention and further embodiments of the aspects described in the previous paragraphs are, for example This will become apparent from the following description taken in conjunction with the accompanying drawings, in which:

[0079] Reference will now be made in detail to specific embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that it is not intended to limit the invention to those embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the scope of the present invention as defined by the claims.

[0080] The inventors have shown that various diseases can be treated using antigen-binding sites that bind to CXCR3 and cause or mediate depletion of cells expressing CXCR3. As a result, the methods described herein find particular use in the prevention or treatment of various diseases associated with CXCR3 expression, including, but not limited to, NASH.

[0081] Overview Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition, group of steps, or group of compositions shall be understood to include one and the plurality (i.e., one or more) of those steps, compositions, group of steps, or group of compositions. Thus, as used herein, the singular forms "a," "an," and "the" include plural aspects, and vice versa, unless the context indicates otherwise. For example, reference to "a" includes one as well as two or more; reference to "an" includes one as well as two or more. including; references to "the" include one as well as two or more, and so forth.

[0082] Those skilled in the art will understand that the present invention is capable of variations and modifications other than those specifically described. It should be understood that the present invention includes all such variations and modifications. The present invention also includes all of the steps, features, compositions and compounds mentioned or shown in this specification, individually or collectively, and includes any combination of any two or more of said steps or features.

[0083] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention, and the present invention is in no way limited to the methods and materials described.

[0084] All patents and publications referenced herein are incorporated by reference in their entirety.

[0085] The present invention is not to be limited in scope by the specific examples described herein, which are intended as illustrative only, and all functionally equivalent products, compositions and methods are clearly within the scope of the invention.

[0086] Any example or embodiment of the present invention herein shall be understood to apply mutatis mutandis to any other example or embodiment of the present invention, unless specifically stated otherwise.

[0087] Unless otherwise defined, all technical and scientific terms used herein are to be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0088] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques used in this disclosure are standard procedures well known to those skilled in the art. Such techniques may be adapted from the methods described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989); T. A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2. d 2, IRL Press (1991), D.M.G. Lover and B.D.H.Mems (eds.), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and1996), and F.M.A.usubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all current revisions), Ed.Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. (eds.) )Current Protocols in Immunology, John Wiley & Sons (including all current revisions) ) and are described and explained throughout the literature.

[0089] The descriptions and definitions of variable regions and portions thereof, immunoglobulins, antibodies and fragments thereof herein may be further clarified by the descriptions in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991, Bork et al., J Mol. Biol. 242, 309-320, 1994, Chothia and Lesk J. Mol Biol. 196:901-917, 1987, Chothia et al. Nature 342, 877-883, 1989 and / or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.

[0090] The term "and / or", e.g., "X and / or Y", shall be understood to mean either "X and Y" or "X or Y", and shall be understood to give clear support for both meanings or for either meaning.

[0091] As used herein, the term "derived from" shall be understood to indicate that a specified integer may be obtained from a particular source, but not necessarily directly from that source.

[0092] For example, references herein to a range of residues are understood to include the endpoints. For example, a reference to "a region comprising amino acids 56-65" is understood in this manner to include the endpoints, i.e., this region includes the sequence of amino acids numbered 56, 57, 58, 59, 60, 61, 62, 63, 64, and 65 in a specified sequence.

[0093] Selected Definitions T cells are classified into several major subclasses, including cytotoxic T cells, which kill virus-infected cells, and two classes of regulatory cells, called helper T cells (Th cells) and suppressor T cells, which act to regulate the activity of other immune cells. During chronic infection, Th cells become at least three phenotypically and functionally distinct effector populations, Th1, Th2, and Th17 T cells. Th1 cells generally produce IFN-γ and IL-2, which are associated with cell-mediated immune responses against various intracellular pathogens, while Th2 cells produce cytokines such as IL-4, IL-5, and IL-13, which are important for controlling extracellular helminth infections. Th17 cells produce isoforms of IL-17, which play an important role in maintaining the mucosal barrier and contributing to pathogen clearance at mucosal surfaces.

[0094] Th1 cells are associated with organ-specific autoimmune diseases, delayed-type hypersensitivity reactions, and transplant rejection. Furthermore, cytokines such as IL-12 and IL-4 play a major role in determining the outcome of Th differentiation into Th1 and Th2 subsets, respectively. For example, in Th1 cells, STAT4 is activated after binding of IL-12 to its cognate receptor, thereby resulting in the production of IFN-γ. Thus, STAT4-deficient mice are defective in Th1 differentiation and do not respond to intracellular pathogens such as Listeria monocytogenes.

[0095] Th1 cells express various chemokine receptors, such as CXCR3, CCR1, and CCR5. In general, CXCR3 is considered to be one of the best markers for Th1 cells. However, CXCR3 is also expressed by NKT cells and some B cells, particularly those cells that are involved in inflammation (Qin et al J. Clin. Invest. 1997).

[0096] CXCR3 is also known as G protein-coupled receptor 9 (GPR9), CD182, CD183, CKR-L2, CMKAR3, GPR9, IP10-R, Mig-R, MigR). CXCR3 is an inflammatory chemokine receptor whose expression is associated with CDr+ type 1 helper (Th1) and CD8+ cytotoxic lymphocytes (CTL). In addition to CXCR3 expression on effector CD4+ and CD8+ T cells, CXCR3 is also highly expressed on innate lymphocytes, such as natural killer (NK) cells and NT cells, as well as plasmacytoid DC and a subset of B cells.

[0097] CXCR3 binds three chemokines: CXCL9 (also known as monokine induced by gamma interferon, or MIG), CXCL10 (interferon-inducible protein of 10 kDa, or IP-10), and CXCL11 I (interferon-inducible T cell alpha chemoattractant, or l-TAC). Binding of CXCR3 to these ligands is itself induced by inflammatory cytokines and mediates cell migration, thereby orchestrating inflammation in the periphery.

[0098] Due to the expression of CXCR3 on effector cells and its role in cell migration and inflammation, there is a need for improved therapies to inhibit cell migration and / or inflammation. For example, migration of activated T cells into the graft after transplantation leads to donor organ damage and is a hallmark of acute and chronic rejection. A wide variety of immunosuppressants are available and are routinely used in the clinic to reduce the incidence of rejection and improve both short-term and long-term graft survival. However, their mechanism of action often results in a complete shutdown of the immune system, and their use relies on a vague understanding. Thus, they are typically associated with side effects such as opportunistic infections, lymphoproliferative disorders, or cardiovascular diseases. Therefore, the development of more specific drugs capable of inducing transplant tolerance and thus eliminating the limitations of high doses of continuous immunosuppressants is a major goal in the transplant field.

[0099] Both IP-10 and MIG belong to the CXC subfamily, but in contrast to IL-8 and other CXC chemokines, which are potent chemoattractants for neutrophils, the primary targets of IP-10 and MIG are lymphocytes, especially effector cells, such as activated or stimulated T lymphocytes and natural killer (NK) cells. Consistently, IP-10 and MIG lack the ELR motif, an essential binding epitope in those CXC chemokines that efficiently induce neutrophil chemotaxis. Furthermore, both recombinant human MIG and recombinant human IP-10 have been reported to induce calcium flux in tumor-infiltrating lymphocytes (TILs). IP-10 has been reported to induce monocyte chemotaxis in vitro, but the receptor involved has not been identified, and human MIG appears to be highly selective and does not show such an effect. IP-10 expression is induced in various tissues in inflammatory diseases, such as psoriasis, fixed drug eruption, cutaneous delayed-type hypersensitivity reaction, tuberculous leprosy, and experimental glomerulonephritis and experimental allergic encephalomyelitis.IP-10 has also been reported to have a potent in vivo antitumor effect that is T cell-dependent, to be an inhibitor of angiogenesis in vivo, and to induce NK cell chemotaxis and degranulation in vitro, suggesting a role as a mediator of NK cell recruitment and degranulation (e.g., in the destruction of tumor cells).The expression patterns of IP-10 and MIG also differ in that their respective expressions are induced by interferon-γ (IFNγ), while IL-8 expression is downregulated by IFNγ.

[0100] Chemokines have recently been recognized as long-sought mediators for lymphocyte recruitment. Several CXC chemokines have been shown to induce lymphocyte chemotaxis. Although we have shown that IP-10 and MIG are active on granulocytes and monocytes, they are also active on granulocytes and monocytes. The situation is different for IP-10 and MIG, which are selective in their actions on lymphocytes, including activated T lymphocytes and NK cells, do not recognize many other chemokines, and bind to CXCR3, a receptor that displays a selective pattern of expression.

[0101] As used herein, reference to CXCR3 refers to a molecule having at least one biochemical or biophysical activity of CXCR3 as described herein. The term "CXCR3" as provided herein includes any of the native forms, homologs, or variants of CXC motif chemokine receptor 3 (CXCR3) protein that maintain the activity of CXCR3 (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150 or 200 consecutive amino acids) compared to the native form.

[0102] For purposes of nomenclature only and without limitation, an exemplary amino acid sequence of human CXCR3 is SEQ ID NO:7, and variants thereof having at least or about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, including naturally occurring variants set forth in SEQ ID NOs:108 and 109, which contain the mutations R292Q and A363T, respectively. Exemplary non-human CXCR3 polypeptides include, but are not limited to, mouse (e.g., SEQ ID NO:5), rat (e.g., SEQ ID NO:8), porcine (e.g., SEQ ID NO:9), bovine (e.g., SEQ ID NO:10), rhesus (e.g., SEQ ID NO:11), and canine (e.g., SEQ ID NO:12) CXCR3, and variants thereof having at least or about 85%, 90%, 91%, 92%, 93%, 94%, 95% 97%, 98% or 99% sequence identity thereto.

[0103] The phrase "inhibiting CXCR3 activity" is understood to mean that the antigen binding site of the present invention inhibits or reduces any one or more activities of CXCR3, including, but not limited to, ligand binding to CXCR3; ligand-induced conformational changes in CXCR3; CXCR3 activation; G protein activation; CXCR3-mediated cell signaling; CXCR3-mediated cell migration, inflammation, tumor growth, angiogenesis or metastasis responses in vitro or in vivo; CXCR3-mediated tumor cell proliferation; and / or CXCR3-mediated lymphocyte recruitment.

[0104] "Inhibiting MIG, IP-10 or I-TAC mediated CXCR3 activity" is understood to mean that the antigen binding site of the invention inhibits or reduces one or more of the above-mentioned activities mediated or induced by MIG, IP-10 and / or I-TAC. Furthermore, the activity is measured using a suitable in vitro, intracellular or in vivo assay, and the activity is blocked or reduced by at least 1%, 5%, 10%, 25%, 50%, 60%, 70%, 80% or 90% or more compared to the CXCR3 activity in the same assay under the same conditions except in the absence of the antigen binding site. Preferably, the CXCR3 activity is mediated or induced by any one or more of the ligands, e.g., MIG, IP-10 and I-TAC.

[0105] As used herein, "depletion" with respect to CXCR3+ cells (i.e., cells that express CXCR3, preferably cells that display cell surface CXCR3) refers to a reduction in the activity of these cells or removal of these cells from a population of cells. Preferably, depletion with respect to CXCR3+ cells refers to a removal of these cells from a population of cells or a reduction in the viability of these cells. Reference to depletion includes complete or partial depletion. Additionally, depletion may refer to permanent The depletion may be continuous or temporary, and may vary in magnitude and / or spatial extent. The depletion may be the result of cell death, such as by apoptosis or necrosis. Preferably, the cell death is mediated by ADCC. Alternatively, the antigen-binding site described herein may itself have little or no ability to reduce the activity or viability of CXCR3+ cells, but it may be conjugated or associated with a compound that has the ability to reduce the activity or viability of cells. Typically, the antigen-binding site that has little or no ability to reduce the activity or viability of CXCR3+ cells is conjugated or associated with a cytotoxic compound.

[0106] CXCR3 in a population before and after exposure to an antigen-binding site or antigen-binding fragment of the invention, or in the absence and presence of an antigen-binding site or antigen-binding fragment of the invention, using any method known in the art (e.g., flow cytometry, immunohistochemistry, etc.). + Depletion can be assessed by measuring the number of cells. After exposure to the antibody or antigen-binding fragment of the invention, CXCR3 + The cells may be depleted by at least or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. An antigen-binding site capable of depletion for CXCR3+ cells may be referred to as a depletable antigen-binding site.

[0107] The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that, by virtue of its origin or source from which it is derived, is not associated with naturally associated components that accompany it in its native state; it is substantially free of other proteins from the same source. A protein may be rendered substantially free of naturally associated components by isolation using protein purification techniques known in the art, or may be substantially purified by such isolation. "Substantially purified" means that the protein is substantially free of contaminants, e.g., at least about 70% or 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% removed from contaminants.

[0108] The term "recombinant" shall be understood to mean a product of artificial genetic recombination. Thus, with respect to a recombinant protein containing an antibody antigen-binding domain, this term does not include naturally occurring antibodies in a subject's body that are the product of natural recombination occurring during B-cell maturation. However, when such an antibody is isolated, it is considered to be an isolated protein containing an antibody antigen-binding domain. Similarly, when a nucleic acid encoding a protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein containing an antibody antigen-binding domain. Recombinant protein also includes a protein expressed by artificial recombinant means when it is present within a cell, tissue, or subject, e.g., where it is expressed.

[0109] The term "protein" shall be understood to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains that are covalently or non-covalently bonded to one another (i.e., a polypeptide complex). For example, a series of polypeptide chains may be covalently bonded using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0110] It will be understood from the previous paragraph that the term "polypeptide" or "polypeptide chain" means a series of consecutive amino acids linked by peptide bonds.

[0111] As used herein, the term "antigen-binding site" is used synonymously with "antigen-binding domain" and refers to a region that binds an antigen, i.e., a VH or VL or both a VH and a VL. The term "antigen-binding domain" should be understood to mean a region of an antibody capable of specifically binding to an Fv, including an antigen-binding domain. The antigen-binding domain need not be in the context of a complete antibody, for example it may be isolated (e.g., a domain antibody) or in another form, such as, for example, an scFv, as described herein.

[0112] For the purposes of this disclosure, the term "antibody" includes proteins capable of specifically binding to one or several closely related antigens (e.g., CXCR3) by means of an antigen-binding domain contained in the Fv. The term includes four-chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, de-immunized antibodies, synhumanized antibodies, half antibodies, bispecific antibodies). Antibodies generally include a constant domain that may be organized into a constant region or constant fragment or crystallizable fragment (Fc). Exemplary forms of antibodies include a four-chain structure as their basic unit. Full-length antibodies include two covalently linked heavy chains (about 50-70 kDa) and two light chains (each about 23 kDa). The light chains generally include a variable region (if present) and a constant domain, and in mammals are either kappa or lambda light chains. Heavy chains generally comprise a variable region and one or two constant domains linked by a hinge region to further constant domains. Mammalian heavy chains are of one of the following types: α, δ, ε, γ, or μ. Each light chain is also covalently linked to one of the heavy chains. For example, two heavy chains as well as heavy and light chains are linked by interchain disulfide bonds and non-covalent interactions. The number of interchain disulfide bonds can vary with different types of antibodies. Each chain has an N-terminal variable region (VH or VL, each about 110 amino acids long) and one or more constant domains at the C-terminus. The constant domain of the light chain (CL, about 110 amino acids long) is aligned and disulfide-linked to the first constant domain of the heavy chain (CH1, 330-440 amino acids long). The light chain variable region is aligned with the variable region of the heavy chain. An antibody heavy chain can include two or more additional CH domains (CH2, CH3, etc.) and can include a hinge region between the CH1 and CH2 constant domains. The antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. In one example, the antibody is a murine (mouse or rat) antibody or a primate (such as human) antibody.In one example, the antibody heavy chain lacks a C-terminal lysine residue, hi one example, the antibody is humanized, synthetic humanized, chimeric, CDR-grafted or deimmunized.

[0113] The terms "full length antibody," "intact antibody," or "complete antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen-binding fragment of an antibody. Specifically, complete antibodies include those having heavy and light chains, including the Fc region. The constant domains may be wild-type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

[0114] As used herein, "variable region" refers to the portion of the light and / or heavy chain of an antibody as defined herein that can specifically bind to an antigen, and includes the amino acid sequences of the complementarity determining regions (CDRs); i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, a variable region includes three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) together with three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0115] As used herein, the term "complementarity determining region" (synonyms are CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable region whose presence is primarily responsible for specific antigen binding. Each variable region domain (VH or VL) typically has three CDRs identified as CDR1, CDR2, and CDR3. The CDRs of VH are also referred to herein as CDR H1, CDR H2, and CDR H3, respectively, where CDR H1 corresponds to CDR 1 of VH, CDR H2 corresponds to CDR 2 of VH, and CDR H3 corresponds to CDR 3 of VH. Similarly, the CDRs of VL are also referred to herein as CDR L1, CDR L2, and CDR L3, respectively, where CDR L1 corresponds to CDR 1 of VL, CDR L2 corresponds to CDR 2 of VL, and CDR L3 corresponds to CDR 3 of VL. In one example, the amino acid positions assigned to the CDRs and FRs are determined according to the Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as the "Kabat numbering system"). In the present invention, the amino acid positions assigned to the CDRs and FRs are defined according to the Enhanced Chothia Numbering Scheme (http: / / www.bioinfo.org.uk / mdex.html). The present invention is not limited to FRs and CDRs defined by the Kabat numbering system, but may be used in conjunction with any standard numbering scheme. or any numbering system, including the numbering system of Chothia and Lesk J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:877-883, 1989; and / or Al-Lazikani et al., J. Mol. Biol. 273:927-948, 1997; Honnegher and Pluekthun J. Mol. Biol. 309:657-670, 2001; or the IMGT system as described in Giudicelliet al., Nucleic Acids Res. 25:206-211 1997. In one example, the CDRs are defined according to the Kabat numbering system. Optionally, the heavy chain CDR2 according to the Kabat numbering system comprises any of the five CDRs listed herein. The terminal amino acids are not included, or any one or more of these amino acids are replaced with another naturally occurring amino acid. In this regard, Padlan et al., FASEB J., 9:133-139, 1995, It is well established that the five C-terminal amino acids of CDR2 are generally not involved in antigen binding.

[0116] "Framework Regions" (FRs) are the variable region residues other than the CDR residues. The FRs of VH are also referred to herein as FR H1, FR H2, FR H3, and FR H4, respectively, where FR H1 corresponds to FR 1 of VH, FR H2 corresponds to FR 2 of VH, FR H3 corresponds to FR 3 of VH, and FR H4 corresponds to FR 4 of VH. Similarly, the FRs of VL are also referred to herein as FR L1, FR L2, FR L3, and FR L4, respectively, where FR L1 corresponds to FR 1 of VL, FR L2 corresponds to FR 2 of VL, FR L3 corresponds to FR 3 of VL, and FR L4 corresponds to FR 4 of VL.

[0117] As used herein, the term "Fv" shall be understood to mean any protein, whether composed of multiple polypeptides or a single polypeptide, in which the VL and VH associate to form a complex having an antigen-binding domain, i.e., capable of specifically binding to an antigen. The VH and VL forming the antigen-binding domain may be in a single polypeptide chain or in different polypeptide chains. Furthermore, the Fv of the present invention (as well as any protein of the present invention) may have multiple antigen-binding domains that may or may not bind to the same antigen. The term shall be understood to encompass fragments derived directly from antibodies as well as proteins corresponding to such fragments produced using recombinant means. In some instances, the VH is not linked to the heavy chain constant domain (CH)1 and / or the VL is not linked to the light chain constant domain (CL). Exemplary Fv-comprising polypeptides or proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFv, diabodies, triabodies, tetrabodies or higher complexes, or any of the above linked to a constant region or domains thereof, e.g., CH2 or CH3 domains, e.g., minibodies. A "Fab fragment" consists of a monovalent antigen-binding fragment of an immunoglobulin and can be produced by digestion of a whole antibody with the enzyme papain to yield a fragment consisting of an intact light chain and a portion of the heavy chain, or can be produced using recombinant means. An "Fab' fragment" of an antibody can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of the heavy chain including the VH and a single constant domain. Two Fab' fragments is obtained for each antibody treated in this manner. Fab' fragments can also be produced by recombinant means. An "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments linked by two disulfide bonds and can be obtained by treating an intact antibody molecule with the enzyme pepsin without subsequent reduction. A "Fab2" fragment is a recombinant fragment comprising two Fab fragments linked, for example, by a leucine zipper or CH3 domain. A "single-chain Fv" or "scFv" is a recombinant molecule comprising an antibody variable fragment (Fv) in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable flexible polypeptide linker.

[0118] As used herein, the term "bind" in reference to the interaction of an antigen-binding site or antigen-binding domain thereof with an antigen means that this interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody recognizes and binds to a particular protein structure, rather than protein in general. If an antibody binds to epitope "A", the presence of a molecule containing epitope "A" (or free, unlabeled "A") will reduce the amount of labeled "A" bound to the antibody in a reaction involving labeled "A" and a protein.

[0119] As used herein, the term "specifically binds" or "binds specifically" shall be understood to mean that the antigen-binding site described herein reacts or associates with a particular antigen or cell expressing it more frequently, more rapidly, for a longer period of time, and / or with a higher affinity than with another antigen or cell. For example, the antigen-binding site binds to CXCR3 (e.g., hCXCR3) with a substantially higher affinity (e.g., 1.5-fold or 2-fold or 5-fold or 10-fold or 20-fold or 40-fold or 60-fold or 80-fold to 100-fold or 150-fold or 200-fold) than other CXCRs. In one example of the present invention, the antigen-binding site "specifically binds" to CXCR3 (preferably human) with at least 1.5-fold or 2-fold or more higher affinity (e.g., 5-fold or 10-fold or 20-fold or 50-fold or 100-fold or 200-fold) than another chemokine receptor, such as CXCR1, CXCR2, and / or C5aR. Generally, but not necessarily, reference to binding refers to specific binding, and it will be understood that each term gives explicit support to the other term.

[0120] As used herein, the term "does not detectably bind" shall be understood to mean that the antigen binding site, e.g., antibody, binds to the candidate antigen at a level less than 10%, or 8%, or 6%, or 5% above background. Background may be the level of binding signal detected in the absence of protein and / or in the presence of a negative control protein (e.g., an isotype control antibody), and / or the level of binding detected in the presence of a negative control antigen. The level of binding is detected using a biosensor assay (e.g., Biacore) in which the antigen binding site is immobilized and contacted with the antigen.

[0121] As used herein, the term "does not bind significantly" shall be understood to mean that the level of binding of an antigen-binding site described herein to a polypeptide is not statistically significantly higher than the background, e.g., the level of binding signal detected in the absence of the antigen-binding site and / or in the presence of a negative control protein (e.g., an isotype control antibody), and / or the level of binding detected in the presence of a negative control polypeptide. The level of binding is detected using a biosensor assay (e.g., Biacore) in which the antigen-binding site is immobilized and contacted with the antigen.

[0122] As used herein, the term "epitope" (synonymous with "antigenic determinant") shall be understood to mean the region of CXCR3 to which an antigen-binding site, including the antigen-binding domain of an antibody, binds. Unless otherwise defined, this term refers to the region of CXCR3 to which an antigen-binding site contacts The term epitope is not necessarily limited to the particular residues or structures that contact the antigen-binding site. For example, the term includes the region spanning the amino acids contacted by the antigen-binding site, and 5-10 (or more), or 2-5, or 1-3 amino acids outside of this region. In some instances, an epitope includes a series of discontinuous amino acids that are located near each other when the antigen-binding site folds, i.e., a "conformational epitope." One of skill in the art will also recognize that the term "epitope" is not limited to peptides or polypeptides. For example, the term "epitope" includes chemically active surface groups such as sugar, phosphoryl, or sulfonyl side chains, and in certain instances, may have specific three-dimensional structural features, and / or specific charge characteristics.

[0123] As used herein, the term "disease" refers to a disruption or interference with normal function and includes a disease or disorder, without being limited to any particular disease.

[0124] As used herein, the terms "preventing," "prevent" or "prevention" include administering an antigen binding site described herein, thereby arresting or preventing the occurrence of at least one symptom of a disease. The term also encompasses treatment of a subject in remission to prevent or prevent recurrence.

[0125] As used herein, the terms "treating," "treat" or "treatment" include administering an antigen binding site described herein, thereby reducing or eliminating at least one symptom of a particular disease or disorder.

[0126] As used herein, the term "subject" shall be understood to mean any animal, including humans, e.g., mammals. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0127] antibody In one example, the antigen binding site or CXCR3 binding protein described herein according to any embodiment is an antibody.

[0128] Anti-CXCR3 antibodies: Various anti-CXCR3 antibodies are commercially available, including but not limited to: 1C6 (anti-human CXCR3) or clone 173 (anti-mouse CXCR3), both of which are available from Biolegend, BD Biosciences, AbCam or other sources. The antibody may be an antibody described in WO 2018 / 119299.

[0129] Methods for producing antibodies are known in the art and / or described in Harlow and Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). Generally, in such methods, CXCR3 (e.g., hCXCR3) or a region thereof (e.g., an extracellular region) or an immunogenic fragment or epitope thereof or a cell expressing and presenting it (i.e., an immunogen) is optionally formulated with any suitable or desired carrier, adjuvant, or pharma- ceutically acceptable excipient and administered to a non-human animal, e.g., a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat, or pig. The immunogen may be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by other known routes.

[0130] The production of polyclonal antibodies can be monitored by sampling the blood of the immunized animal at various times after immunization. One or more further immunizations can be given as necessary to obtain the desired antibody titer. The process of boosting and titering is repeated until a suitable titer is obtained. When a desired level of immunogenicity is obtained, the immunized animal is bled and the serum isolated and stored, and / or the animal is used to produce monoclonal antibodies (mAbs). )

[0131] Monoclonal antibodies are one exemplary form of antibody contemplated by the present invention. The term "monoclonal antibody" or "mAb" refers to a homogeneous antibody population capable of binding to the same antigen, e.g., the same epitope within an antigen. The term is not intended to be limited with respect to the origin of the antibody or the method by which it is made.

[0132] For production of mAbs, any one of a number of well-known techniques can be used, such as, for example, the procedures exemplified in U.S. Pat. No. 4,196,265, supra, or Harlow and Lane (1988). It is possible.

[0133] For example, a suitable animal is immunized with an immunogen under conditions sufficient to stimulate antibody-producing cells.Rodents such as rabbits, mice and rats are exemplary animals.For example, mice that do not express murine antibodies but are genetically modified to express human antibodies can also be used to produce the antibodies of the present invention (e.g., as described in WO2002 / 066630).

[0134] Following immunization, somatic cells with the potential to produce antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells are obtained from biopsies of the spleen, tonsils or lymph nodes, or from a peripheral blood sample. The B cells from the immunized animal are then fused with immortal myeloma cells, generally derived from the same species as the animal immunized with the immunogen.

[0135] The hybrids are amplified by culture in selective media containing drugs that block de novo synthesis of nucleotides in tissue culture media. Exemplary drugs are aminopterin, methotrexate and azaserine.

[0136] The amplified hybridomas are subjected to functional selection for antibody specificity and / or titer, for example, by flow cytometry and / or immunohistochemistry and / or immunoassays (e.g., radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunoassays, etc.).

[0137] Alternatively, to generate cell lines secreting MAbs, ABL-MYC technology (NeoClone, Madison WI) was used. 53713, USA) is used (e.g., as described in Largaespada et al, J. Immunol. Methods. 197:85-95, 1996).

[0138] Antibodies can also be produced or isolated by screening a display library, e.g., a phage display library, as described, e.g., in U.S. Patent No. 6,300,064 and / or U.S. Patent No. 5,885,793. For example, the present inventors have isolated fully human antibodies from a phage display library.

[0139] The antibody of the invention may be a synthetic antibody, for example, the antibody is a chimeric antibody, a humanized antibody, a human antibody, a synthetic humanized antibody, a primatized antibody or a deimmunized antibody.

[0140] Proteins containing antibody-binding domains Single Domain Antibodies In some instances, the proteins of the invention are or comprise single domain antibodies (used interchangeably with the terms "domain antibody" or "dAb"). Single domain antibodies are single polypeptide chains that comprise all or a portion of the heavy chain variable region of an antibody. In particular instances, single domain antibodies are human single domain antibodies (Domantis, Inc., Walt ham, MA; see, e.g., U.S. Patent No. 6,248,516).

[0141] Diabodies, triabodies, tetrabodies In some instances, the proteins of the invention are or comprise diabodies, triabodies, tetrabodies or higher order protein complexes such as those described in WO 98 / 044001 and / or WO 94 / 007921.

[0142] For example, a diabody is a protein that includes two associated polypeptide chains, each of which has the structure V L -XV H Or V H -XV L where V L is an antibody light chain variable region, and V H is an antibody heavy chain variable region, and X is a V H and VL A linker that contains insufficient residues to allow association of the V of one polypeptide chain (or to form an Fv) or is absent. H V of another polypeptide chain L to form an antigen-binding domain, i.e., an Fv molecule capable of specifically binding to one or more antigens. L and V H may be the same in each polypeptide chain, or V L and V H can be different in each polypeptide chain, thereby forming a bispecific diabody (ie, containing two Fvs with different specificities).

[0143] Single chain Fv (scFv) Those skilled in the art will appreciate that scFvs are composed of V H and V L The V domain, together with the scFv, forms the desired structure for antigen binding (i.e., the V domain of a single polypeptide chain). H and V L V allows V to associate with each other to form Fv H and V L It will be appreciated that the linker may include a polypeptide linker between the nucleotides 1 and 2. For example, the linker may include more than 12 amino acid residues, with (Gly4Ser)3 being one of the more preferred linkers for scFvs.

[0144] The present invention also contemplates disulfide-stabilized Fvs (or diFvs or dsFvs), in which a single cysteine ​​residue is located at the V H FR and V L and this cysteine ​​residue is linked by a disulfide bond to generate a stable Fv.

[0145] Alternatively, or in addition, the invention encompasses a dimeric scFv, i.e., a protein comprising two scFv molecules non-covalently or covalently linked, for example, by a leucine zipper domain (e.g., from Fos or Jun), or the two scFvs are linked by a peptide linker of sufficient length to allow both scFvs to form and bind to the antigen, as described, for example, in US Patent Publication No. 20060263367.

[0146] Heavy Chain Antibodies Heavy chain antibodies are structurally different from many other forms of antibodies in that they contain heavy chains but no light chains. Thus, these antibodies are also called "heavy chain-only antibodies". Heavy chain antibodies are found, for example, in camelids and cartilaginous fish (also called IgNAR).

[0147] The heavy chain variable region (V H The light chain variable region (called the “V domain”) present in conventional four-chain antibodies L The variable regions present in native heavy chain antibodies are generally referred to as "V domains" in camelid antibodies to distinguish them from the variable regions present in native heavy chain antibodies (called "V domains" in camelid antibodies). HH In IgNARs, these are referred to as "domains" and in IgNARs, V-NARs.

[0148] General descriptions of camelid-derived heavy chain antibodies and variable regions thereof and methods for their production and / or isolation and / or use can be found, inter alia, in the following references WO 94 / 04678, WO 97 / 49805 and WO 97 / 49805.

[0149] A general description of cartilaginous fish-derived heavy chain antibodies and their variable regions, as well as methods for their production and / or isolation and / or use, can be found, inter alia, in WO 2005 / 118629.

[0150] Other antibodies and proteins containing their antigen-binding domains The present invention also provides (i) "key and hole" bispecific proteins as described in U.S. Pat. No. 5,731,168; (ii) heteroconjugate proteins (e.g., as described in U.S. Pat. No. 4,676,980); (iii) heteroconjugate proteins produced using chemical cross-linkers (e.g., as described in U.S. Pat. No. 4,676,980); and (iv) Fab3 (e.g., as described in EP19930302894) Other antibodies and proteins comprising antigen-binding domains thereof, such as, are contemplated.

[0151] Mutations to proteins Percent identity of nucleic acids or polypeptides is calculated using GAP (Needleman and Wunsch. Mol. Biol. 48, 443-453, 1970) with a gap creation penalty of 5 and a gap extension penalty of 0.3. ) analysis (GCG program). The query sequence is at least 50 residues long, and the GAP analysis aligns the two sequences over a region of at least 50 residues. For example, the query sequence is at least 100 residues long, and the GAP analysis aligns the two sequences over a region of at least 100 residues. For example, the two sequences are aligned over their entire lengths.

[0152] The present invention also contemplates nucleic acids that hybridize under stringent hybridization conditions to nucleic acids encoding antigen-binding sites described herein. "Moderate stringency" is defined herein as hybridization and / or washing in 2xSSC buffer, 0.1% (w / v) SDS, at a temperature ranging from 45°C to 65°C, or equivalent conditions. "High stringency" is defined herein as hybridization and / or washing in 0.1xSSC buffer, 0.1% (w / v) SDS, or at a lower salt concentration, and at a temperature of at least 65°C, or equivalent conditions. Reference herein to a particular level of stringency encompasses equivalent conditions using washing / hybridization solutions other than SSC known to those of skill in the art. For example, methods for calculating the temperature at which the strands of a double-stranded nucleic acid dissociate (also known as the melting temperature, or Tm) are known in the art. A temperature similar (e.g., within 5° C. or within 10° C.) or equal to the Tm of the nucleic acid is considered high stringency. Moderate stringency should be considered to be within 10° C.-20° C. or 10° C.-15° C. of the calculated Tm of the nucleic acid.

[0153] The present invention also contemplates mutant forms of the antigen binding sites described herein that contain one or more conservative amino acid substitutions compared to the sequences described herein. In some examples, the antigen binding site contains up to 10, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain and / or hydropathicity and / or hydrophilicity.

[0154] Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamic acid), and cyclic side chains (e.g., cyclic acid ... Hydropathic indexes include, for example, polar side chains (e.g., threonine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). The hydropathic index is described, for example, in Kyte and Doolittle J. Mol. Biol., 157:105-132, 1982, and the hydrophilicity index is described, for example, in U.S. Pat. No. 4,554,101. It is being done.

[0155] The present invention also contemplates non-conservative amino acid mutations. For example, substitution of a charged amino acid with another charged amino acid, and substitution with a neutral or positively charged amino acid are of particular interest. In some instances, the antigen-binding site contains 10 or less, for example, 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 non-conservative amino acid substitutions.

[0156] In one example, the mutations occur within the FRs of the antigen-binding domain of the antigen-binding site described herein. In another example, the mutations occur within the CDRs of the antigen-binding site described herein.

[0157] Exemplary methods for generating mutant forms of an antigen-binding site include: DNA mutagenesis (Thie et al., Methods Mol. Biol. 525:309-322, 2009) or R NA (Kopsidas et al., Immunol. Lett. 107:163-168, 2006; Kopsidas et al. BMC Biotechnology, 7:18, 2007; and WO 1999 / 058661); Introduction of a nucleic acid encoding a polypeptide into a mutagenized cell, e.g., XL-1Red, XL-mutS and XL-mutS-Kanr bacterial cells (Stratagene); DNA shuffling (e.g., as disclosed in Stemmer, Nature 370:389-91, 1994); and Site-directed mutagenesis (e.g., Dieffenbach (ed) and Dveksler (ed) (PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995).

[0158] Exemplary methods for determining the biological activity of the mutant antigen binding sites described herein will be apparent to one of skill in the art and / or are described herein, e.g., for antigen binding. For example, methods for determining antigen binding, competitive inhibition of binding, affinity, association, dissociation and therapeutic effect are described herein.

[0159] Constant region The present invention encompasses antigen-binding sites and / or antibodies described herein that comprise the constant region of an antibody, including antigen-binding fragments of an antibody fused to Fc.

[0160] The sequences of constant regions useful for producing the proteins of the invention can be obtained from several different sources. In some instances, the constant region of the protein or a portion thereof is derived from a human antibody. The constant region or a portion thereof can be derived from any antibody class, including IgM, IgG, IgD, IgA, and IgE, as well as any antibody isotype, including IgG1, IgG2, and IgG3.

[0161] In one example, the Fc region of the constant region has an enhanced ability to induce effector function, e.g., compared to a native or wild-type human IgG1 or IgG3 Fc region. In one example, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). Methods for assessing the level of effector function of an Fc region containing protein are known in the art and / or described herein.

[0162] In any embodiment of the invention, the antigen binding site comprises an Fc region that has been modified to have an enhanced ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Preferably, the enhanced ability to induce ADCC is conferred by mutation, deletion or modification of amino acids in the Fc region that interact with the Fc receptor. Preferably, the mutated, deleted or modified amino acids are at positions 239, 330 and / or 332 or equivalent positions 239, 330 and / or 332 according to SEQ ID NO: 1 (wherein alanine is at position 118). Preferably, the amino acids are mutated to S239D, A330L and I332E. Typically, the Fc comprises, consists essentially of or consists of the amino acid sequence shown in SEQ ID NO:3.

[0163] In any embodiment of the invention, the antigen binding site comprises an Fc region that has not been modified to have a reduced ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC), preferably wherein the amino acids at or equivalent to positions 234, 235 and / or 331 according to SEQ ID NO:1 (wherein alanine is at position 118) are not F, E and / or S, respectively. In other words, the amino acid at position 234 is not F, the amino acid at position 235 is not E and / or the amino acid at position 331 is not S.

[0164] In any embodiment of the invention, the antigen binding site does not include an Fc region comprising, consisting essentially of or consisting of the amino acid sequence set forth in SEQ ID NO:2.

[0165] In any embodiment of the invention, the antigen-binding site described herein may itself have little or no ability to reduce the activity or viability of CXCR3+ cells, but it may be conjugated or associated with a compound that has the ability to reduce the activity or viability of the cells. For example, the Fc region may be conjugated or associated with a compound that has the ability to reduce the activity or viability of the cells, such as a native or wild-type human IgG1 or IgG3. It may have a reduced ability or function to induce effector functions compared to the Fc region, but in this situation the antigen binding site is conjugated or associated with a cytotoxic compound.

[0166] One example of an Fc region with reduced effector function is an IgG4 Fc region (i.e. derived from an IgG4 constant region), e.g., a human IgG4 Fc region. The sequences of suitable IgG4 Fc regions will be apparent to the skilled artisan and / or available in publicly accessible databases (e.g., available from the National Center for Biotechnology Information).

[0167] In another example, the Fc region is a region modified to have reduced effector function, i.e., a "non-immunostimulatory Fc region." For example, the Fc region is an IgG1 Fc region containing substitutions at one or more positions selected from the group consisting of 268, 309, 330, and 331. In another example, the Fc region is an IgG1 Fc region containing one or more of the following mutations E233P, L234V, L235A, and a deletion of G236, and / or one or more of the following mutations A327G, A330S, and P331S (Armour et al., Eur J Immunol. 29:2613-2624, 1999; Shields et al., J BiolChem. 276(9):6591-604, 2001). Further examples of stimulatory Fc regions are described, for example, in Dall'Acqua et al., J Immunol. 177:1129-1138 2006; and / or Hezareh J Virol; 75:12161-12168, 2001).

[0168] In another example, the Fc region can comprise at least one C domain, e.g., from an IgG4 antibody. H 2 domains and at least one C from an IgG1 antibody Ha chimeric Fc region comprising three domains, wherein the Fc region is selected from the group consisting of 240, 262, 264, 266, 297, 299, 307, 309, 323, 399, 409 and 427 (EU numbering). (e.g., as described in WO 2010 / 085682). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.

[0169] In any of these examples, the antigen binding site with reduced effector function is conjugated or associated with a compound that can reduce the activity or viability of a cell.

[0170] Further modifications The present invention also contemplates further modifications to the antibodies or antigen binding sites, including the Fc region or constant region.

[0171] For example, the antibody comprises one or more amino acid substitutions that increase the half-life of the protein. For example, the antibody comprises an Fc region that comprises one or more amino acid substitutions that increase the affinity of the Fc region to neonatal Fc region (FcRn). For example, the Fc region has an increased affinity for FcRn at lower pH, e.g., about pH 6.0, thereby promoting Fc / FcRn binding in endosomes. In one example, the Fc region has an increased affinity for FcRn at about pH 6 compared to its affinity at about pH 7.4, thereby promoting re-release of Fc into blood after cell recycling. These amino acid substitutions are useful for extending the half-life of the protein by reducing clearance from blood.

[0172] Exemplary amino acid substitutions include T250Q and / or M428L or T252A, T254S and T266F or M252Y, S254T and T256E or H433K and N434F according to the EU numbering system. Additional or alternative amino acid substitutions are described, for example, in U.S. Patent Application Publication No. 20070135620 or U.S. Patent No. 7,083,784.

[0173] Protein production In one example, an antigen binding site described herein according to any of the Examples is produced by culturing a hybridoma under conditions sufficient to produce the protein, e.g., as described herein and / or known in the art.

[0174] Recombinant Expression In another example, the antigen binding site described herein according to any of the embodiments is recombinant.

[0175] In the case of recombinant proteins, the nucleic acid encoding it can be cloned into an expression construct or vector, which is then transfected into a host cell that does not naturally produce the protein, such as an E. coli cell, a yeast cell, an insect cell, or a mammalian cell, such as a Simian COS cell, a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a myeloma cell. Exemplary cells used to express proteins are CHO cells, myeloma cells, or HEK cells. Molecular cloning techniques to achieve these ends are known in the art and are described, for example, in Ausubel et al., (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all current editions) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A wide variety of Cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Methods for producing recombinant antibodies are also known in the art, see, for example, U.S. Patent No. 4,816,567 or U.S. Patent No. 5,530,101.

[0176] After isolation, the nucleic acid is operably linked and inserted into a promoter or expression vector in an expression construct for further cloning (amplification of the DNA) or for expression in a cell-free system or in a cell.

[0177] As used herein, the term "promoter" should be interpreted in its broadest sense and includes the transcriptional regulatory sequences of genomic genes, including the TATA box or initiation elements, which are required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter the expression of the nucleic acid, for example, in response to developmental and / or external stimuli or in a tissue-specific manner. In the context of this specification, the term "promoter" is also used to refer to a recombinant, synthetic or fusion nucleic acid, or derivatives thereof, that confers, activates or enhances the expression of a nucleic acid to which it is operably linked. Exemplary promoters may contain additional copies of one or more specific regulatory elements to further enhance expression and / or alter the spatial and / or temporal expression of said nucleic acid.

[0178] As used herein, the term "operably linked" means positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter.

[0179] Many vectors are available for expression in cells. The components of a vector generally include, but are not limited to, one or more of the following: signal sequence, protein coding sequence (e.g., obtained from the information provided herein), enhancer element, promoter, and transcription termination sequence. Those skilled in the art will recognize the appropriate sequence for protein expression. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, alpha-factor leader, or acid phosphatase leader) or mammalian secretion signals (e.g., herpes simplex gD signal).

[0180] Exemplary promoters active in mammalian cells include the cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1-alpha promoter (EF1), small nuclear RNA promoters (U1a and U1b), alpha-myosin heavy chain promoter, simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), adenovirus major late promoter, β-actin promoter; hybrid regulatory elements comprising the CMV enhancer / β-actin promoter or immunoglobulin promoter or active fragments thereof. Examples of useful mammalian host cell lines are the monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 cells or 293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

[0181] Exemplary promoters suitable for expression in yeast cells, such as yeast cells selected from the group including Pichia pastoris, Saccharomyces cerevisiae, and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PHO5 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

[0182] Means for introducing isolated nucleic acids or expression constructs containing same into cells for expression are known to those of skill in the art. The technique used for a given cell will depend on known established techniques. Means for introducing recombinant DNA into cells include, inter alia, microinjection, DEAE-dextran mediated transfection, liposome mediated transfection using, for example, lipofectamine (Gibco, MD, USA) and / or cellfectin (Gibco, MD, USA), PEG mediated DNA uptake, electroporation and microparticle bombardment using, for example, DNA coated tungsten or gold particles (Agracetus Inc., WI, USA).

[0183] Host cells used to produce proteins may be cultured in a variety of media, depending on the type of cell used. Ham's F10 (Sigma), Minimum Essential Medium (MEM), (Sigma) Commercially available media such as RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM, Sigma) were used. The commercially available media are suitable for culturing mammalian cells. Media for culturing the other cell types described herein are known in the art.

[0184] Protein isolation Methods for isolating proteins are known in the art and / or described herein.

[0185] If the antigen-binding site is secreted into the culture medium, the supernatant from such an expression system is first concentrated using a commercially available protein concentration filter, e.g., an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of incidental contaminants. Alternatively, or in addition, the supernatant may be filtered and / or separated from the cells expressing the protein, e.g., using continuous centrifugation.

[0186] Antigen binding sites prepared from cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., Protein A affinity chromatography or Protein G chromatography), or any combination of the above. These methods are known in the art and described, for example, in WO 99 / 57134 or in Ed Harlow and David Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).

[0187] Those skilled in the art will also recognize that proteins can be modified to include tags for facilitating purification or detection, such as polyhistidine tags, such as hexahistidine tags, or influenza virus hemagglutinin (HA) tags, or simian virus 5 (V5) tags, or FLAG tags, or glutathione S-transferase (GST) tags. The resulting proteins are then purified using methods known in the art, such as affinity purification. For example, proteins that include hexahis tags are purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds to the hexahis tag and is immobilized on a solid or semi-solid support, washing the sample to remove unbound proteins, and then eluting the bound proteins. Alternatively, or in addition, a ligand or antibody that binds to the tag is used in the affinity purification method.

[0188] Assay of antigen-binding site activity Binding to CXCR3 and its mutants It will be apparent to those skilled in the art from the disclosure herein that the antigen-binding site described herein binds to CXCR3. Methods for evaluating binding to proteins are known in the art and are described, for example, in Scopes (Protein purification: principles and principles) and the like. This method is described in the Journal of the American Chemical Society, Third Edition, Springer Verlag, 1994. Generally, it involves immobilizing the antigen binding site and contacting it with labeled antigen (CXCR3). After washing to remove non-specifically bound proteins, the amount of label and, consequently, bound antigen is detected. Of course, the antigen binding site may be labeled and the antigen may be immobilized. Panning-type assays may also be used. Alternatively, or in addition, surface plasmon resonance assays may be used.

[0189] Optionally, the dissociation constant (Kd), association constant (Ka) and / or affinity constant (K) of the immobilized antigen binding site for CXCR3 or an epitope thereof are D ) is determined. D " is measured in one example by a radioactively or fluorescently labeled CXCR3 ligand binding assay. In the case of "Kd", the assay equilibrates the antigen binding site with a minimal concentration of labeled CXCR3 or its epitope in the presence of a titration series of unlabeled CXCR3. After washing to remove unbound CXCR3 or its epitope, the amount of label, which is an indication of the Kd of the protein, is determined.

[0190] In another example, Kd, ​​Ka or K D The present invention relates to a method for detecting CXCR3 or a region thereof using a surface plasmon resonance assay, for example, using a BIAcore surface plate having immobilized CXCR3 or a region thereof or an immobilized antigen binding site. It is measured using Raman resonance (BIAcore, Inc., Piscataway, NJ).

[0191] Measurement of inhibitory activity In some examples of the present invention, the protein is capable of inhibiting CXCR3 activity.

[0192] A variety of assays are known in the art for assessing the ability of a protein to inhibit or reduce ligand signaling through a receptor that results in a functional response.

[0193] In one example, the antigen-binding site inhibits migration of immune cells expressing CXCR3 (e.g., neutrophils) cultured in the presence of a CXCR3 ligand (e.g., MIG, IP-10, and I-TAC). Methods for assessing migration are known in the art and described herein. An antigen-binding site that inhibits migration compared to levels observed in the absence of the antigen-binding site is considered to inhibit or reduce CXCR3 activity, particularly CXCR3-mediated signaling and migration. Other assays for measuring the inhibitory activity of anti-CXCR3 antibodies include calcium flux assays, radioligand binding assays, cAMP assays, and beta-arrestin recruitment assays. Any of the assays described herein may include one or more CXCR3 ligands, including MIG, IP-10, and I-TAC.

[0194] Disease being treated The antigen binding sites of the present invention are useful in the treatment or prevention of any disease associated with or caused by the presence or overexpression of CXCR3.

[0195] The antigen binding sites of the present invention are useful in the treatment or prevention of any disease associated with or caused by Th1 cells.

[0196] The antigen-binding sites of the present invention are useful in the treatment or prevention of diseases associated with or caused by acute or chronic inflammation, such as fibrosis, fatty liver disease (including NASH) and transplant rejection (graft-versus-host disease, GVHD). The antigen-binding sites of the present invention are preferably used in the treatment or prevention of autoimmune inflammatory diseases.

[0197] In yet other embodiments, the antigen-binding site of the invention is characterized by inflammation. It may be used to reduce fibrosis associated with autoimmune diseases.

[0198] "Fibrosis", "Fibrotic disease" or "fibroproliferative disease" refers to the formation of excess fibrous connective tissue during the repair process upon injury. Scarring is the result of persistent fibrosis clearing the affected organ or tissue structure. Fibrosis progresses further as a result of abnormal repair processes that do not clear the scar tissue that has formed. Fibrosis is found in a variety of tissues, including the heart, lungs, liver, skin, blood vessels and kidneys. Examples of fibrosis are described herein and include pulmonary fibrosis, liver cirrhosis, systemic sclerosis, progressive kidney disease and myocardial fibrosis associated with various cardiovascular diseases.

[0199] A subject can be identified as having fibrosis by determining whether the subject has organ dysfunction, scarring, an alteration in normal extracellular matrix balance, increased collagen deposition, increased collagen volume fraction, differentiation of fibroblasts into myofibroblasts, decreased levels of matrix metalloproteinases and increased levels of tissue inhibitors of matrix metalloproteinases, increased levels of either the N-terminal or C-terminal propeptide of type I procollagen (PINP or PICP) and decreased levels of the C-terminal telopeptide of type I collagen (CTP or CITP), increased collagen deposition and cardiac dysfunction as measured by various non-invasive imaging techniques, renal dysfunction as measured by increased proteinuria and albuminuria, decreased glomerular filtration rate, a doubling of plasma creatinine levels.

[0200] According to a preferred embodiment of the invention, liver fibrosis is due to chronic liver disease, hepatitis B virus infection, hepatitis C virus infection, hepatitis D virus infection, schistosomiasis, alcoholic or non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, obesity, diabetes, protein malnutrition, coronary artery disease, autoimmune hepatitis, cystic fibrosis, alpha-1-antitrypsin deficiency, primary biliary cirrhosis, drug reactions and exposure to toxins.

[0201] The term "liver fibrosis" refers to the formation or development of excess fibrous connective tissue (fibrosis) in the liver, which leads to the development of scar (fibrotic) tissue. The scar tissue replaces healthy tissue through the process of fibrosis, leading to subsequent cirrhosis of the liver. Liver fibrosis can be associated with nonalcoholic steatohepatitis (NASH).

[0202] Liver fibrosis includes, but is not limited to, cirrhosis and related diseases such as chronic viral hepatitis, nonalcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis (ASH), nonalcoholic steatohepatitis (NASH), primary biliary cirrhosis (PBC), biliary cirrhosis, and autoimmune hepatitis. Pulmonary fibrosis includes idiopathic pulmonary fibrosis (IPF) or idiopathic fibrosing alveolitis, chronic fibrosing interstitial pneumonia, interstitial lung disease (ILD), and diffuse parenchymal lung disease (DPLD), myocardial fibrosis, congestive heart failure, cardiomyopathies, impaired cardiac function after myocardial infarction; peripheral vascular disease; rheumatoid arthritis; glaucoma; age-related macular degeneration (wet AMD and dry AMD); emphysema, chronic obstructive pulmonary disease (COPD); multiple sclerosis; chronic asthma may also be prevented, treated, or ameliorated using the compositions, methods, or uses described herein.

[0203] Fatty Liver Disease and NASH The present invention also relates to methods for preventing the onset of, preventing the progression of, or treating fatty liver disease in an individual at risk of developing, or exhibiting symptoms of, fatty liver disease.

[0204] In certain preferred embodiments, the present invention provides a method for preventing the onset or progression of NASH in individuals at risk of developing or exhibiting symptoms of NASH. or a method for treating NASH.

[0205] Fatty liver disease, which develops without alcohol abuse, is increasingly recognized as a major health burden, especially when an inflammatory component is involved, such as in nonalcoholic steatohepatitis (NASH). Estimates based on imaging and biopsy studies suggest that approximately 20%-30% of adults in the United States and other Western countries have excess fat accumulation in the liver. Approximately 10% of these individuals, or a mere 2%-3% of adults, are estimated to meet the current diagnostic criteria for NASH. Persistent liver damage leads to progressive fibrosis and cirrhosis in approximately 30% of NASH patients. Diagnostic criteria for NASH continue to evolve and rely on histological findings of steatosis, hepatocellular injury (ballooning of hepatocytes, Mallory bodies), and patterns of fibrosis.

[0206] As used herein, NAFLD refers to non-alcoholic fatty liver disease. NAFLD, which is considered a non-life-threatening disease, is differentiated from NASH, which is a more severe form of NAFLD and is associated with increased mortality. The terms "non-alcoholic fatty liver disease" or "NAFLD", "non-alcoholic steatohepatitis" or "NASH" and "simple fatty liver" are each used with the meaning currently accepted by the scientific community. In general, NAFLD exists as a spectrum of histological changes. All stages of NAFLD have in common the accumulation of fat (steatosis) in hepatocytes. Simple fatty liver refers to hepatic steatosis without significant inflammation and hepatocyte injury, while NASH shows inflammation and hepatocyte injury, and sometimes fibrosis.

[0207] Those skilled in the art are familiar with methods for determining whether an individual suffers from or is at risk of developing non-alcoholic steatohepatitis (NASH).In particular, those skilled in the art are familiar with methods for determining whether an individual has symptoms or signs of non-alcoholic fatty liver disease (including whether an individual suffers from or is at risk of any of the subtypes of fatty liver disease, such as steatosis, NASH, or NAFDL), and in particular for distinguishing between life-threatening NASH and milder NAFLD.

[0208] The term "fatty liver disease" is well known in the art. Preferably, the term refers to damage to the liver. Preferably, said damage is the result of excess triacylglycerides accumulating in the liver and forming large vacuoles. Symptoms associated with fatty liver disease are well known from standard textbooks of medicine such as Stedman's or Pschyrembel. Fatty liver disease is a condition that is associated with the development of a fatty liver disease caused by alcoholism. The fatty liver disease in the present invention may be caused by drug abuse, diabetes, nutritional deficiencies and incorrect diet, drug toxicity or genetic predisposition. Fatty liver disease in the present invention also includes its more severe forms, in particular steatosis, NASH or NAFDL. The symptoms associated with these diseases are also well known to physicians and are described in detail in standard textbooks on medicine.

[0209] Liver biopsy remains the gold standard or standard in the evaluation of the etiology and extent of liver diseases such as NAFLD and NASH. Percutaneous liver biopsy is the preferred method for determining NAFLD and distinguishing NASH from NAFLD. Other biopsy methods are typically even more invasive, including intravenous and laparoscopic liver biopsy. The American Gastroenterological Association has published detailed recommendations on how to grade NAFLD, including NASH, into macrovesicular steatosis grade, necroinflammatory activity grade and fibrosis stage (American Gastroenterological Association 2002, Gastroenterology 123: 1705-25; Brunt 1999, Am J Gastroenterol. 94: 2467-74, Brunt 2010, Nat Rev Gastroenterol Hepatol. 7: 195-203).

[0210] Although simple fatty liver appears to be a relatively benign condition, it can progress to NASH over time. Therefore, individuals at risk for NASH may be diagnosed with simple fatty liver. In addition to its association with cardiovascular complications, NAFLD can result in liver-related morbidity and mortality. The risk of developing cirrhosis is higher in the presence of NASH, which is more likely in the presence of the following features: Type 2 diabetes (T2DM) Obesity (Body Mass Index [BMI]>30kg / m2) Age over 50 Serum aminotransferases (ALT or AST) greater than twice the upper limit of normal.

[0211] Thus, if a person is diagnosed with NAFLD and has one or more of the above characteristics, they may be considered to be at risk for NASH. A definitive diagnosis of NAFLD depends on three factors: 1) Evidence of fatty infiltration by either imaging (ultrasound, magnetic resonance imaging [MRI]) or histology (liver biopsy) 2) Exclusion of significant alcohol intake 3) exclusion of other reasons for hepatic steatosis (e.g., medications, surgery, metabolic disorders).

[0212] In addition to the above, signs of complications of cirrhosis may also be considered, including signs of portal hypertension (splenomegaly, increased portal vein size, varices) or other complications such as HCC, portal vein thrombosis, or ascites. The risk of fibrosis and progressive liver disease in NAFLD increases with the severity of insulin resistance. The number of features of the metabolic syndrome (obesity, combined hyperlipidemia, (type 2) diabetes mellitus, and hypertension) can be used to estimate the risk of insulin resistance. The presence of three or more features of the metabolic syndrome predicts the presence of NASH rather than simple fatty liver, especially when these include central obesity and type 2 diabetes mellitus (T2DM). Furthermore, family history plays a role: individuals with first-degree relatives with T2DM have a 90% chance of developing T2DM and thus NASH. Central obesity can be assessed using waist circumference, measured with the patient in an upright position at the end of expiration, at the narrowest midpoint between the lowest rib and the iliac crest.

[0213] Non-invasive means to estimate the extent of fibrosis include transient elastography (FibroScan®), acoustic radiation force impulse (ARFI), and non-invasive biomarker algorithms such as NAFLD Fibrosis Score, FibroTest, and Hepascore.

[0214] Those skilled in the art will also be familiar with methods for determining whether an individual has been successfully treated to prevent further progression of NASH, ameliorate the symptoms of NASH, or prevent the onset of NASH in at-risk individuals. Successful treatment or prevention can be determined by measuring an improvement in, or a reduction in the rate of progression or worsening of, any one or more of the symptoms described above in connection with NASH.

[0215] Transplant rejection (graft-versus-host disease) The methods of the invention also have utility in treating or preventing the onset or progression of, or reducing the severity of, GVHD in individuals in need of cell transplantation.

[0216] GVHD is an inflammatory disease generated by T cells in the donor graft that recognize histocompatible and other tissue antigens of the host, and is mediated by various effector cells and inflammatory cytokines. GVHD manifests in both acute and chronic forms. The most common symptomatic organs are the skin, liver, and gastrointestinal tract (including the oral and oropharyngeal regions). GVHD can involve other organs, such as the lungs.

[0217] Treatment of GVHD is generally successful in only 50-75% of cases; the remainder of patients generally do not survive. The risk and severity of this immune-mediated disease is directly related to the degree of mismatch between the host and the donor of hematopoietic cells. For example, GVHD develops in up to 30% of recipients of human leukocyte antigen (HLA)-matched sibling bone marrow, up to 60% of recipients of HLA-matched unrelated donor bone marrow, and in a higher percentage of recipients of HLA-mismatched bone marrow. Patients with mild intestinal GVHD exhibit anorexia, nausea, vomiting, abdominal pain, and diarrhea, while patients with severe GVHD are disabled by these symptoms. If untreated, symptoms of intestinal GVHD persist and often progress; spontaneous remission is rare. In its most severe form, GVHD leads to necrosis and desquamation of large portions of the epithelial cells of the intestinal mucosa, an often fatal disease. Symptoms of acute GVHD are usually present within 100 days of transplantation. Symptoms of chronic GVHD are usually present somewhat later, up to 3 years after allogeneic HCT, and are often preceded by a history of acute GVHD.

[0218] Two distinct types of GVHD are recognized clinically: acute and chronic. The acute form of the disease usually develops within the first 3 months after transplantation. The incidence of acute GVHD is estimated at 30-50% of patients receiving transplants from HLA-matched sibling donors and 50-70% of patients receiving HLA-matched unrelated transplants. Severe acute GVHD (grades III-IV) occurs in up to 20% of recipients of related donors and up to 35% of recipients of unrelated donors. Severe acute GVHD is associated with a poor prognosis, with long-term survival rates of 25% for grade III and 5% for grade IV.

[0219] Those skilled in the art will be able to identify individuals who require treatment or prevention of GVHD, such as by identifying patient groups most likely to receive cell transplants and patients at risk for acute GVHD or showing symptoms of chronic GVHD. For example, chronic GVHD develops in up to 60% of patients who receive HLA-matched sibling bone marrow transplants and 70% of patients who receive alternative donor bone marrow transplants who survive for more than 100 days. Symptoms of chronic GVHD are usually present for 3 months to 2 years after allogeneic transplantation, with approximately two-thirds developing within the first 12 months. Ultimately, less than 20% of transplanted patients will develop either acute or chronic GVHD.

[0220] composition In some instances, the antigen binding sites described herein may be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, bucally, vaginally, via an implanted reservoir, in a dosage formulation containing a conventional non-toxic pharma- ceutically acceptable carrier, or by any other convenient dosage form. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques.

[0221] Methods for preparing antigen-binding sites in a suitable form (e.g., a pharmaceutical composition) for administration to a subject are known in the art and include, for example, those methods described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and US Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

[0222] The pharmaceutical compositions of the invention are particularly useful for parenteral administration, e.g., intravenous administration or administration into a cavity or lumen of an organ or joint. Compositions for administration generally comprise a solution of the antigen-binding site dissolved in a pharma- ceutically acceptable carrier, e.g., an aqueous carrier. A variety of aqueous carriers, e.g., buffered saline, etc., may be used. The compositions may contain pharma- ceutically acceptable auxiliary agents required to mimic physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the antigen-binding site of the invention in these formulations may vary widely, depending primarily on fluid volume, viscosity, weight, etc., depending on the particular method of administration selected and the needs of the patient. The carrier is selected based on the following: water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as mixed oils and ethyl oleate can also be used. Liposomes can also be used as carriers. The vehicle can contain small amounts of additives that enhance isotonicity and chemical stability, such as buffers and preservatives.

[0223] Once formulated, the antigen-binding sites described herein are administered in a manner compatible with the dosage formulation and in such amount as will be therapeutically / prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the types of injections described above, although other pharma- ceutically acceptable forms are also contemplated, such as tablets, pills, capsules or other solids for oral administration, suppositories, pessaries, nasal drops or sprays, aerosols, inhalants, liposomal forms, and the like. Pharmaceutical "sustained release" capsules or compositions may also be used. Sustained release formulations are generally designed to provide a constant drug level over an extended period of time and may be used to deliver the antigen-binding sites of the present invention.

[0224] WO 2002 / 080967 describes compositions and methods for administering aerosolized compositions containing antibodies, for example for the treatment of asthma, which are also suitable for administration of the antigen-binding sites of the invention.

[0225] Dosage and timing Suitable dosages of the antigen-binding sites of the present invention will vary depending on the specific antigen-binding site, the disease being treated, and / or the subject being treated. Determining suitable dosages is within the capabilities of a skilled physician, for example by starting with a suboptimal dosage and gradually modifying the dosage to determine an optimal or useful dosage. Alternatively, data from cell culture assays or animal studies can be used to determine appropriate dosages for treatment / prophylaxis, where a suitable dose is determined to be greater than or equal to the ED of the active compound with little or no toxicity. 50 The therapeutically / prophylactically effective dose may be estimated initially from cell culture assays. The dose may be greater than or equal to the IC50 or IC60 as determined in cell culture. 50 The compound may be formulated in animal models to achieve a circulating plasma concentration range that includes the compound (i.e., the concentration or amount of compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0226] In some instances, the methods of the present invention comprise administering a prophylactically or therapeutically effective amount of a protein described herein.

[0227] The term "therapeutically effective amount" is an amount that, when administered to a subject in need of treatment, improves the subject's prognosis and / or condition and / or reduces or inhibits one or more symptoms of a clinical condition described herein to a level below that observed and recognized as a clinical diagnostic or clinical characteristic of the condition. The amount administered to a subject will depend on the specific characteristics of the disease being treated, the type and stage of the disease being treated, the method of administration, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, and weight. Those skilled in the art will be able to determine appropriate dosages depending on these and other factors. Thus, the term should not be construed to limit the invention to a specific amount, e.g., weight or amount of protein, but rather, the invention encompasses any amount of antigen binding site sufficient to obtain a desired result in a subject.

[0228] As used herein, the term "prophylactically effective amount" shall be understood to mean an amount of protein sufficient to prevent or inhibit or delay the onset of one or more detectable symptoms of a clinical condition. Those skilled in the art will appreciate that such an amount is, for example, It will be recognized that the amount of antigen binding site administered will vary depending on the particular antigen binding site and / or the particular subject and / or the type or severity or level of disease and / or predisposition to disease (genetic or otherwise). Thus, this term should not be construed to limit the invention to a particular amount, e.g., weight or amount of antigen binding site, but rather, the invention encompasses any amount of antigen binding site sufficient to achieve a given result in a subject.

[0229] kit The present invention further includes kits comprising one or more of the following: (i) an antigen-binding site described herein or an expression construct encoding same; (ii) a cell of the invention; (iii) a conjugate of the present invention; or (iii) A pharmaceutical composition of the present invention.

[0230] In the case of a kit for detecting CXCR3, the kit may, for example, further comprise a detection means linked to the antigen-binding site of the invention.

[0231] In the case of a kit for therapeutic / prophylactic use, the kit may further comprise a pharma- ceutically acceptable carrier.

[0232] Optionally, kits of the invention are packaged with instructions for use of the methods described herein according to any of the Examples.

[0233] [Table 1]

[0234] [Table 2]

[0235] [Table 3]

[0236] [Table 4]

[0237] [Table 5] EXAMPLES

[0238] Working Example Example 1 This example describes a method for producing CXCR3 antibodies.

[0239] The mice were stimulated with 5 mM butyric acid 20 hours before harvesting and then 2 × 10 7Monoclonal antibodies reactive with human CXCR3 (hCXCR3) are produced using L1.2 / hCXCR3 transfected cells by immunizing C57BL / 6 mice a total of 5-6 times at 2-week intervals. The final immunization is injected intravenously in PBS. Four days later, the spleen is removed and the cells are fused with the SP2 / 0 cell line using standard methods. Hybridomas are grown in DMEM (Gibco / Invitrogen) containing 10% Fetalclone (HyClone), 1x HAT supplement (Sigma Aldrich) and mouse IL-6. After 10-14 days of growth, culture supernatants are harvested for primary screening.

[0240] Immunofluorescence staining and analysis using a FACSCalibur (BD Biosciences) were used to identify human CXC R3-transfected L1.2 cells and non-transfected L1.2 cells, or L1.2 cells transfected with unrelated or similar receptors such as CXCR1, CXCR2 or C5aR, are used to identify monoclonal antibodies that react with CXCR3. Monoclonal antibody staining of cells is performed using standard procedures as previously described (Lee et al., 2006, Nat. Biotech. 24:1279-1284).

[0241] Antibody production involved growing hybridomas in tissue culture flasks and harvesting the culture medium. In some experiments, the concentration of antibody in the culture supernatant may be sufficient to continue without further purification. Production of selected antibodies is scaled up and monoclonal antibodies are purified by protein G chromatography, concentrated, and buffer exchanged into PBS. Monoclonal antibody concentrations are determined using a total IgG ELISA.

[0242] L1.2 transfectants expressing high levels of hCXCR3 are used to immunize mice, and monoclonal antibodies that react with hCXCR3-transfected L1.2 cells are first identified by flow cytometry. To ensure clonality, selected hybridomas are subcloned using dilution plating into 384-well plates. The specificity of the cross-reactivity of the subclones with L1.2 / hCXCR3 transfectants and non-transfected L1.2 cells is confirmed by flow cytometry.

[0243] Example 2 This example describes methods for determining the receptor binding specificity of CXCR3 antibodies.

[0244] Indirect immunofluorescence staining and flow cytometry are used to assess the reactivity of mAbs on transfected cells. Cells are washed once with PBS and resuspended in 100 μl of PBS containing 2% (wt / vol) BSA and 0.1% (wt / vol) sodium azide (staining buffer) and purified antibodies. After 30 min at 4° C., cells are washed twice with staining buffer and incubated with 50 μl of PE-conjugated anti-human IgG (Jackson ImmunoResearch Laboratories) for humanized mAb detection or mouse m mAb, diluted 1:500 in staining buffer. Resuspend in 50 μl of PE-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories) for Ab detection. After 20 min incubation at 4° C., cells were stained. Wash twice with buffer and analyze on an LSRII flow cytometer. , to exclude dead cells.

[0245] Example 3 This example describes a method for epitope mapping CXCR3 antibodies.

[0246] To determine the regions in CXCR3 that are recognized by anti-CXCR3 mAbs, epitope mapping studies are performed. First, biotinylated peptides corresponding to the N-terminal region and the first, second and third extracellular loops of CXCR3 are used in ELISA.

[0247] Next, overlapping biotinylated peptides spanning the entire N-terminal region of human CXCR3 are synthesized and used in more detailed epitope mapping studies. Briefly, a multi-well plate is coated with streptavidin, washed, and then the biotinylated peptides are added to separate wells and incubated to facilitate binding of the peptides to the plate. Different anti-mCXCR3 antibodies are tested in parallel with the newly developed mAb by adding each antibody to a well of the plate and incubating the plate. Isotype controls and buffer only are included as negative controls. After washing, the appropriate conjugated antibody is added and the plate is incubated. The plate is washed again and the binding of the antibodies to the immobilized peptides is visualized.

[0248] Example 4 This example describes a method for measuring inhibition of T cell migration by anti-CXCR3 antibodies.

[0249] Human CD4+ T cells were precipitated and cultured in migration medium (MM=RPMI 1640, 0.5% BSA). Wash in 10 7 Tissue culture inserts (Becton Dickinson & Co., Mountain View, Calif.) were placed in each well of a 24-well tissue culture plate. The inserts are placed in a 24-well tissue culture plate to form upper and lower chambers separated by a polyethylene terephthalate membrane with 3 mm diameter holes. Chemotactic CXCR3 ligands, CXCL9 (MIG), CXCL10 (IP-10) or CXCL11 (I-TAC), (diluted in assay medium) are added to 600 μl of assay medium in a 24-well tissue culture plate. One million cells in 100 μl are pre-incubated with the antibody for 30 min. Purified mAbs are added to the upper chamber of the wells and the cells are allowed to migrate through the lower chamber for 4 h in a 5% CO2, 37°C incubator. The inserts are removed after migration and the cells are counted by an LSRII cytometer (BD Biosciences). A setting of 30 s is used. Relative cell numbers are obtained by obtaining events between the intervals.

[0250] Example 5 This example describes the use of a depleting anti-CXCR3 antibody to treat NASH.

[0251] MCD diet for inducing NASH C57 / BL6 mice were fed a control diet or an MCD diet for 5 weeks. The MCD diet is low in choline and methionine and is used to model the induction and progression of NASH. The MCD diet was developed based on the prior art (e.g., Machado et al., 2015, PLoS One;10(5):e0127991). The results were as described in (see reference).

[0252] Antibody treatment Five weeks later, mice were treated with either an isotype control antibody or a depleting anti-CXCR3 antibody.

[0253] histopathology Liver tissue was processed and stained using standard procedures. Hemolysin and eosin staining was used to visualize fat deposition. Picrosirius red staining was used to visualize collagen deposition (thereby determining the extent of fibrosis).

[0254] Steatosis was scored based on the percentage of total affected area and severity was graded into the following categories: 0 (<5%), 1 (5-33%), 2 (34-66%) and 3 (>66%).

[0255] Inflammation is assessed by counting the number of inflammatory foci per field using 100x magnification.

[0256] Measurement of NKT cells and intrahepatic CD8 T cells Measurement of NKT and intrahepatic CD8+ T cells Livers were sectioned and liver lymphocytes were purified using a Ficoll gradient. Cells were stained with anti-CD45, anti-TCRβ; anti-NK1.1; anti-CD8 and anti-CXCR3 antibodies. A Fortessa instrument (BD Biosciences) and FlowJo software were used for acquisition and data analysis.

[0257] Measurement of serum alanine aminotransferase levels Alanine aminotransferase (ALT) activity in serum was measured using an ALT activity colorimetric assay according to the manufacturer's protocol (Sigma).

[0258] Results: Treatment with CXCR3-depleting antibodies prevents NASH As shown in Figures 4 and 5, the livers of mice fed the MCD diet and isotype antibody had significant fatty deposits at the end of the study compared to mice on the control (regular) diet.

[0259] Livers of mice fed an NASH-inducing MCD diet with anti-CXCR3-depleting antibody treatment were significantly protected from the development of fatty deposits.

[0260] FIG. 4 also shows that mice fed anti-CXCR3 depleting antibodies have reduced levels of liver fibrosis compared to mice fed isotype antibodies with the MCD diet.

[0261] Thus, the results shown in Figures 4 and 5 demonstrate that treatment with a depleting anti-CXCR3 antibody inhibits hepatic steatosis and fibrosis in a recognized model of NASH.

[0262] Figure 6 shows that treatment with anti-CXCR3 mAb depletes NKT cells and activated intrahepatic CD8 T cells. MCD diet increases the frequency of CD8+ CXCR3+ T cells and NKT cells; NKT cells are CXCR3+ and are depleted after treatment with Fc-modified anti-CXCR3 mAB.

[0263] Increased serum levels of ALT indicate liver damage. Figure 7 shows that treatment with anti-CXCR3 antibodies reduces the release of alanine aminotransferase into the serum and reduces monocyte infiltration in the liver in a recognized model of NASH.

[0264] In summary, these data suggest that treatment with CXCR-depleting antibodies may improve NASH-inducing dietary outcomes: Inhibits the formation of fatty deposits (hepatic steatosis); Inhibits collagen deposition and fibrosis, Depletion of NKT cells and activated intrahepatic CD8+ T cells · Reduces the release of alanine aminotransferase into the serum (indicating improved liver function); Reduces monocyte infiltration in the liver This indicates that.

[0265] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features described or apparent from the text or drawings, all of which different combinations constitute various alternative aspects of the invention.

Claims

1. Use of a depleting antigen binding protein that binds to CXCR3 for the manufacture of a medicament for treating or slowing the progression of fatty liver disease in a subject, wherein the antigen binding protein is a depleting antibody capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC).

2. The use described in claim 1, wherein the fatty liver disease is caused by or is a result of alcoholism.

3. The use described in claim 1, wherein the fatty liver disease is caused or results from dietary causes other than alcohol.

4. The use described in claim 1, wherein the fatty liver disease is non-alcoholic fatty liver disease (NAFLD), and the NAFLD is simple fatty liver or simple fatty liver including one or more signs of inflammation and fibrosis.

5. The use described in claim 1, wherein the fatty liver disease is characterized by hepatic steatosis, elevated circulating or hepatic triglyceride levels and / or elevated circulating or hepatic cholesterol levels.

6. The use described in claim 1, wherein the fatty liver disease is non-alcoholic steatohepatitis (NASH).

7. The use described in any one of claims 1 to 6, wherein the medicament is for reducing or treating inflammation in the subject.

8. The use of any one of claims 1 to 7, wherein the medicament is for treating or preventing accumulation of lipid deposits in the liver of the subject.

9. Use of a depletable antigen binding protein that binds to CXCR3 for the manufacture of a medicament for treating or preventing steatohepatitis in a subject.

10. The fatty liver disease is alcoholic fatty liver disease (ASH) or non-alcoholic fatty liver disease. (NASH).

11. The use of any one of claims 1 to 10, wherein the antigen binding protein binds or specifically binds to CXCR3 and inhibits CXCR3 activity.

12. The use of any one of claims 1 to 11, wherein the antigen-binding protein is a depleting antibody capable of causing or mediating a reduction in the activity or viability of cells expressing CXCR3.

13. The use of any one of claims 1 to 12, wherein the antigen binding protein has been modified to provide a cell depletion capability or to enhance an existing capability of the antigen binding protein to deplete cells expressing CXCR3.

14. The use of any one of claims 1 to 13, wherein the antigen binding protein is capable of depleting cells selected from CXCR3+ / CD4+ T cells, CXCR3+ / CD8+ T cells and / or CXCR3+ / CD19+ B cells.

15. The use of any one of claims 1 to 14, wherein the antigen binding protein inhibits one or more of: ligand binding to CXCR3; ligand-induced conformational change in CXCR3; CXCR3 activation; G protein activation; CXCR3-mediated cell signaling; CXCR3-mediated cell migration, inflammation, tumor growth, angiogenesis or metastasis responses in vitro or in vivo; CXCR3-mediated tumor cell proliferation; and / or CXCR3-mediated inflammatory cell recruitment, leukocyte (e.g. neutrophils, eosinophils, mast cells or T cells) migration, integrin activation, chemotactic migration and Th1 cell maturation.

16. The use of any one of claims 1 to 15, wherein the antigen binding protein does not detectably bind to or does not substantially bind to CXCR1, CXCR2, and / or C5aR.

17. The use of any one of claims 1 to 16, wherein the antigen-binding protein binds to the first and / or second N-terminal loop in CXCR3.

18. The use of any one of claims 1 to 17, wherein the antigen-binding protein comprises an Fc region that has been modified so as to have an enhanced ability to induce ADCC.

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