Monoclonal antibodies against human tim-3

Abstract

To provide anti-human Tim-3 IgV domain-specific antibodies and fragments thereof, as well as methods of using such antibodies and / or fragments.SOLUTION: The present invention provides an anti-human Tim-3 IgV domain-specific antibody and a fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to an IgV domain of a human Tim-3 (T cell immunoglobulin and mucin-domain containing-3) or IgV mucin-stalk of the human Tim-3, and comprises a specific sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 720,234, filed Aug. 21, 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that was submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created on August 21, 2019, is filed as SequenceListing.txt and is 15.6KB in size. [Background technology]

[0003] The disclosures of all publications, patents, patent applications and books referenced herein are hereby incorporated by reference in their entireties into this specification in order to more fully describe the state of the art to which this invention pertains.

[0004] T-cell immunoglobulin mucin domain-containing 3 (Tim-3), also known as Hepatitis A virus cellular receptor 2 (HAVCR2), is a type I transmembrane protein and an inhibitory receptor expressed in IFNγ-producing T cells, Foxp3+ Treg cells, and innate immune cells such as macrophages and dendritic cells. Tim-3 can suppress immune responses by interacting with its ligands. Tim-3 contains an N-terminal immunoglobulin (IgV) domain, a mucin domain containing an O-linked glycosylation site, a stalk domain between the transmembrane and mucin domains with N-linked sugars, a transmembrane domain, and a cytoplasmic tail (Monney 2002). Our laboratory previously reported that the IgV domain of Tim-3 is a functional domain that can bind carbohydrates (Cao 2007). The Tim-3 IgV domain contains a unique CC'-FG groove, a structure characteristic of all Tim family proteins but not found in other members of the immunoglobulin superfamily, and two noncanonical disulfide bonds (Cao 2007). Some researchers have reported that immune escape against PD-1 blockade treatment is associated with upregulation of other immune checkpoints, including Tim-3 (Koyama 2016). In addition to carbohydrates (Cao 2007; Wilker 2007), other molecules have been reported to be ligands for Tim-3, including galectin-9 (Zhu 2005), phosphatidylserine (DeKruyff 2010), HMGB1 (high mobility group protein box 1) (Chiba 2012), and LILRB2 (leukocyte immunoglobulin-like receptor B2) (WO 2016 / 111947).

[0005] Summary of the Invention binds to the IgV domain of human Tim-3 (T cell immunoglobulin mucin domain containing 3) or the IgV mucin stalk of human Tim-3; a) GYSFTGYTIN (SEQ ID NO: 1) (complementarity determining region (CDR) 1 (CDR1)); LFNPYNGGTT (SEQ ID NO:2) (CDR2); ARRYYGYDAMDY (SEQ ID NO:3) (CDR3); or GFNIKDYYMH (SEQ ID NO:7) (CDR1); WIDPENDNTIY (SEQ ID NO:8) (CDR2); ARDFGYVAWLVY (SEQ ID NO:9) (CDR3); and b) KSSQSVLYSSNQKNHLA (SEQ ID NO: 4) (CDR1); WASTRES (SEQ ID NO:5) (CDR2); HQYLSSYT (SEQ ID NO:6) (CDR3); or KASQNVDTAVA (SEQ ID NO: 10) (CDR1); SASNRYT (SEQ ID NO: 11) (CDR2); QQYSSYPT (SEQ ID NO: 12) (CDR3) and a light chain comprising one or more of the following:

[0006] binds to the IgV domain of human Tim-3 or the IgV mucin stalk of human Tim-3; GYSFTGYTIN (SEQ ID NO: 1) (CDR1); LFNPYNGGTT (SEQ ID NO:2) (CDR2); ARRYYGYDAMDY (SEQ ID NO:3) (CDR3); and a heavy chain comprising KSSQSVLYSSNQKNHLA (SEQ ID NO: 4) (CDR1); WASTRES (SEQ ID NO:5) (CDR2); HQYLSSYT (SEQ ID NO: 6) (CDR3) and a light chain comprising the antibody or antigen-binding fragment thereof.

[0007] binds to the IgV domain of human Tim-3 or the IgV mucin stalk of human Tim-3; GFNIKDYYMH (SEQ ID NO:7) (CDR1); WIDPENDNTIY (SEQ ID NO:8) (CDR2); ARDFGYVAWLVY (SEQ ID NO: 9) (CDR3) and a heavy chain comprising KASQNVDTAVA (SEQ ID NO: 10) (CDR1); SASNRYT (SEQ ID NO: 11) (CDR2); QQYSSYPT (SEQ ID NO: 12) (CDR3) and a light chain comprising the antibody or antigen-binding fragment thereof.

[0008] K D The present invention provides isolated antibodies that bind to the IgV domain of human Tim-3 or the IgV mucin stalk of human Tim-3 with an affinity of less than 10.0 nM.

[0009] One or more GYSFTGYTIN (SEQ ID NO: 1) (CDR1); LFNPYNGGTT (SEQ ID NO:2) (CDR2); ARRYYGYDAMDY (SEQ ID NO: 3) (CDR3) The present invention provides a nucleic acid encoding the heavy chain of an antibody comprising:

[0010] One or more GFNIKDYYMH (SEQ ID NO:7) (CDR1); WIDPENDNTIY (SEQ ID NO:8) (CDR2); ARDFGYVAWLVY (SEQ ID NO: 9) (CDR3) The present invention provides a nucleic acid encoding the heavy chain of an antibody comprising:

[0011] One or more KSSQSVLYSSNQKNHLA (SEQ ID NO: 4) (CDR1); WASTRES (SEQ ID NO:5) (CDR2); HQYLSSYT (SEQ ID NO: 6) (CDR3) The present invention provides a nucleic acid encoding the light chain of an antibody comprising:

[0012] One or more KASQNVDTAVA (SEQ ID NO: 10) (CDR1); SASNRYT (SEQ ID NO: 11) (CDR2) QQYSSYPT (SEQ ID NO: 12) (CDR3) The present invention provides a nucleic acid encoding the light chain of an antibody comprising:

[0013] Host cells containing one or more of the nucleic acids described herein are provided.

[0014] Provided is an antibody or fragment thereof as described herein linked or conjugated to a therapeutic agent.

[0015] A method of inhibiting human Tim-3 in a subject is provided, comprising administering an antibody or fragment thereof described herein in an amount effective to inhibit human Tim-3.

[0016] A method is provided for inhibiting Tim-3-mediated T cell suppression in a subject, comprising administering an antibody or fragment thereof described herein in an amount effective to inhibit Tim-3-mediated T cell suppression.

[0017] Methods are provided for treating cancer in a subject comprising administering an antibody or fragment thereof described herein effective to treat the cancer in the subject.

[0018] Provided is a method for detecting human Tim-3 positive cells in a subject, comprising administering an antibody or fragment thereof described herein conjugated to a detectable marker in an amount effective to label human Tim-3 positive cells, and detecting the presence of the marker in the subject, thereby detecting human Tim-3 positive cells in the subject.

[0019] Provided is an isolated nucleic acid molecule encoding an antibody or fragment thereof described herein. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is cDNA. In some embodiments, the nucleic acid is RNA.

[0020] In some embodiments, the antigen-binding fragment of the antibody is a Tim-3 IgV domain-binding fragment of the antibody. In some embodiments, the antigen-binding fragment of the antibody is a human Tim-3 IgV domain-binding fragment of the antibody.

[0021] Vectors are provided that encode the nucleic acid molecules described herein.Host cells are provided that comprise the nucleic acid molecules described herein or the vectors described herein.

[0022] A method for producing an anti-Tim-3 IgV domain antibody or a Tim-3 IgV domain-binding fragment thereof comprises culturing a host cell described herein under conditions in which the host cell produces the anti-Tim-3 IgV domain antibody or a Tim-3 IgV domain-binding fragment thereof.

[0023] Provided is a pharmaceutical composition comprising an anti-Tim-3 IgV domain antibody or Tim-3 IgV domain binding fragment thereof described herein and a pharma- ceutically acceptable excipient.

[0024] A method is provided for reducing the activity of Tim-3 in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an anti-Tim-3 IgV domain antibody or Tim-3 IgV domain binding fragment thereof described herein. [Brief description of the drawings]

[0025] [Figure 1] ELISA shows that 50B5 binds to the Tim-3-IgV protein and to the extracellular protein of Tim-3 (the IgV mucin stalk), but not to the human IgG control protein. [Diagram 2] FACS shows that 50B5 binds to cells expressing human Tim-3, but not mouse Tim-3. 50B5: thick line, mouse IgG1 isotype control: shaded histogram. [Diagram 3] Kinetic parameters of 50B5 by surface plasmon resonance. [Figure 4]A: In mixed lymphocyte reactions, dividing but not non-dividing CD4 and CD8 T cells expressed Tim-3 on day 4. B: In mixed lymphocyte reactions, mAb 50B5 significantly enhanced IFNγ production from CD4 and CD8 T cells on day 4; N=8; **P<0.01. [Diagram 5] FACS shows that 15B4 binds to cells expressing human Tim-3. 15B4: bold line, mouse IgG1 isotype control: shaded histogram. [Figure 6] Kinetic parameters by surface plasmon resonance. BEST MODE FOR CARRYING OUT THEINVENTION

[0026] binds to the IgV domain of human Tim-3 (T cell immunoglobulin mucin domain containing 3) or the IgV mucin stalk of human Tim-3; a) GYSFTGYTIN (SEQ ID NO: 1) (CDR1); LFNPYNGGTT (SEQ ID NO:2) (CDR2); ARRYYGYDAMDY (SEQ ID NO:3) (CDR3); or GFNIKDYYMH (SEQ ID NO:7) (CDR1); WIDPENDNTIY (SEQ ID NO:8) (CDR2); ARDFGYVAWLVY (SEQ ID NO:9) (CDR3); and b) KSSQSVLYSSNQKNHLA (SEQ ID NO: 4) (CDR1); WASTRES (SEQ ID NO:5) (CDR2); HQYLSSYT (SEQ ID NO:6) (CDR3); or KASQNVDTAVA (SEQ ID NO: 10) (CDR1); SASNRYT (SEQ ID NO: 11) (CDR2); QQYSSYPT (SEQ ID NO: 12) (CDR3) and a light chain comprising one or more of the following:

[0027] binds to the IgV domain of human Tim-3 or the IgV mucin stalk of human Tim-3; GYSFTGYTIN (SEQ ID NO: 1) (CDR1); LFNPYNGGTT (SEQ ID NO:2) (CDR2); ARRYYGYDAMDY (SEQ ID NO:3) (CDR3); and a heavy chain comprising KSSQSVLYSSNQKNHLA (SEQ ID NO: 4) (CDR1); WASTRES (SEQ ID NO:5) (CDR2); HQYLSSYT (SEQ ID NO: 6) (CDR3) and a light chain comprising the antibody or antigen-binding fragment thereof.

[0028] binds to the IgV domain of human Tim-3 or the IgV mucin stalk of human Tim-3; GFNIKDYYMH (SEQ ID NO:7) (CDR1); WIDPENDNTIY (SEQ ID NO:8) (CDR2); ARDFGYVAWLVY (SEQ ID NO:9) (CDR3); and a heavy chain comprising KASQNVDTAVA (SEQ ID NO: 10) (CDR1); SASNRYT (SEQ ID NO: 11) (CDR2); QQYSSYPT (SEQ ID NO: 12) (CDR3) and a light chain comprising the antibody or antigen-binding fragment thereof.

[0029] In some embodiments, the light and heavy chain framework regions have greater than 85% identity to human framework regions.

[0030] In some aspects, the light and heavy chain framework regions are human. DThe present invention provides an isolated antibody or antigen-binding fragment thereof that binds to the IgV domain of human Tim-3 or the IgV mucin stalk of human Tim-3 with an affinity of less than 10.0 nM.

[0031] In some embodiments, the antibody is D In some embodiments, the antibody binds to the IgV domain of human Tim-3 or the IgV mucin stalk of human Tim-3 with an affinity of less than 1.0 nM. D binds to the IgV domain of human Tim-3 or the IgV mucin stalk of human Tim-3 with an affinity of less than 0.5 nM.

[0032] In some embodiments, the antibody does not bind to mouse Tim-3.

[0033] In some embodiments, the antibody inhibits binding of human Tim-3 to phosphatidylserine expressed in dexamethasone-treated Jurkat T cells.

[0034] In some embodiments, the isolated antibody or antigen-binding fragment thereof has a human Fc region sequence.

[0035] In some aspects, the isolated antibody or antigen-binding fragment thereof comprises: GYSFTGYTIN (SEQ ID NO: 1) (CDR1) LFNPYNGGTT (SEQ ID NO: 2) (CDR2) ARRYYGYDAMDY (SEQ ID NO: 3) (CDR3) The heavy chain comprises one or more of:

[0036] In some aspects, the isolated antibody or antigen-binding fragment thereof comprises: KSSQSVLYSSNQKNHLA (SEQ ID NO: 4) (CDR1) WASTRES (SEQ ID NO:5) (CDR2) HQYLSSYT (SEQ ID NO: 6) (CDR3) and a light chain comprising one or more of:

[0037] In some aspects, the isolated antibody or antigen-binding fragment thereof comprises: GFNIKDYYMH (SEQ ID NO: 7) (CDR1) WIDPENDNTIY (SEQ ID NO: 8) (CDR2) ARDFGYVAWLVY (SEQ ID NO: 9) (CDR3) The heavy chain comprises one or more of:

[0038] In some aspects, the isolated antibody or antigen-binding fragment thereof comprises: KASQNVDTAVA (SEQ ID NO: 10) (CDR1) SASNRYT (SEQ ID NO: 11) (CDR2) QQYSSYPT (SEQ ID NO: 12) (CDR3) and a light chain comprising one or more of:

[0039] In some aspects, the isolated antibody or fragment thereof is chimeric or humanized.

[0040] In some embodiments, the isolated antibody or fragment thereof is selected from the group consisting of a monoclonal antibody, an scFv, a Fab fragment, a Fab' fragment, and a F(ab)' fragment. Note that, strictly speaking, an scFv is a fusion protein and not an antibody fragment, but as used herein, unless otherwise specified, an antibody fragment includes an scFv.

[0041] One or more GYSFTGYTIN (SEQ ID NO: 1) (CDR1); LFNPYNGGTT (SEQ ID NO:2) (CDR2); ARRYYGYDAMDY (SEQ ID NO: 3) (CDR3) The present invention provides a nucleic acid encoding the heavy chain of an antibody comprising:

[0042] One or more GFNIKDYYMH (SEQ ID NO:7) (CDR1); WIDPENDNTIY (SEQ ID NO:8) (CDR2); ARDFGYVAWLVY (SEQ ID NO: 9) (CDR3) The present invention provides a nucleic acid encoding the heavy chain of an antibody comprising:

[0043] One or more KSSQSVLYSSNQKNHLA (SEQ ID NO: 4) (CDR1); WASTRES (SEQ ID NO:5) (CDR2); HQYLSSYT (SEQ ID NO: 6) (CDR3) The present invention provides a nucleic acid encoding the light chain of an antibody comprising:

[0044] One or more KASQNVDTAVA (SEQ ID NO: 10) (CDR1); SASNRYT (SEQ ID NO: 11) (CDR2) QQYSSYPT (SEQ ID NO: 12) (CDR3) The present invention provides a nucleic acid encoding the light chain of an antibody comprising:

[0045] Host cells containing one or more of the nucleic acids described herein are provided.

[0046] Provided is an antibody or fragment thereof as described herein linked or conjugated to a therapeutic agent.

[0047] In some aspects, the therapeutic agent is a cytotoxic agent, a radioisotope, an immunomodulatory agent, or a second antibody.

[0048] A method of inhibiting human Tim-3 in a subject is provided, comprising administering an antibody or fragment thereof described herein, or a human Tim-3-binding fragment thereof, in an amount effective to inhibit human Tim-3.

[0049] In some embodiments, the subject is suffering from cancer.

[0050] Provided is a method for inhibiting Tim-3-mediated T cell suppression in a subject, comprising administering an antibody or fragment thereof described herein, or a human Tim-3-binding fragment thereof, in an amount effective to inhibit Tim-3-mediated T cell suppression.

[0051] Methods are provided for treating cancer in a subject, comprising administering an antibody or fragment thereof described herein in an amount effective to treat the cancer in the subject.

[0052] In some embodiments, the cancer is a human Tim-3 positive cancer.

[0053] Provided is a method for detecting human Tim-3 positive cells in a subject, comprising administering an antibody or fragment thereof described herein conjugated to a detectable marker in an amount effective to label human Tim-3 positive cells, and detecting the presence of the marker in the subject, thereby detecting human Tim-3 positive cells in the subject.

[0054] In some embodiments, the label is detected by imaging.

[0055] In some embodiments, the cell is a cancer cell.

[0056] In some embodiments, the cancer is a hematological malignancy, hi some embodiments, the cancer is lung cancer, gastric cancer, head and neck cancer, schwannoma, melanoma, or follicular B-cell non-Hodgkin's lymphoma.

[0057] In some embodiments, the cancer comprises a solid tumor.

[0058] In some embodiments, the subject is receiving anti-PD-1, anti-PD-L1, or anti-CTLA4 therapy.

[0059] In some embodiments, the anti-Tim-3 IgV domain antibody or Tim-3 IgV domain binding fragment thereof is selected from the group consisting of: (i) human germline IGHV1-2 * 02, IGHV1-2 * 04, IGHV1-2 * 05, IGHV1-18 * 04, IGHV1-69-2 * 01, IGHV1-46 * 01, IGHD5-12 * 01, IGHD5-24 * 01, IGHD6-25 *01, IGHJ3 * 01, IGHJ4 * 01, IGHJ4 * 03, IGHJ6 * 01, IGHJ6 * and / or (ii) a VH framework comprising the framework sequence of human germline IGKV1-13. * 02, IGKV1-27 * 01, IGKV3-7 * 02, IGKV4-1 * 01, IGKV1D-13 * 02, IGKV3D-7 * 01, IGKJ1 * 01, IGKJ2 * 01, IGKJ4 * 01, IGKJ4 * 02 framework sequence.

[0060] In some embodiments, the anti-Tim-3 IgV domain antibody or Tim-3 IgV domain binding fragment thereof binds the Tim-3 IgV domain at about 1×10 -9 M ~ approx. 1×10 -12 Binding affinity of M (K D ) to join them.

[0061] In some embodiments, the anti-Tim-3 IgV domain antibody or Tim-3 IgV domain binding fragment thereof is a monoclonal antibody.

[0062] In some embodiments, the anti-Tim-3 IgV domain antibody or Tim-3 IgV domain binding fragment thereof is a recombinant antibody.

[0063] In some embodiments, the anti-Tim-3 IgV domain antibody or Tim-3 IgV domain binding fragment thereof has human framework regions.

[0064] In some embodiments, the anti-Tim-3 IgV domain antibody or Tim-3 IgV domain binding fragment thereof has human constant regions.

[0065] In some embodiments, anti-Tim-3 IgV domain antibodies are provided.

[0066] In some embodiments, Tim-3 IgV domain binding fragments of antibodies are provided.

[0067] In some embodiments, the Tim-3 IgV domain binding fragment of the antibody is a Fab, F(ab)2 or scFv.

[0068] Provided is an isolated nucleic acid molecule encoding an anti-Tim-3 IgV domain antibody or a Tim-3 IgV domain binding fragment thereof described herein. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is cDNA. In some embodiments, the nucleic acid is RNA.

[0069] Vectors are provided that encode the nucleic acid molecules described herein.Host cells are provided that comprise the nucleic acid molecules described herein or the vectors described herein.

[0070] A method for producing an anti-Tim-3 IgV domain antibody or a Tim-3 IgV domain-binding fragment thereof comprises culturing a host cell described herein under conditions such that the host cell produces the anti-Tim-3 IgV domain antibody or the Tim-3 IgV domain-binding fragment thereof.

[0071] Compositions are provided that include an anti-Tim-3 IgV domain antibody or Tim-3 IgV domain binding fragment thereof described herein and a carrier. In one embodiment, the composition is a pharmaceutical composition and the carrier is a pharmaceutical carrier. Also provided are pharmaceutical compositions that include an antibody or binding fragment thereof described herein and a pharma- ceutical acceptable excipient.

[0072] As used herein, the term "antibody" refers to an intact antibody, i.e., having complete Fc and Fv regions. "Fragment" refers to a portion of an antibody or portions of an antibody linked together, such as, but not limited to, Fab, F(ab)2, single chain Fv (scFv), which is an antigen-binding portion of an antibody but not the whole antibody, that competes with the intact antibody for specific binding. In this case, the antigen is the human Tim-3 IgV domain.

[0073] Such fragments can be prepared, for example, by cleavage of intact antibodies or recombinantly. See Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989), incorporated herein by reference in its entirety). Antigen-binding fragments may be produced by recombinant DNA techniques, by enzymatic or chemical cleavage of intact antibodies, or by molecular biology techniques. In some embodiments, the fragment is a Fab, Fab', F(ab')2, Fd, Fv, a complementarity determining region (CDR) fragment, a single chain antibody (scFv) (a light chain variable domain (VL) and a heavy chain variable domain (VH) linked via a peptide linker). In some embodiments, the scFv comprises a variable domain framework sequence having a sequence identical to the human variable domain FR1, FR2, FR3, or FR4. In some embodiments, the scFv comprises a linker peptide having 5-30 amino acid residues. In one embodiment, the scFv comprises a linker peptide comprising one or more glycine, serine, and threonine residues.

[0074] In some embodiments, the linker of the scFv is 10-25 amino acids in length. In some embodiments, the peptide linker comprises glycine, serine and / or threonine residues (see, e.g., Bird 1988 and Huston 1988, which are incorporated herein by reference in their entireties) or comprises a polypeptide comprising at least a portion of an antibody sufficient to specifically bind to the human Tim-3 IgV domain, including diabodies. From the N-terminus to the C-terminus, the mature light and heavy chain variable domains comprise the FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 regions. The assignment of amino acids to each domain follows the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk 1987 or Chothia 1989, which are incorporated herein by reference in their entireties. As used herein, the term "polypeptide" includes natural or artificial proteins, fragments of proteins, and polypeptide analogs. Polypeptides may be monomeric or polymeric. As used herein, an Fd fragment refers to a fragment of an antibody consisting of the VH and CH1 domains, an Fv fragment consists of the V1 and VH domains of a single chain of an antibody, and a dAb fragment (Ward 1989, incorporated herein by reference in its entirety) consists of the VH domain. In some embodiments, the fragment is at least 5, 6, 8, or 10 amino acids in length. In other embodiments, the fragment is at least 14, at least 20, at least 50, or at least 70, 80, 90, 100, 150, or 200 amino acids in length.

[0075] The term "monoclonal antibody" as used herein refers to a population of substantially homogeneous antibodies, i.e., the individual antibodies are identical except for minor variations that may be present, e.g., naturally occurring mutations. Thus, the term "monoclonal" refers to the characteristic of an antibody that is not a mixture of different antibodies. In certain embodiments, such monoclonal antibodies typically include antibodies that include a peptide sequence that binds to a human Tim-3 IgV domain antibody. In contrast to polyclonal antibodies, which include antibodies against different antigenic determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant of an antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are usually not contaminated by other immunoglobulins. Thus, once their sequences have been identified, monoclonal antibodies can be prepared by non-hybridoma techniques, e.g., recombinantly.

[0076] In aspects of the invention described herein, the antibody is isolated. As used herein, the term "isolated antibody" refers to an antibody that, based on its origin or derivation, has one, two, three, or all of the following: (1) is free from association with coexisting elements in its natural state, (2) is free from other proteins of the same species, (3) is produced in a cell of a different species, and (4) does not occur in nature without human intervention.

[0077] In certain aspects, the antibody is humanized. "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from a non-human immunoglobulin. In certain aspects, a humanized antibody is a human immunoglobulin (recipient antibody) in which the recipient's hypervariable regions (HVRs) (or CDRs) have been replaced by residues from the CDRs of a non-human species (donor antibody), such as mouse, rat, rabbit or non-human primate, that has the desired specificity, affinity, and / or capacity. In certain aspects, the antibody has one, two, three, four, five or all six CDRs 1-3 of the heavy and light chains of the murine antibodies (mAb15B4 and mAb50B5) described herein. In preferred aspects, framework (FR) residues of the murine mAb are replaced by corresponding human immunoglobulin variable domain framework (FR) residues. These may be further modified in embodiments to further improve antibody performance. Furthermore, in certain aspects, a humanized antibody may comprise residues that are not present in the recipient antibody or in the donor antibody. In some embodiments, a humanized antibody does not contain residues that are not present in the recipient antibody or in the donor antibody. In general, a humanized antibody can comprise substantially all of at least one, and typically two, variable domains in which all or in some embodiments substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin (e.g., mAb 15B4 and mAb 50B5 described herein) and all or in some embodiments substantially all of the FRs are human immunoglobulin sequences. A humanized antibody can optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin. See, e.g., Jones 1986; Riechmann 1988; Presta 1992; Vaswani & Hamilton 1998; Harris 1995; Hurle & Gross 1994, and U.S. Patent Nos. 6,982,321 and 7,087,409, which are incorporated herein by reference in their entireties. In embodiments where the humanized antibody comprises residues that are not present in the recipient antibody or in the donor antibody, the Fc region of the antibody is modified as described in WO 99 / 58572, which is incorporated herein by reference in its entirety.

[0078] Techniques for humanizing monoclonal antibodies are well known and are described, for example, in U.S. Patent Nos. 4,816,567, 5,807,715, 5,866,692, 6,331,415, 5,530,101, 5,693,761, 5,693,762, 5,585,089, and 6,180,370, which are incorporated herein by reference in their entireties. Many "humanized" antibody molecules have been described that contain antigen-binding sites derived from non-human immunoglobulins, including antibodies in which rodent or modified rodent V-regions and their complementarity determining regions (CDRs) are fused with human constant domains. See, for example, Winter 1991; Lobuglio 1989; Shaw 1987, and Brown 1987, which are incorporated herein by reference in their entireties. Other publications describe rodent hypervariable regions or CDRs that are grafted into human framework regions (FRs) before fusing with an appropriate human antibody constant domain. See, for example, Riechmann 1988; Verhoeyen 1988 and Jones 1986, which are incorporated herein by reference in their entirety. Another publication describes rodent CDRs with rodent engineered framework regions - EP 0519596, which is incorporated herein by reference in its entirety. These "humanized" molecules are designed to minimize unwanted immune responses against non-human rodent antibodies that limit the therapeutic duration and efficacy of the antibodies in humans. The constant regions of the antibodies can be engineered to be immunologically inert (e.g., do not trigger complement lysis). See, for example, WO 99 / 58572; UK Patent Application No. 9809951.8. Other methods for humanizing antibodies that can be used are described in Daugherty 1991; U.S. Patent Nos. 6,180,377; 6,054,297; 5,997,867; 5,866,692; 6,210,671 and 6,350,861, and WO 01 / 27160, which are incorporated herein by reference in their entireties.

[0079] Other forms of humanized antibodies have one or more or all of the CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 or CDR H3) altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more original antibodies. In one embodiment, the antibodies of the invention are humanized mAb 15B4 and mAb 50B5 with one or more or all of the CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 or CDR H3) altered relative to the original antibody. In one embodiment, the antibodies of the invention are humanized mAb 15B4 and mAb 50B5 with CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2 or CDR H3) that are not altered relative to the original antibody.

[0080] In some aspects, the antibodies or fragments herein can be recombinantly derived, such as, for example, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, or antibodies isolated from a transgenic animal (e.g., a mouse) carrying human immunoglobulin genes.

[0081] In certain aspects, the anti-Tim-3 IgV domain antibodies described herein are capable of specifically binding or specifically bind to the human Tim-3 IgV domain. As used herein, the terms "capable of specifically binding" or "specifically binds" refer to the property of an antibody or fragment thereof to bind to a (specific) antigen with a dissociation constant of less than 1 μM, preferably less than 1 nM, and most preferably less than 10 pM. In certain aspects, the K d In one embodiment, the K of the antibody (or fragment) to the Tim-3 IgV domain is less than 10.0 nM. d In one embodiment, the K of the antibody (or fragment) to the Tim-3 IgV domain is less than 1.0 nM. d In one embodiment, the K of the antibody (or fragment) to the Tim-3 IgV domain is less than 0.5 nM. d is 0.1 nM or less.

[0082] "K d The term "dissociation constant" as used herein refers to the dissociation constant of an antibody-antigen interaction. The K d Alternatively, one method to determine binding affinity is to measure the binding affinity of a monofunctional Fab fragment of an antibody. (The affinity constant is the reciprocal of the dissociation constant.) Monofunctional Fab fragments can be obtained by papain digestion of an antibody (e.g., IgG) or by recombinant expression. The affinity of anti-human Tim-3 IgV domain antibody fragments can be determined, for example, by surface plasmon resonance (BIAcore3000® Surface Plasmon Resonance (SPR) System, BIAcore Inc., Piscataway NJ). CM5 chips are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiinide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted in 10 mM sodium acetate (pH 4.0) and injected onto the activated chip at 0.005 mg / mL. Use variable flow times across the chip to achieve two antigen densities: 100-200 response units (RU) for detailed kinetic studies and 500-600 RU for screening assays. Serial dilutions of purified Fab (estimated K d A volume of 0.1-10x the binding protein (0.1-10x the binding protein) is injected at 100 μl / min for 1 min, allowing a dissociation time of up to 2 h. The concentration of the Fab protein is determined by ELISA and / or SDS-PAGE electrophoresis using a Fab of known concentration (determined by amino acid analysis). The binding rate constant (k on ) and dissociation rate constant (k off ) is obtained by fitting the data to a 1:1 Langmuir binding model (see Karlsson et al. "Kinetic and concentration analysis using BIA technology, Methods: A Companion to Methods Enzymol 6:99-110 (1994), which is incorporated herein by reference in its entirety) using the BIAevaluation program. The equilibrium dissociation constant (K d ) value is koff / k on This method is suitable for determining the binding affinity of an antibody or a fragment thereof to an antigen. Other methods known in the art, such as ELISA, can also be used.

[0083] An epitope that "specifically binds" to an antibody or polypeptide is a term well understood in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for longer, and / or with higher affinity. An antibody "specifically binds" or "preferentially binds" if it binds to a target with higher affinity, avidity, more readily, and / or for longer than it binds to other substances. For example, an antibody that specifically or preferentially binds to the human Tim-3 IgV domain is an antibody that binds to this epitope with higher affinity, avidity, more readily, and / or for longer than it binds to other Tim-3 epitopes or non-Tim-3 epitopes. From this definition, it can also be understood that, for example, an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding.

[0084] The term "competing" antibodies is used herein to mean that a primary antibody or antigen-binding portion thereof binds to an epitope in a manner similar to that of a secondary antibody or antigen-binding portion thereof, such that binding of the primary antibody to its cognate epitope is detectably reduced in the presence of the secondary antibody compared to its absence. Binding of the secondary antibody to the epitope may, but need not, be detectably reduced in the presence of the primary antibody. That is, a primary antibody can inhibit binding of a secondary antibody to an epitope without the secondary antibody inhibiting binding of the primary antibody to the epitope. And, if each antibody detectably inhibits binding of the other antibody to the epitope or ligand, regardless of the degree to which they do so, the antibodies are said to "cross-compete" with each other for binding to their respective epitopes. The present invention includes both competing and cross-competing antibodies. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational changes, or binding to a common epitope or portion thereof), one of skill in the art, based on the teachings herein, will understand that such competing and / or cross-competing antibodies are included in and may be useful in the methods described herein.

[0085] Depending on the amino acid sequence of the constant domain of the heavy chain, antibodies (immunoglobulins) can be assigned to different classes. The antibody or fragment thereof can be, for example, any of the following antibodies or fragments: IgG, IgD, IgE, IgA or IgM, respectively. In one embodiment, the antibody is an immunoglobulin G. In one embodiment, the antibody fragment is a fragment of an immunoglobulin G. In one embodiment, the antibody is an IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgG4. In one embodiment, the antibody comprises a sequence from human IgG1, human IgG2, human IgG2a, human IgG2b, human IgG3 or human IgG4. Combinations of these antibody subclasses can also be used. One factor to consider when choosing the type of antibody to use is the serum half-life of the antibody. For example, the serum half-life of IgG is generally 23 days, IgA 6 days, IgM 5 days, IgD 3 days, and IgE 2 days (Abbas AK, Lichtman AH, Pober JS. Cellular and Molecular Immunology, 4th edition, WB Saunders Co., Philadelphia, 2000, which is incorporated herein by reference in its entirety).

[0086] The "variable region" or "variable domain" of an antibody refers to the N-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are generally the most variable parts of the antibody and contain the antigen binding site. The term "variable" refers to the fact that the amino acid sequence of certain parts of the variable domain varies widely between antibodies and is used in the binding and specificity of the antibody to a particular antigen. However, this sequence variation is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called hypervariable regions (HVRs) (or CDRs) of the light and heavy chain variable domains. The highly conserved parts of the variable domain are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FR regions that mainly adopt a beta-sheet structure, and these FRs are connected by three looped CDRs that connect to and in some cases form part of the beta-sheet structure. The CDRs of each chain are held in close proximity by the FR regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0087] The "light chains" of antibodies (immunoglobulins) of any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0088] "Framework" or "FR" refers to the variable domains other than the HVRs as defined herein.

[0089] The term "hypervariable region" or "HVR" as used herein refers to a region of an antibody variable domain that is hypervariable in sequence and / or forms loops. Generally, an antibody is composed of six HVRs, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 show the most diversity among the six HVRs, and H3 in particular is thought to play a special role in conferring high specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, natural camelid antibodies consisting of only heavy chains are functional and stable even without light chains (see, for example, Hamers-Casterman 1993; Sheriff & Constantine 1996). Several HVR descriptions are in use and are included herein. The Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), incorporated herein by reference in its entirety). There are CDRs 1, 2 and 3 for the heavy and light chains, respectively. Alternatively, Chothia is based on the position of the structural loops (Chothia & Lesk 1987). The AbM HVRs are a compromise between the Kabat HVRs and the Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on the analysis of available complex crystal structures. The HVR may be the following "extended HVRs": for VL, 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), 89-97 or 89-96 (L3), and for VH, 26-35 (H1), 50-65 or 49-65 (H2), 93-102, 94-102 or 95-102 (H3).Within each of these definitions, the variable domain residues are numbered according to Kabat et al., supra.

[0090] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including wild-type sequence Fc regions and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region may vary, the Fc region of a human IgG heavy chain is usually defined as from Cys226 or Pro230 to its C-terminus. The C-terminal lysine of the Fc region may be removed, for example, during production or purification of the antibody, or can be removed by recombination of the nucleic acid encoding the antibody heavy chain. Thus, as used herein, an intact antibody may be an antibody that either includes or does not include a C-terminal lysine.

[0091] A composition or pharmaceutical composition comprising an antibody, scFv or fragment of an antibody described herein preferably comprises a stabilizer to prevent loss of activity or structural integrity of the protein due to denaturation, oxidation or aggregation during storage and transport. The composition or pharmaceutical composition may comprise one or more isotonicity agents such as salts, surfactants and sugars, which may contribute to overcoming the problem of aggregation. When the composition or pharmaceutical composition of the present invention is used as an injection, it is desirable that the pH is approximately neutral pH, and it is also advantageous to minimize the amount of surfactant to avoid air bubbles in the formulation, which may be harmful upon injection into a subject. In some embodiments, the composition or pharmaceutical composition is liquid, stably holds a high concentration of bioactive antibody in solution, and is suitable for inhalation or parenteral administration. In some embodiments, the composition or pharmaceutical composition is suitable for intravenous, intramuscular, intraperitoneal, intradermal and / or subcutaneous injection. In some embodiments, the composition or pharmaceutical composition is liquid, minimizing the risk of air bubble formation and anaphylactoid side effects. In some embodiments, the composition or pharmaceutical composition is isotonic. In some embodiments, the pH of the composition or pharmaceutical composition is 6.8 to 7.4.

[0092] In one aspect, the ScFv or antibody fragments described herein are lyophilized and / or freeze dried and reconstituted at the time of use.

[0093] Examples of pharma- ceutically acceptable carriers include, but are not limited to, phosphate buffered saline, sterile water (including water for injection (USP)), emulsions such as o / w emulsions, and various wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal saline (0.9%), e.g., 0.9% sodium chloride solution (USP). Compositions containing such carriers are prepared by well-known methods (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990 and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000, which are incorporated herein by reference in their entirety). In non-limiting examples, the compositions can include one or more of disodium hydrogen phosphate, potassium chloride, potassium dihydrogen phosphate, polysorbate 80 (e.g., 2-[2-[3,5-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl 9-(E)-octadecenoate), disodium edetate hydrate, sucrose, sodium dihydrogen phosphate monohydrate, and disodium hydrogen phosphate dihydrate.

[0094] The antibodies or antibody fragments, or compositions or pharmaceutical compositions described herein may be lyophilized or provided in any suitable form, including, but not limited to, injectable or inhalable solutions, gels, and tablets.

[0095] The term "Fc domain" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including wild-type sequence Fc regions and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc domain may vary, the Fc domain of a human IgG heavy chain is usually defined as from Cys226 or Pro230 to its C-terminus. The C-terminal lysine of the Fc domain can be removed, for example, by recombination of the nucleic acid encoding it. In some embodiments, an antibody comprises an Fc domain. In some embodiments, the sequence of the Fc domain is identical to or has 99% or more similarity to a human IgG1 Fc domain. In some embodiments, the sequence of the Fc domain is identical to or has 99% or more similarity to a human IgG2 Fc domain. In some embodiments, the sequence of the Fc domain is identical to or has 99% or more similarity to a human IgG3 Fc domain. In some embodiments, the sequence of the Fc domain is identical to or has 99% or more similarity to a human IgG4 Fc domain. In some embodiments, the Fc domain is not mutated. In some embodiments, the Fc domain is mutated at the CH2-CH3 domain interface to increase the affinity of IgG for FcRn at acidic conditions (Dall'Acqua 2006; Yeung 2009). In some embodiments, the sequence of the Fc domain is identical to the human IgG1 Fc domain.

[0096] In some aspects, the invention includes modifications to the variable regions described herein. For example, the invention includes antibodies that contain functionally equivalent variable regions and CDRs that do not significantly affect their properties, as well as variants with enhanced or decreased activity and / or affinity. For example, the amino acid sequence may be mutated to obtain an antibody with a desired binding affinity to human Tim-3 IgV. Modification of polypeptides is routine in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides in which amino acid residues have been conservatively substituted, polypeptides with deletions or additions of one or more amino acids that do not significantly inhibit activity or that mature (enhance) the affinity of the polypeptide for a ligand, or polypeptides with chemical analogs.

[0097] Amino acid sequence insertions include N- and / or C-terminal fusions of polypeptides containing from 1 to 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or an antibody fused to an epitope tag. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the half-life of the antibody in the blood.

[0098] Substitutional variants have at least one amino acid residue in the antibody molecule removed and another residue inserted in its place. Sites of greatest interest for substitutional mutagenesis include the hypervariable regions, although framework modifications are also contemplated. Conservative substitutions are shown in Table 1 under the "Conservative Substitutions" column. If such substitutions alter biological activity, more substantial substitutions, such as those shown in Table 1 under the "Representative Substitutions" column, or as described below for amino acid classes, may be made and the products screened.

[0099] [Table 1]

[0100] Substantial alteration of the biological properties of the antibody is achieved by selecting substitutions that differ significantly in their impact on (a) the structure of the polypeptide backbone in the substituted region, e.g., beta-sheet or helical structure, (b) the charge or hydrophobicity of the target site, or (c) maintaining the bulk of the side chains. Naturally occurring residues are divided into the following groups based on common side chain properties: (1) Nonpolar: Norleucine, Met, Ala, Val, Leu, Ile; (2) uncharged polar: Cys, Ser, Thr, Asn, Gln; (3) Acidic (negative charge): Asp, Glu; (4) Basic (positive charge): Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro, and (6) Aromatic: Trp, Tyr, Phe, His.

[0101] Non-conservative substitutions are made by exchanging an amino acid from one of these groups for another group.

[0102] One example of substitutions that may be made is changing one or more potentially chemically reactive cysteines in the antibody to another residue, such as, but not limited to, alanine or serine. For example, non-canonical cysteines may be substituted. Substitutions may be made in the CDR or framework regions of the variable domain or in the constant region of the antibody. In some embodiments, the cysteines are canonical. Cysteine ​​residues that are not involved in maintaining the proper conformation of the antibody may also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bond(s) may be added to improve the stability of the antibody, especially when the antibody is an antibody fragment such as an Fv fragment.

[0103] The antibody may be modified, for example, in the heavy and / or light chain variable domains, for example to alter its binding properties. Alterations in the variable regions may alter binding affinity and / or specificity. In some embodiments, no more than 1-5 conservative amino acid substitutions are made in the CDR domains. In other embodiments, no more than 1-3 conservative amino acid substitutions are made in the CDR domains. For example, the K of an antibody against the human Tim-3 IgV domain may be modified. D To increase or decrease k off Mutations may be made in one or more CDR regions to increase or decrease the binding affinity or to alter the binding specificity of the antibody. The technique of site-directed mutagenesis is well known in the art.

[0104] Modifications or mutations can also be made in the framework or constant regions to increase the half-life of the anti-human Tim-3 IgV domain antibody. See, for example, WO 00 / 09560. Mutations can also be made in the framework or constant regions to alter the immunogenicity of the antibody, to provide sites for covalent or non-covalent binding to another molecule, or to alter properties such as complement binding, FcR binding, and antibody-dependent cellular cytotoxicity. According to the present invention, a single antibody may have mutations in one or more CDRs or framework regions of the variable domain or in the constant region.

[0105] In some embodiments, the antibodies described herein are recombinantly produced, hi some embodiments, the fusion proteins are produced in eukaryotic expression systems.

[0106] In one embodiment, the fusion protein produced in a eukaryotic expression system contains glycosylation at the residue corresponding to Asn297 of Fc.

[0107] In certain embodiments, a composition or pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein is substantially pure with respect to the antibody or antigen-binding fragment thereof. A composition or pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein is "substantially pure" with respect to the antibody or antigen-binding fragment thereof if at least 60%-75% of a sample of the composition or pharmaceutical composition is a single antibody or antigen-binding fragment thereof. A substantially pure composition or pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein can comprise 60%, 70%, 80% or 90%, more usually about 95%, and preferably greater than 99% of a single antibody with respect to the antibody. Purity or homogeneity can be tested by various methods well known in the art, such as polyacrylamide gel electrophoresis or HPLC.

[0108] In one embodiment, human Tim3 has the sequence: MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIGIYIGAGICAGLALALIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQPLGCRFAMP (SEQ ID NO: 13).

[0109] In one embodiment, the sequence of the IgV domain of the antibody is SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPA (SEQ ID NO: 22) Includes.

[0110] As used herein, "and / or" as used herein, for example, A and / or B, includes the forms (i) A, (ii) B, and (iii) A and B.

[0111] All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated in the specification or clearly contradicted by context.

[0112] The present invention can be further understood by the following detailed experiments. EXAMPLES

[0113] Generation of mAbs that specifically bind to the IgV domain of human Tim-3: Tim-3 IgV-Ig fusion protein was obtained by fusing the human IgG1Fc tag of the plasmid pMT / BiP to the human Tim-3 IgV coding region (S22-A132) (Zhao 2013). The fusion protein was expressed in the S2 system and then purified. Mice were immunized with the Tim-3 IgV-Ig fusion protein, and hybridomas were obtained by standard methods of fusing splenocytes with NSO myeloma cells.

[0114] Description of mAb 50B5: The mAb 50B5 was obtained. It is an IgG1 with a kappa chain. In an ELISA assay, it bound to the Tim-3-IgV protein and the extracellular protein of Tim-3 (the IgV mucin stalk), but not to human IgG protein (Figure 1). FACS confirmed that 50B5 bound to human Tim-3 expressed on cells, but not to mouse Tim-3 (Figure 2).

[0115] Surface plasmon resonance analysis revealed that the binding affinity of mAb 50B5 to human Tim-3 protein is K D The binding activity of mAb 50B5 was 0.13 nM (Figure 3). To determine the inhibitory potential of mAb 50B5, we performed a binding assay and found that mAb 50B5 prevented the binding of human Tim-3 to phosphatidylserine expressed on dexamethasone-treated Jurkat T cells. Since Tim-3 is an inhibitory receptor expressed on IFNγ-producing T cells, we confirmed the antagonistic potential of mAb 50B5 by a mixed lymphocyte reaction. Mature dendritic cells differentiated from monocytes obtained from PBMC of one donor were incubated with purified T cells obtained from PBMC of another donor for 4 days. Divided CD4 and CD8 T cells expressing Tim-3 on their surface were found, but non-divided CD4 and CD8 T cells expressing Tim-3 were not found (Figure 4A). In a mixed lymphocyte reaction, mAb 50B5 or mouse IgG1 (control) was added, and it was found that mAb 50B5 increased IFNγ production by more than 3-fold compared to the control IgG1 (FIG. 4B).

[0116] Taken together, these results indicate that mAb 50B5 is an antagonistic antibody against human Tim-3.Finally, the mAb 50B5 hybridoma was sequenced and found to have unique VH and VL sequences.

[0117] [ka]

[0118] Description of mAb 15B4: Another mAb, 15B4, was obtained by the same method as described above. mAb 15B4 is an IgG1 with a kappa chain and binds to human Tim-3 expressed on cells (Figure 5). Surface plasmon resonance analysis showed that the binding affinity of mAb 15B4 to human Tim-3 protein was K D The binding activity of mAb 50B5 was 0.32 nM (Figure 6). Binding assays were performed to determine the inhibitory potency of mAb 50B5 and found that mAb 15B4 prevented the binding of human Tim-3 to phosphatidylserine expressed on dexamethasone-treated Jurkat T cells. Finally, the 15B4 hybridoma was sequenced and found to have unique VH and VL sequences.

[0119] [ka]

[0120] References Bird et al. Science 242(4877):423-426 (1988) Brown et al. Cancer Res 47(13):3577-3583 (1987) Cao et al. Immunity 26(3):311-321 (2007) Chiba et al. Nat Immunol 13(9):832-842 (2012) Chothia & Lesk J Mol Biol 196(4):901-917 (1987) Chothia et al. Nature 342(6252):877-883 (1989) Dall'Acqua et al. J Biol Chem 281(33):23514-23524 (2006) Daugherty et al. Nucleic Acids Res 19(9):2471–2476 (1991) DeKruyff et al. J Immunol 184(4):1918–1930 (2010) Hamers-Casterman et al. Nature 363(6428):446-448 (1993) Harris Biochem Soc Trans 23(4):1035–1038 (1995) Hurle & Gross Curr Opin Biotechnol 5(4):428-433 (1994) Huston et al. Proc Natl Acad Sci USA 85(16):5879-5883 (1988) Jones et al. Nature 321(6069):522-525 (1986) Koyama et al. Nat Commun 7:10501 (2016) Lobuglio et al. Proc Natl Acad Sci USA 86(11):4220-4224 (1989) Monney et al. Nature 415(6871):536-541 (2002) Presta Curr Opin Biotechnol 3(4):394-398 (1992) Riechmann et al. Nature 332(6162):323-327 (1988) Shaw et al. J Immunol 138(12):4534–4538 (1987) Sheriff & Constantine Nat Struct Biol 3(9):733-736 (1996) Vaswani & Hamilton Ann Allergy Asthma Immunol 81(2):105-115 (1998) Verhoeyen et al. Science 239(4847):1534-1536 (1988) Ward et al. Nature 341(6242):544-546 (1989) Wilker et al. Int Immunol 19(6):763-773 (2007) Winter et al. Nature 349(6307):293-299 (1991) Yeung et al. J Immunol 182:7663-7671 (2009) Zhao et al. Proc Natl Acad Sci USA 110(24):9879-9884 (2013) Zhu et al. Nat Immunol 6(12):1245-1252 (2005)

Claims

1. An antibody or antigen-binding fragment thereof that binds to the IgV domain of human Tim-3 (T cell immunoglobulin mucin domain containing 3) or the IgV mucin stalk of human Tim-3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of CDR1 to 3 set forth in SEQ ID NO: 15 and a light chain comprising the amino acid sequence of CDR1 to 3 set forth in SEQ ID NO: 17, and the antibody is IgG1, IgG2, IgG3 or IgG4.

2. The antibody or antigen-binding fragment thereof of claim 1 , wherein the framework regions of the light chain and the heavy chain have 85% or more identity to human framework regions.

3. The antibody or antigen-binding fragment thereof of claim 2 , wherein the framework regions of the light chain and the heavy chain are human framework regions.

4. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, having a human Fc region sequence.

5. An antibody or an antigen-binding fragment thereof described in any one of claims 1 to 3, wherein the antibody is IgG4.

6. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is chimeric or humanized.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, an scFv, a Fab fragment, a Fab' fragment and an F(ab)' fragment.

8. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3 linked or conjugated to a therapeutic agent.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein the therapeutic agent is a cytotoxic agent, a radioisotope, an immunomodulatory agent or a secondary antibody.

10. A nucleic acid encoding an antibody or an antigen-binding fragment thereof described in any one of claims 1 to 9.

11. A host cell containing the nucleic acid described in claim 10.

12. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in any one of claims 1 to 9 and a pharma- ceutically acceptable excipient, diluent and / or carrier.

13. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 for treating cancer in a subject.

14. The pharmaceutical composition of claim 13, wherein the cancer is a hematological malignancy.

15. The pharmaceutical composition of claim 13 , wherein the cancer comprises a solid tumor.

16. The pharmaceutical composition of claim 13, wherein the cancer comprises lung cancer, gastric cancer, head and neck cancer, neurinoma, melanoma or follicular B-cell non-Hodgkin's lymphoma.

17. The pharmaceutical composition of any one of claims 13 to 16, wherein the subject is undergoing anti-PD-1, anti-PD-L1 or anti-CTLA4 therapy.

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