Anti-human tim3 antibodies for in vitro diagnostics

Abstract

The disclosure provides binding agents (e.g., antibodies) against a human Hepatitis A virus cellular receptor 2 protein (TIM3), as well as kits and methods for using the same (e.g., immunoassays) as part of a companion diagnostic and for other applications. In some aspects, the binding agents described herein may be used in assays for detecting Non-Small Cell Lung Cancer (NSCLC) and / or other types of lung cancer, Head and Neck Squamous Cell Carcinoma (HNSCC), Hepatocellular Carcinoma (HCC) or other types of liver cancer, Renal cell carcinoma, malignant melanoma, gastro-intestinal cancer, colorectal cancer, urothelial carcinoma and other types of bladder cancer, mamma carcinoma and / or other types of breast cancer, ovarian cancer, cervical cancer, prostate cancer, pancreatic cancer, lymphoma / leukemia, malignant mesothelioma, or a cancer in another organ or cell type.

Description

AFS Ref. : 039062.00181ANTI-HUMAN TIM3 ANTIBODIES FOR IN VITRO DIAGNOSTICSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority to U.S. Provisional Application No. 63 / 693,104, filed September 10, 2024, the content of which is hereby incorporated by reference in its entirety.SEQUENCE LISTING

[0002] The present application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on September 7, 2025, is named 039062.00181 ST26. xml and is 19 KB in size.TECHNICAL FIELD

[0003] This disclosure generally relates to binding agents (e.g., antibodies) against a human T-cell immunoglobulin and mucin-domain containing-3 (TIM3) protein, as well as kits and methods for using the same (e.g., immunoassays) as part of a companion diagnostic and for other applications.BACKGROUND

[0004] The T-cell immunoglobulin and mucin-domain containing-3 protein (abbreviated herein as “TIM3”) is a type-I transmembrane protein that functions as a key regulator of immune responses. The canonical protein sequence of TIM has been assigned GenBank Accession No. GenBank Accession No. NP_116171.3 (SEQ ID NO: 1). TIM3 was originally identified as a receptor expressed on interferon-y-producing CD4+ and CD8+ T cells and shown to induce T cell death or exhaustion after binding to galectin-9. Later studies have reported that TIM3 expression is also important in regulating the activities of many other immune cell types, e.g., macrophages, monocytes, dendritic cells, mast cells, and natural killer cells). See Han et al., “Tim-3: an activation marker and activation limiter of innate immune cells.” Frontiers in Immunology 4 (2013): 449 (2013). TIM3 expression has been associated with many types of chronic diseases, including cancer. For example, TIM3+ T cells have been detected in patients with advanced melanoma, non-small cell lung cancer, breast cancer, kidney cancer, and follicular B-cell non-Hodgkin lymphoma. Moreover, the presence of TIM3+ regulatory T cells have been described as an effective indicator of lung cancer progression. SeeAFS Ref.: 039062.00181Anderson, “Tim-3: an emerging target in the cancer immunotherapy landscape.” Cancer Immunology Research 2.5 (2014): 393-398.

[0005] In view of these findings, TIM3 has emerged as a diagnostic or prognostic biomarker for various types of cancer. See, e.g., Chen, et al. “Prospects of TIM-3 as a Promising Diagnostic and Prognostic Biomarker for Cancer.” Discovery Medicine 31.162: 15-20 (2021), which sum rizes research findings which show that TIM3 is useful as a diagnostic biomarker for acute myeloid leukemia (AML), prostate cancer, lung cancer, colorectal cancer, glioma, and hepatic carcinoma, and as a prognostic biomarker for prostate cancer, lung cancer, colorectal cancer, and other solid tumors. Studies have also shown a close relationship between TIM3 and inhibitory receptors, such as PD-1. For example, many tumor-specific T cells express both PD-1 and TIM3, and these T cells have been shown to be dysfunctional compared to T cells that express only PD-1 or TIM3. See Fourcade, et al. “Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8+ T cell dysfunction in melanoma patients.” Journal of Experimental Medicine 207.10 (2010): 2175-2186.

[0006] In view of this clinical significance, TIM3 has emerged as a potential diagnostic or prognostic biomarker for various cancers, and as a target for anticancer therapy. See Lu et al., “Promising immunotherapy targets: TIM3, LAG3, and TIGIT joined the party .” Molecular Therapy Oncology 32.1 (2024); Chen et al. (2021).BRIEF SUMMARY OF THE DISCLOSURE

[0007] Antibodies and other binding agents specific for TIM3 are commercially available. However, there remains a need in the art for additional high affinity TIM3-binding agents. In alternative embodiments, provided herein are chimeric or recombinant antibodies (Abs), or an antigen (Ag) binding fragments thereof, or a monomeric or dimeric antigen binding protein, capable of specifically binding to a human TIM3 polypeptide, or a fragment thereof, including human TIM3 polypeptides (or fragments thereof) expressed on the surface of lymphocytes such as activated T cells that have infiltrated tumors, or human TIM3 polypeptides expressed on tumor infiltrating lymphocytes (TILs). In some aspects, the TIM3 polypeptide may be expressed on the surface of tumor cells.

[0008] In a first general aspect, the disclosure provides a chimeric or a recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigenbinding protein, capable of specifically binding to a human TIM3 polypeptide having an amino acid sequence comprising or consisting of SEQ ID NO: 1, or a fragment thereof. In some aspects, a fragment may comprise or consist of at least, at most or exactly 10, 15, 20, 25, 30,AFS Ref.: 039062.0018135, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 contiguous amino acids of SEQ ID NO: 1, or an amino acid sequence comprising or consisting of a number of contiguous amino acids of SEQ ID NO: 1 within a range defined by any pair of the foregoing lengths.

[0009] In some aspects, the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein is fabricated as or in the form of an antigen-binding fragment, a F(ab')2, a Fab', a single-chain variable fragment (scFv), an (SCFV)2, a minibody, a diabody, or a tetrabody, or as any other type of recombinant antigenbinding polypeptide or peptide construct known in the art (e.g., as an scFv comprising one or more of the complementarity determining regions, “CDRs” sequences described herein).

[0010] In some aspects, the chimeric or recombinant antibody (Ab), or antigen-binding- fragment thereof, or monomeric or dimeric antigen-binding protein comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO:8; and / or a light chain variable domain having the amino acid sequence of SEQ ID NO:9.

[0011] In some aspects, the chimeric or recombinant antibody (Ab), or antigen-binding- fragment thereof, or monomeric or dimeric antigen-binding protein comprises: (a) a heavy chain variable domain and a constant domain having SEQ ID NO: 14; (b) a heavy chain signal peptide having SEQ ID NO: 12; (c) a heavy chain variable domain and a signal peptide sequence having SEQ ID NO: 16; (d) a heavy chain variable domain and a constant domain and a signal peptide sequence having SEQ ID NO: 10; (e) a light chain variable domain and a constant domain having SEQ ID NO: 15; (f) a light chain signal peptide having SEQ ID NO: 13; (g) a light chain variable domain and signal peptide sequence having SEQ ID NO: 17; (h) a light chain variable domain and a constant domain and a signal peptide sequence having SEQ ID NO: 11;

[0012] In some aspects, the chimeric or recombinant antibody (Ab), or antigen-binding- fragment thereof, or monomeric or dimeric antigen-binding protein comprises: (1) a heavy chain variable domain comprising one or more CDRs having (a) an amino acid sequence selected from SEQ ID NOs:2, 3, or 4; or (b) an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions compared to any one of SEQ ID NOs: 2, 3, 4, optionally wherein the one or more substitutions are conservative substitutions; and / or (2) a light chain variable domain comprising one or more CDRs having (a) an amino acid sequence selected from SEQ ID NOs:5, 6, or 7; or (b) an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions compared to any one of SEQ ID NOs: 5, 6, or 7, optionally wherein the one or more substitutions are conservative substitutions.AFS Ref.: 039062.00181

[0013] In some aspects, the chimeric or recombinant antibody (Ab), or antigen-binding- fragment thereof, or monomeric or dimeric antigen-binding protein includes a heavy chain variable domain, wherein the sequence of the heavy chain variable domain comprises SEQ ID NO: 8 having at least one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve conservative amino acid substitutions, wherein the heavy chain variable domain is capable of specifically binding to the human TIM3 polypeptide having the amino acid sequence of SEQ ID NO: 1, or a fragment thereof, when either unpaired or paired with a light chain variable domain.

[0014] In some aspects, the chimeric or recombinant antibody (Ab), or antigen-binding- fragment thereof, or monomeric or dimeric antigen-binding protein includes a light chain variable domain, wherein the sequence of the light chain variable domain comprises SEQ ID NO: 9 having at least one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve conservative amino acid substitutions, wherein the light chain variable domain is capable of specifically binding to the human TIM3 polypeptide having the amino acid sequence of SEQ ID NO: 1, or a fragment thereof, when either unpaired or paired with a heavy chain variable domain.

[0015] In some aspects, the chimeric or recombinant antibody (Ab), or antigen-binding- fragment thereof, or monomeric or dimeric antigen-binding protein comprises a heavy chain variable domain, wherein the sequence of the heavy chain variable domain has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity compared to SEQ ID NO:8.

[0016] In some aspects, the chimeric or recombinant antibody (Ab), or antigen-binding- fragment thereof, or monomeric or dimeric antigen-binding protein comprises a light chain variable domain, wherein the sequence of the light chain variable domain has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity compared to SEQ ID NO:9.

[0017] In some aspects, the chimeric or recombinant antibody (Ab), or antigen-binding- fragment thereof, or monomeric or dimeric antigen-binding protein comprises a heavy chain variable domain, wherein the sequence of the heavy chain variable domain and the amino acid sequence of SEQ ID NO: 8 have a Z score of from about 2 to about 8, or a Z score of at least 8, when aligned using distance matrix alignment.

[0018] In some aspects, the chimeric or recombinant antibody (Ab), or antigen-binding- fragment thereof, or monomeric or dimeric antigen-binding protein comprises a light chainAFS Ref.: 039062.00181 variable domain, wherein the sequence of the light chain variable domain and the amino acid sequence of SEQ ID NO: 9 have a Z score of from about 2 to about 8, or a Z score of at least 8, when aligned using distance matrix alignment.

[0019] In some aspects, the chimeric or recombinant antibody (Ab), or antigen-binding- fragment thereof, or monomeric or dimeric antigen-binding protein comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:8; or CDR1 amino acid (aa) residues 25-32, CDR2 aa residues 50-56, and / or CDR3 aa residues 94-104, of SEQ ID NO:8.

[0020] In some aspects, the chimeric or recombinant antibody (Ab), or antigen-binding- fragment thereof, or monomeric or dimeric antigen-binding protein comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:9; or CDR1 amino acid (aa) residues 27-32, CDR2 aa residues 50-52, and / or CDR3 aa residues 89-100, of SEQ ID NO:9.

[0021] In some aspects, the chimeric or recombinant antibody (Ab), or antigen-binding- fragment thereof, or monomeric or dimeric antigen-binding protein, comprises an antibody heavy chain selected from an IgM, IgG, IgA or IgE isotype heavy chain, and / or a light chain selected from a kappa or a lambda light chain.

[0022] In some aspects, the chimeric or recombinant antibody (Ab), or antigen-binding- fragment thereof, or monomeric or dimeric antigen-binding protein, further comprises, or is bound to or covalently conjugated to, a detectable agent or a binding moiety. In some aspects, the detectable agent comprises: an enzyme, a biotin, a fluorescent or chemiluminescent label, a fluorophore, sulfoindo-cyanine, nile red, rhodamine, perylene, fluorenyl, coumarin, 7- methoxycoumarin (Mca), dabcyl, [2-(4-nitro-2,l,3-benzoxadiazol-7- yl)aminoethyl]trimethylammonium (NBD), Nile blue, Tamra, boron-dipyrromethene (BODIPY), or derivatives thereof), a dye, a radioisotope, a quantum dot or photoluminescent aqueous nanocrystal, a hapten or an antibody binding epitope or domain. In some aspects, the enzyme is a peroxidase, an alkaline phosphatase, or a beta-galactosidase. In some aspects, the peroxidase is a horse radish peroxidase (HRP). In some aspects, the hapten comprises a biotin, theophylline, digoxigenin, carborane, fluorescein or bromodeoxyuridine. In some aspects, the dye comprises a cyanine dye; or Cy3 or Cy5. In some aspects, the fluorophore comprises dansyl, fluorescein or carboxyfluorescein (FAM) or 6-FAM. In some aspects, the binding moiety comprises a glutathione S-transferase (GST) or ligandin tag, a polyhistidine (poly-his) tag, a chitin binding protein (CBP), a STREP-TAG™ (SEQ ID NO: 18) peptide tag, a FLAG tag (SEQ ID NO: 19) peptide tag, or a maltose binding protein.AFS Ref.: 039062.00181

[0023] In a second general aspect, the disclosure provides a recombinant nucleic acid molecule encoding any chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, described herein. In some aspects, the recombinant nucleic acid molecule further comprises and is operatively linked to a transcriptional regulatory element. In some aspects, the transcriptional regulatory element comprises a promoter (e.g., an inducible or a constitutive promoter).

[0024] In some aspects, the recombinant nucleic acid molecule further comprises sequence encoding an additional protein or peptide moiety or domain. In some aspects, the additional protein or peptide moiety or domain comprises a purification moiety or domain to aid in the purification or isolation of the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, encoded by the recombinant nucleic acid. In some aspects, the additional protein or peptide moiety or domain comprises: a glutathione S-transferase (GST) or ligandin tag, a polyhistidine (poly-his) tag, a chitin binding protein (CBP), a STREP-TAG™ (SEQ ID NO: 18) peptide tag, a FLAG tag (SEQ ID NO: 19) peptide tag, or a maltose binding protein.

[0025] In some aspects, the recombinant nucleic acid molecule further comprises sequence encoding a protease cleavage site positioned between the purification moiety or domain and the sequence encoding the chimeric or a recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein.

[0026] In a third general aspect, the disclosure provides an expression cassette, vector, recombinant virus, artificial chromosome, cosmid or plasmid comprising any of the recombinant nucleic acid molecules described herein.

[0027] In a fourth general aspect, the disclosure provides a cell comprising any chimeric or a recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein described herein, any recombinant nucleic acid molecule described herein, or an expression cassette, vector, recombinant virus, artificial chromosome, cosmid or plasmid described herein. In some aspects, the cell is a bacterial, fungal, mammalian, yeast, insect, avian or plant cell.

[0028] In a fifth general aspect, the disclosure provides a method for generating an polyclonal antibody, or for generating a polyclonal immune serum, that is specific for or specifically binds to a human TIM3 polypeptide, the method comprising administering to or immunizing a mammal or an avian species with a peptide or polypeptide, or an epitope, comprising the amino acid sequence of SEQ ID NO: 1, or a fragment thereof.AFS Ref.: 039062.00181

[0029] In a sixth general aspect, the disclosure provides a method for detecting the presence of a human TIM3 polypeptide in a cell, a tissue, an organ, or a portion thereof, comprising: (a) contacting the cell, tissue, organ, or portion thereof with any chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein described herein, or encoded by a recombinant nucleic acid molecule described herein, and (b) detecting the specific binding of the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, with a human TIM3 polypeptide having the amino acid sequence of SEQ ID NO:1, or a fragment thereof, in the cell, tissue, organ, or portion thereof. In some aspects, the contacting comprises use of an immunohistochemistry (IHC) assay.

[0030] In some aspects, the method further comprises contacting the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein with a detectable agent to indicate or signal the specific binding of the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein to the human TIM3 polypeptide or the fragment thereof. In some aspects, the detectable agent specifically binds to the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein. In some aspects, the detectable agent is or comprises an antibody or an antigen-binding fragment or a secondary antibody thereof that specifically binds to the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein which is bound to the human TIM3 polypeptide. In some aspects, the detectable agent is or comprises an antibody or an antigen-binding fragment or a secondary antibody thereof that specifically binds to a hapten or tag attached or conjugated to the chimeric or recombinant antibody (Ab) or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein.

[0031] In some aspects, the antibody or an antigen-binding fragment or the secondary antibody further comprises or has attached or conjugated thereto a second detectable agent or an enzyme. In some aspects, the enzyme is an alkaline phosphatase, a beta-galactosidase or a peroxidase. In some aspects, the antibody or an antigen-binding fragment or the secondary antibody further comprises or has attached or conjugated thereto a biotin, a fluorescent or chemiluminescent label, a fluorophore, sulfoindo-cyanine, nile red, rhodamine, perylene, fluorenyl, coumarin, 7-m ethoxy coumarin (Mca), dabcyl, [2-(4-nitro-2,l,3-benzoxadiazol-7- yl)aminoethyl]trimethylammonium (NBD), Nile blue, Tamra, boron-dipyrrometheneAFS Ref.: 039062.00181(BODIPY), or derivatives thereof), a dye, a radioisotope, a quantum dot or photoluminescent aqueous nanocrystal, a hapten. In some aspects, the dye comprises a cyanine dye, or Cy3 or Cy5. In some aspects, the hapten comprises a biotin, theophylline, digoxigenin, carborane, fluorescein or bromodeoxyuridine.

[0032] In a seventh general aspect, the disclosure provides a method for diagnosing a TIM3 protein-expressing cancer, comprising: detecting the expression or presence of a TIM3 polypeptide or fragment thereof, in a cell, tissue, or organ sample, or a portion thereof, by contacting the cell, tissue, organ sample, or portion thereof with any chimeric or recombinant antibody or antigen-binding fragment thereof described herein, or encoded by any recombinant nucleic acid molecule described herein, and detecting whether or not the chimeric or recombinant antibody specifically binds to the human TIM3 polypeptide or fragment thereof in the cell, tissue, organ sample, or portion thereof, wherein the detecting of specific binding indicates the expression or presence of the human TIM3 polypeptide or fragment thereof, in the cell, tissue, or organ sample, or the portion thereof.

[0033] In some aspects, the cell is an activated T cell, an activated T cell that has infiltrated a tumor, or a tumor infiltrating lymphocyte (TIL).

[0034] In some aspects, the detecting of specific binding indicates the expression or presence of the human TIM3 polypeptide or fragment thereof, in the cell, tissue or organ sample, or portion thereof, thereby characterizing the cancer in detail. In some aspects, the cancer is selected from the group consisting of a Non-Small Cell Lung Cancer (NSCLC) and / or other types of lung cancer, Head and Neck Squamous Cell Carcinoma (HNSCC), Hepatocellular Carcinoma (HCC) or other types of liver cancer, Renal cell carcinoma, malignant melanoma, gastro-intestinal cancer, colorectal cancer, urothelial carcinoma and other types of bladder cancer, mamma carcinoma and / or other types of breast cancer, ovarian cancer, cervical cancer, prostate cancer, pancreatic cancer, lymphoma / leukemia, malignant mesothelioma, or cancer in other organs or cell types.

[0035] In some aspects, the detection comprises using or conducting an immunohistochemistry (IHC) assay or a flow cytometry. In some aspects, the conducting or using of the flow cytometry comprises use of a fluorescence-activated cell sorter (FACS) or an impedance flow cytometer. In some aspects, the cell, tissue, organ sample, or portion thereof is or is derived from a biopsy from a patient.

[0036] In an eighth general aspect, the disclosure provides a method for treating, ameliorating or preventing a cancer comprising first characterizing a cancer in detail, in anAFS Ref.: 039062.00181 individual in need thereof, using any of the methods described herein, followed by treatment of the individual in need thereof. In some aspects, the cancer is selected from the group consisting of: Non-Small Cell Lung Cancer (NSCLC) and / or other types of lung cancer, Head and Neck Squamous Cell Carcinoma (HNSCC), Hepatocellular Carcinoma (HCC) or other types of liver cancer, Renal cell carcinoma, malignant melanoma, gastro-intestinal cancer, colorectal cancer, urothelial carcinoma and other types of bladder cancer, mamma carcinoma and / or other types of breast cancer, ovarian cancer, cervical cancer, prostate cancer, pancreatic cancer, lymphoma / leukemia, malignant mesothelioma or cancer in other organs or cell types.

[0037] In a ninth general aspect, the disclosure describes use of a chimeric or a recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigenbinding protein according to any exemplary aspect described herein, or encoded by any of the recombinant nucleic acid molecules described herein, for characterizing a cancer, or treating, ameliorating or preventing a cancer. The disclosure further provides a chimeric or a recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein according to any exemplary aspect described herein, or encoded by any of the recombinant nucleic acid molecules described herein, for use in characterizing a cancer, or treating, ameliorating or preventing a cancer.

[0038] In a tenth general aspect, the disclosure provides a kit comprising any chimeric or a recombinant antibody (Ab), or antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein described herein, or encoded by any of the recombinant nucleic acid molecules described herein. In some aspects, the kit comprises components and / or instructions needed for an immunohistochemistry (IHC) assay (e.g., according to any of the detection methods described herein). In some aspects, the chimeric or the recombinant antibody (Ab), the antigen-binding fragment thereof, or the monomeric or dimeric antigen-binding protein, is substantially purified or isolated.

[0039] In an eleventh general aspect, the disclosure provides a product of manufacture comprising any chimeric or a recombinant antibody (Ab), or antigen-binding fragment thereof, described herein, or encoded by any recombinant nucleic acid molecule described herein. In some aspects, the product of manufacture comprises or is fabricated as or manufactured as a slide, a well, a chip, a biochip, an array, a tray, a dish or a microtiter plate or dish. In some aspects, the chimeric or the recombinant antibody (Ab), the antigen-binding fragment thereof, or the monomeric or dimeric antigen-binding protein, is substantially purified or isolated, or is in the form of an unpurified or partially purified culture supernatant.AFS Ref.: 039062.00181

[0040] In a twelfth general aspect, the disclosure provides a phage or a phagemid comprising or expressing on its surface any chimeric or recombinant antibody (Ab), antigenbinding fragment thereof, or monomeric or dimeric antigen-binding protein described herein, or encoded by any of the recombinant nucleic acid molecules described herein.

[0041] To the accomplishment of the foregoing and related ends, the one or more aspects comprise the features hereinafter fully described and particularly pointed out in the claims. The following description and the annexed drawings set forth in detail certain illustrative features of the one or more aspects. These features are indicative, however, of but a few of the various ways in which the principles of various aspects may be employed, and this description is intended to include all such aspects and their equivalents.DESCRIPTION OF DRAWINGS

[0042] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0043] The drawings set forth herein illustrate and describe exemplary aspects of the disclosure and are not meant to limit the scope of the invention as defined by the claims.

[0044] FIG 1 IHC staining of lymphocytes in germinal center, mantle zone, T zones and crypt epithelia of normal tonsil. TIM3, clone 1C12 (right) and TIM3 reference (left).

[0045] FIG 2. IHC staining of tumor infiltrating lymphocytes (TILs) in liver hepatocellular carcinoma (HCC). TIM3, clone 1C12 (right) and TIM3 reference (left).

[0046] FIG 3 IHC staining of tumor infiltrating lymphocytes (TILs) in lung non small cell carcinoma (NSCLC). TIM3, clone 1C12 (right) and TIM3 reference (left).

[0047] FIG 4 IHC staining of tumor infiltrating lymphocytes (TILs) in lung non small cell carcinoma (NSCLC). TIM3, clone 1C12 (right) and TIM3 reference (left).

[0048] FIG 5. IHC staining of tumor infiltrating lymphocytes (TILs) in melanoma. TIM3, clone 1C12 (right) and TIM3 reference (left).

[0049] FIG 6 IHC single staining with CD8 (magenta) left, and double staining with TIM3, clone 1C12 (DAP) and CD8 (magenta) right. Normal tonsil mantle zone and germinal center. Blue arrow illustrate possibly co-localized of TIM 3 and CD8+ positive T cells.

[0050] FIG 7. IHC single staining with TIM3, clone 1C12 (DAP) left, and double staining with TIM3, clone 1C12 (DAP) and PD-L1 (magenta) right. Normal tonsil epithelium. Blue arrow possibly co-localized TIM3 andPD-Ll in activated T cell, green arrow PD-L1 positive epithelial cells.AFS Ref.: 039062.00181

[0051] FIG 8. IHC single staining with TIM3, clone 1C12 (magenta) left, and double staining with TIM3, clone 1C12 (magenta) and CD8 (DAP) right. Lung NSCLC. Blue arrow possibly co-localized TIM3 and CD8 positive activated T cells among TILs.

[0052] FIG 9. IHC single staining with PD-L1 (magenta) left, and double staining with TIM3, clone 1C12 (DAP) and PD-L1 (magenta) right. Lung NSCLC. Blue arrow possible colocalized TIM 3 and PD-L1 in activated T cell and macrophage in TILs, yellow arrow TIM 3 positive T cell, green arrow PD-L1 positive tumor cells.

[0053] FIG 10. IHC single staining with CD8 (magenta) left, and double staining with TIM3, clone 1C12 (DAP) and CD8 (magenta) right. Liver HCC. Blue arrow possibly colocalized TIM 3 and CD8+ positive T cells in TILs. Red arrow negative tumor cells.

[0054] FIG 11 IHC single staining with CD8 (magenta) left, and double staining with TIM3, clone 1C12 (DAP) and CD8 (magenta) right. Melanoma. Blue arrow possibly colocalized TIM3 and CD8 positive T cells among TILs, red arrow negative tumor cells.DETAILED DESCRIPTION

[0055] In alternative embodiments, provided are chimeric or a recombinant antibodies (Ab), or antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins, that can specifically bind to a human TIM3 polypeptide (SEQ ID NO: 1) or a fragment thereof. In alternative embodiments, provided are products of manufacture and kits comprising the chimeric or a recombinant Abs, or antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins, as provided herein, or nucleic acid molecules encoding them, and methods for making and using them. In alternative embodiments, chimeric or a recombinant antibodies (Ab), or antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins as provided herein are used for in vitro diagnostics, for example, by immunohistochemistry (IHC), for example, in IHC protocols to characterize, and / or treat a cancer, for example aNon-Small Cell Lung Cancer (NSCLC) and / or other types of lung cancer, Head and Neck Squamous Cell Carcinoma (HNSCC), Hepatocellular Carcinoma (HCC) or other types of liver cancer, Renal cell carcinoma, malignant melanoma, gastro-intestinal cancer, colorectal cancer, urothelial carcinoma and other types of bladder cancer, mamma carcinoma and / or other types of breast cancer, ovarian cancer, cervical cancer, prostate cancer, pancreatic cancer, lymphoma / leukemia, malignant mesothelioma, or a cancer in another organ or cell type, by their ability to specifically bind to activated T cells that have infiltrated a tumor, for example, including tumor infiltrating lymphocytes (TILs). Thus, the chimeric or a recombinant antibodies (Ab), or antigen binding fragments thereof, or monomeric or dimeric antigenAFS Ref.: 039062.00181 binding proteins as provided herein can be used as a companion diagnostic for the diagnosis and treatment of cancer by specifically staining TILs.

[0056] Expression of Recombinant Chimeric Antibodies

[0057] In alternative embodiments, chimeric and / or recombinant antibodies (Abs), the antigen binding fragments thereof, or the monomeric or dimeric antigen binding proteins are provided herein, including exemplary chimeric or recombinant anti-human TIM3 Abs comprising the heavy chain variable domain of SEQ ID NO: 8, and / or light chain variable domain of SEQ ID NO:9, with or without a signal peptide, can be expressed as a recombinant Ab using, for example, a plasmid or any expression vehicle encoding the respective heavy and light chains, or the heavy chain and the light chain can be encoded in separate expression vehicles.

[0058] In some embodiments, the heavy and light chains can be (cis- or trans-) expressed from any plasmid, cosmid, recombinant virus or equivalent vector, for example, from a pTT5™ vector(s) (National Research Council Canada, NRC-CNRC, Canada) or equivalents.

[0059] In alternative embodiments, the expression vehicles (such as a plasmid) containing exemplary Ab-encoding nucleic acid(s) as provided herein are expressed in in vitro expression systems or are expressed in cultured tissues, cells or organoids, which can be a bacterial, fungal, mammalian, yeast, insect or plant cell expression systems, or hybrid or synthetic expression system. For example, exemplary Ab-encoding nucleic acid(s) can be expressed in a human embryonic kidney (HEK) cell such as an HEK293- 6E cell. In alternative embodiment, the vector or vectors expressing exemplary Ab- encoding nucleic acid(s), for example, exemplary heavy and / or light chains, are episomal or are chromosomally integrated, for example, in a stable cell line capable of synthesizing, optionally inducibly synthesizing, the heavy and / or light chains.

[0060] In alternative embodiments, provided are nucleic acid molecules encoding chimeric or recombinant Abs as provided herein. Nucleic acid molecules as provided herein can be made, isolated and / or manipulated by, for example, cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, and the like. Nucleic acid molecules used to practice embodiments as provided herein, whether RNA, cDNA, genomic DNA, vectors, viruses or hybrids thereof, may be isolated from a variety of sources, genetically engineered, amplified, and / or expressed / generated recombinantly. Recombinant polypeptides generated from these nucleic acid molecules can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including bacterial, fungal,AFS Ref.: 039062.00181 mammalian, yeast, insect or plant cell expression systems, or hybrid or synthetic expression systems.

[0061] Alternatively, these nucleic acid molecules can be synthesized in vitro by well- known chemical synthesis techniques, as described in, for example, Martin et al, ACS Synth. Biol. (2017) 6, 7, 1370-1379; Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acid Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373- 380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; U.S. Patent No. 4,458,066.

[0062] Techniques for the manipulation of nucleic acid molecules, such as, for example, subcloning, labeling probes (for example, random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, for example, Sambrook, ed., Molecular Cloning: A Laboratory Manual (2ndEd.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); Current Protocols In Molecular Biology, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); Laboratory Techniques In Biochemistry And Molecular Biology. Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

[0063] Another useful means of obtaining and manipulating nucleic acid molecules used to practice embodiments as provided herein comprises screening and re-cloning inserts isolated or amplified from, for example, genomic clones or cDNA clones. Sources of nucleic acid molecules include recombinant nucleic acid sequences, genomic or cDNA libraries contained and / or expressed in, for example, mammalian artificial chromosomes (MACs), see, for example, U.S. Patent Nos. 5,721,118; 6,025,155; human artificial chromosomes, see, for example, Rosenfeld (1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC); bacterial artificial chromosomes (BAC); Pl artificial chromosomes, see, for example, Woon (1998) Genomics 50:306-316; Pl-derived vectors (PACs), see, for example, Kern (1997) Biotechniques 23: 120-124; cosmids, recombinant viruses, phages or plasmids.

[0064] In alternative embodiments, nucleic acid molecules as provided herein are operably linked to transcriptional regulatory elements, including promoters, with can be constitutive or inducible transcriptional regulatory elements.

[0065] In alternative aspects, provided are “expression cassettes” comprising a nucleotide sequence as provided herein, for example encoding a chimeric or recombinant antibody as provided herein. Expression cassettes can include at least a transcriptional regulatory element,AFS Ref.: 039062.00181 for example, a promoter, operably linked with an antibody coding sequence, and optionally can also include transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used, for example, enhancers.

[0066] In alternative aspects, expression cassettes used to practice embodiments as provided herein include plasmids, expression vectors, recombinant viruses, any form of recombinant “naked DNA” vector, and the like. In alternative aspects, a “vector” used to practice embodiments as provided herein can comprise a nucleic acid molecule that can infect, transfect, transiently or permanently transduce a cell. In alternative aspects, a vector used to practice embodiments as provided herein can be a naked nucleic acid molecule, or a nucleic acid molecule complexed with protein or lipid. In alternative aspects, vectors used to practice embodiments as provided herein can comprise viral or bacterial nucleic acid molecules and / or proteins, and / or membranes (for example, a cell membrane, a viral lipid envelope, etc.).

[0067] In alternative aspects, vectors used to practice embodiments as provided herein can include, but are not limited to replicons (for example, RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated. Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (for example, plasmids, viruses, and the like, see, for example, U.S. Patent No. 5,217,879), and can include both the expression and non-expression plasmids. In alternative aspects, the vector used to practice embodiments as provided herein can be stably replicated by the cells during mitosis as an autonomous structure, or can be incorporated within the host’s genome.

[0068] In alternative aspects, “promoters” used to practice embodiments as provided herein include all sequences capable of driving transcription of a coding sequence in a cell, for example, a bacterial, yeast, fungal, plant, insect (for example, baculovirus) or mammalian cell. Thus, promoters used in the constructs include cv.s-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and / or rate of transcription of a gene. For example, a promoter used to practice embodiments as provided herein can be a cv.s-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences can interact with proteins or other biomolecules to carry out (turn on / off, regulate, modulate, etc.) transcription.

[0069] Constitutive” promoters used to practice embodiments as provided herein can be those that drive expression continuously under most environmental conditions and states ofAFS Ref.: 039062.00181 development or cell differentiation. “Inducible” or “regulatable” promoters used to practice embodiments as provided herein can direct expression of a nucleic acid sequence as provided herein under the influence of environmental conditions or developmental conditions. Examples of environmental conditions that may affect transcription by inducible promoters used to practice embodiments as provided herein include the presence of an inducing factor administered to a cell.

[0070] In alternative embodiments, antibodies used to practice embodiments as provided herein can comprise any “mimetic” and / or “peptidomimetic” form. In alternative embodiments, peptides and polypeptides used to practice embodiments as provided herein can comprise synthetic chemical compounds which have substantially the same structural and / or functional characteristics of the natural polypeptide, for example, a chimeric or recombinant antibody as provided herein. The mimetic used to practice embodiments as provided herein can be either entirely composed of synthetic, non- natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic’s structure and / or activity. Routine experimentation will determine whether a mimetic is effective for practicing the invention, for example, if a mimetic composition is effective in specifically binding to a human TIM3 polypeptide. Methodologies detailed herein and others known to persons skilled in the art may be used to select or guide one to choose effective mimetic for practicing the compositions and / or methods as provided herein.

[0071] Polypeptide mimetic compositions for practicing embodiments as provided herein can comprise any combination of non-natural structural components. In alternative aspects, mimetic compositions for practicing embodiments as provided herein can comprise one or all of the following three structural groups: a) residue linkage groups other than the natural amide bond (“peptide bond”) linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, for example, a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like. For example, a polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds.

[0072] Purification and Isolation of Recombinant Proteins

[0073] In alternative embodiments, chimeric or the recombinant antibodies, antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins, are substantiallyAFS Ref.: 039062.00181 purified or isolated, and optionally the substantially purified or isolated forms are the forms used in immunohistochemistry methodologies and / or as reagents, kits and / or products of manufacture as provided herein.

[0074] In alternative embodiments, chimeric or the recombinant antibodies, antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins, are substantially purified or isolated using: physicochemical fractionation, for example, using differential precipitation, size-exclusion or solid-phase binding of immunoglobulins based on size, charge or other shared chemical characteristics of antibodies in typical samples; class-specific affinity, for example, solid-phase binding of particular antibody classes (for example, IgG or IgM) by immobilized biological ligands (for example, proteins, lectins, and the like) that have specific affinity to immunoglobulins, and this can purify all antibodies of the target class without regard to antigen specificity; or antigen- specific affinity, for example, affinity purification of only those antibodies in a sample that bind to a particular antigen molecule through their specific antigen-binding domains, where this purifies all antibodies that bind the antigen without regard to antibody class or isotype.

[0075] In alternative embodiments, chimeric or the recombinant antibodies, antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins, are substantially purified or isolated using standard isolation methodologies such as chromatography, for example, Ion Exchange (IEX) Chromatography, Hydrophobic Interaction Chromatography (HIC), countercurrent chromatography, immunoaffinity and / or size exclusion chromatography.

[0076] In alternative embodiments, chimeric or the recombinant antibodies, antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins, are generated in bioreactors, for example, a perfusion bioreactor, using continuous expression and purification processes, for example, as described by Vogg et al., n Methods Mol Biol. 2018; vol 1850: 147- 178, or using stirred-tank or rocking bioreactor systems, followed by purification.

[0077] Products of manufacture and Kits

[0078] In some aspects, products of manufacture and kits comprising at least one antihuman TIM3 Ab, or an antigen-binding fragment thereof, are provided, as well as methods of using the same; optionally, the products of manufacture and kits can further comprise some or all reagents needed to perform an IHC, and optionally can comprise instructions for practicing any of the methods described herein. Such products of manufacture and kits may comprise one or more anti-human TIM3 Abs disclosed herein (e.g., recombinant or chimeric Abs), or antigen-binding fragments thereof.AFS Ref.: 039062.00181

[0079] In alternative embodiments, products of manufacture have attached thereto or affixed (optionally covalently bound) on or onto chimeric or a recombinant antibodies (Abs), or antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins as provided herein, and optionally products of manufacture as provided herein are or comprise arrays, biochips, slides, trays, dishes (for example, microtiter dishes), phages or phagemids.

[0080] Immunohistochemistry

[0081] In alternative embodiments, immunohistochemistry methodologies and / or reagents used to practice compositions, products of manufacture, kits or methods as provided herein can include or comprise or comprise use of any IHC protocol, IHC armamentarium, device and / or image or data analysis system, for practicing IHC or IHC reagents known in the art, for example, as described in U.S. patent nos. (USPNs) 10,565,479 (describing methods for identifying blurred areas in digital images of stained tissue); 10,564,076 (describing systems for analytical ( or IHC) sample preparation); 10,551,395 (describing an automated histological staining system); 10,551,378 (describing a tissue staining method); 10,504,224 (describing a digital tissue image analysis system for IHC); 10,501,777 (describing simultaneous, multiplexed detection and quantification of protein expression in IHC); 10,488,340 (describing method for extracting an image of a target fluorophore in a biological material); 10,453,195 (describing methods of detecting tissue areas of interest using digital pathology imaging); 10,438,381 (describing devices, systems and methods for generating a digital image of a tissue section); 10,416,176 (describing methods for processing specimens in an automated histological staining system); 10,393,633 (describing methods for processing and inhibiting the degradation of an IHC sample); 10,217,011 (describing handling of IHC slides); 10,209,165 (describing automated or semi-automated methods for assessing the quality of staining of a specimen containing cells); 10,126,216 (describing methods for fixing tissue samples for IHC); 9,423,322.

[0082] In alternative embodiments, chimeric or the recombinant antibodies, antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins, in IHC protocols, or kits, as provided herein are substantially purified or isolated or are in the form of an unpurified or partially purified culture supernatant.

[0083] In alternative embodiments, methods as provided herein can use or comprise reagents for detecting or visualizing an antibody-antigen interaction using any products or methods know in the art, for example, and IHC protocol or reagents.AFS Ref.: 039062.00181

[0084] In alternative embodiments, methods as provided herein comprise use of chromogenic immunohistochemistry (CIH), wherein a primary antibody (for example, chimeric or a recombinant antibodies (Ab), or antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins as provided herein) or secondary antibody (for example, where the secondary antibody binds to (the primary antibody) chimeric or a recombinant antibodies (Ab), or antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins as provided herein after they have specifically bound to, paired with, associated with, or configured with a LAG-3 epitope or polypeptide) is conjugated to an enzyme, such as peroxidase (or immunoperoxidase), for example, a horseradish peroxidase (HRP), that can catalyze a color-producing reaction.

[0085] In alternative embodiments, methods as provided herein comprise use of immunofluorescence, where a primary or a secondary antibody is tagged to a fluorophore, such as fluorescein or fluorescein isothiocyanate (FITC), a triarylmethane dye such as rhodamine or rhodamine derivatives (for example, tetramethylrhodamine (TRITC), rhodamine 6G, rhodamine 123, rhodamine B, carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TMR), sulforhodamine 101), aminomethylcoumarin acetate (AMCA), ALEXA™ or DYLIGHT™ fluors. 3,3’-Diaminobenzidine (DAB) also can be used.

[0086] In alternative embodiments, methods as provided herein comprise use of a direct method or one-step staining method where a primary antibody (for example, chimeric or a recombinant antibodies (Ab), or antigen binding fragments thereof, or monomeric or dimeric antigen binding proteins as provided herein) is labeled and reacts directly with an antigen, for example, in a tissue sections. While this technique utilizes only one antibody and therefore is simple and rapid, the sensitivity may be lower due to little signal amplification.

[0087] In alternative embodiments, methods as provided herein comprise use of an indirect method where an unlabeled primary antibody (first layer) binds to a target antigen (human TIM3, or a fragment thereof), for example, in a tissue or organ, and a labeled secondary antibody (second layer) then is reacted with the primary antibody. The secondary antibody can be against the isotype, for example, IgG, of the animal species in which the primary antibody is derived. This method can be more sensitive than direct detection strategies because of signal amplification due to the binding of several secondary antibodies to each primary antibody if the secondary antibody is conjugated to a detecting agent such as a fluorescent or enzyme reporter.AFS Ref.: 039062.00181

[0088] In alternative embodiments, further amplification is achieved if the secondary antibody is conjugated to several detecting molecules, for example, biotin molecules, which can recruit complexes of avidin-, streptavidin- or NEUTRA VIDIN™ protein- bound enzyme.

[0089] In alternative embodiments, the IHC is performed on tissue sections or tissue biopsies, for example, paraformaldehyde (PF A) fixed tissues or organs, or formalin-fixed paraffin-embedded tissues. In alternative embodiments, a tissue is sliced or used whole. Before sectioning, the tissue sample can be embedded in a medium, for example, paraffin wax or cryomedia. Tissue sections can be sliced on a variety of instruments, most commonly using a microtome, cryostat, or vibratome. Specimens can be sliced at a range of about 3 pm to 5 urn. The slices can be mounted on slides, dehydrated using alcohol washes of increasing concentrations (for example, 50%, 75%, 90%, 95%, 100%), and cleared using a detergent like xylene before being imaged under a microscope.

[0090] Depending on the method of fixation and tissue preservation, the sample may require additional steps to make the TIM3 epitopes available for antibody binding, including deparaffinization and antigen retrieval. For formalin-fixed paraffin-embedded tissues, antigenretrieval is often necessary, and can comprise pre-treating the sections with heat or proteases.

[0091] In alternative embodiments, the IHC is performed using an ENVISION DUOFLEX DOUBLESTAIN SYSTEM™ (EnVision DuoFLEX Doublestain System) (Agilent, San Jose, CA), which allows for staining of two or more markers on a single slide. In alternative embodiments, the IHC is performed using an EnVision FLEX HRP Magenta, High pH (Dako Omnis) system, and binding can be visualized by EnVision FLEX HRP Magenta Chromogen. In alternative embodiments, the IHC is performed using EnVision FLEX Mini Kit, High pH, which is a high-sensitivity visualization system intended for use in IHC together with Dako AUTOSTAINER™ instruments, this dual link system detects primary mouse and rabbit antibodies and the reaction is visualized by 3,3’-Diaminobenzidine (DAB) chromogen (DAB forms a water-insoluble brown precipitate when oxidized, for example, by a peroxidase).

[0092] Methods of Use

[0093] In some aspects, the TIM3 antibodies (or antigen-binding fragments thereof), as well as the kits and products of manufacture comprising the same, described herein, may be used in clinical or diagnostic applications. For example, a TIM3 antibody, or antigen-binding fragment thereof, may be used to detect a TIM3 polypeptide via an in vitro IHC assay in order to determine whether a subject has a cancer or other disease, or is likely to develop cancer or a disease. Potential applications included, e.g., use as a companion diagnostic to identify patientsAFS Ref. : 039062.00181 that could benefit from a TIM3 -targeting antibody-drug conjugate (ADC) therapy. Potential indications, include without limitation a Non-Small Cell Lung Cancer (NSCLC) and / or other types of lung cancer, Head and Neck Squamous Cell Carcinoma (HNSCC), Hepatocellular Carcinoma (HCC) or other types of liver cancer, Renal cell carcinoma, malignant melanoma, gastro-intestinal cancer, colorectal cancer, urothelial carcinoma and other types of bladder cancer, mamma carcinoma and / or other types of breast cancer, ovarian cancer, cervical cancer, prostate cancer, pancreatic cancer, lymphoma / leukemia, malignant mesothelioma, or a cancer in another organ or cell type.

[0094] Exemplary aspects of the disclosure.1. A chimeric or a recombinant antibody (“Ab”), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, capable of specifically binding to a human T-cell immunoglobulin and mucin-domain containing-3 (“TIM3”) polypeptide having an amino acid sequence comprising or consisting of SEQ ID NO: 1, or a fragment thereof.2. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of aspect 1, fabricated as or in the form of an antigen-binding fragment, a F(ab')2, a Fab', a single-chain variable fragment (“scFv”), a (SCFV)2, a minibody, a diabody, or a tetrabody.3. The chimeric or recombinant antibody (Ab), or antigen-binding-fragment thereof, or monomeric or dimeric antigen-binding protein, of aspect 1 or aspect 2, wherein the sequence of the heavy chain variable domain is:QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYDMTWVRQAPGKGLEWIGIIASTGTTYY ASWAKGRFPISKTSSTTVDLMITSLTAADTATYFCARAHYSDGWYFDLWGQGTLVT VSS (SEQ ID NO:8).AFS Ref. : 039062.001814. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of any of aspects 1 to 3, wherein the sequence of the light chain variable domain is: AYDMTQTPASVEVAVGGTVTIKCQASQSIYSYLAWYQKKPGQPPKLLIYGTSTLESG VPSRFSGSGSGTEFTLTISGVQCDDAATYYCQQGYSTRDVFKIFGGGTEVVVK (SEQ ID N0:9).5. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of any of aspects 1 to 4, wherein the sequence of the heavy chain variable domain is: QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYDMTWVRQAPGKGLEWIGIIASTGTTYY ASWAKGRFPISKTSSTTVDLMITSLTAADTATYFCARAHYSDGWYFDLWGQGTLVT VSS (SEQ ID NO: 8), and the sequence of the light chain variable domain is: AYDMTQTPASVEVAVGGTVTIKCQASQSIYSYLAWYQKKPGQPPKLLIYGTSTLESG VPSRFSGSGSGTEFTLTISGVQCDDAATYYCQQGYSTRDVFKIFGGGTEVVVK (SEQ ID N0:9).6. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of any of aspects 1 to 5, having an amino acid sequence further comprising:(a) a heavy chain variable domain and a constant domain having SEQ ID NO: 14;(b) a heavy chain signal peptide having SEQ ID NO: 12;(c) a heavy chain variable domain and a signal peptide sequence having SEQ ID NO: 16;(d) a heavy chain variable domain and a constant domain and a signal peptide sequence having SEQ ID NO: 10;(e) a light chain variable domain and a constant domain having SEQ ID NO: 15;(f) a light chain signal peptide having SEQ ID NO: 13;(g) a light chain variable domain and signal peptide sequence having SEQ ID NO: 17;(h) a light chain variable domain and a constant domain and a signal peptide sequence having SEQ ID NO: 11;AFS Ref.: 039062.00181(i) a heavy chain variable domain comprising one or more complementarity determining regions (CDRs) having an amino acid sequence selected from SEQ ID NOs:2, 3, or 4; or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions compared to any one of SEQ ID NOs: 2, 3, 4, optionally wherein the one or more substitutions are conservative substitutions; and / or(j) a light chain variable domain comprising one or more complementarity determining regions (CDRs) having an amino acid sequence selected from SEQ ID NOs:5, 6, or 7; or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions compared to any one of SEQ ID NOs: 5, 6, or 7, optionally wherein the one or more substitutions are conservative substitutions.7. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of any of aspects 1 to 6, wherein the sequence of the heavy chain variable domain comprises SEQ ID NO:8 having at least one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve conservative amino acid substitutions, wherein the heavy chain variable domain is capable of specifically binding to the human TIM3 polypeptide having the amino acid sequence of SEQ ID NO:1, or a fragment thereof, when either unpaired or paired with a light chain variable domain.8. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of any of aspects 1 to 7, wherein the sequence of the light chain variable domain comprises SEQ ID NO:9 having at least one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve conservative amino acid substitutions, wherein the light chain variable domain is capable of specifically binding to the human TIM3 polypeptide having the amino acid sequence of SEQ ID NO:1, or a fragment thereof, when either unpaired or paired with a heavy chain variable domain.9. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of any of aspects 1 to 8, wherein the sequence of the heavy chain variable domain has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity compared to SEQ ID NO:8.10. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of any of aspects 1 to 9, wherein the sequence of the light chain variable domain has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity compared to SEQ ID NO:9.AFS Ref.: 039062.0018111. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of any of aspects 1 to 10, wherein the sequence of the heavy chain variable domain and the amino acid sequence of SEQ ID NO: 8 have a Z score of from about 2 to about 8, or a Z score of at least 8, when aligned using distance matrix alignment.12. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of any of aspects 1 to 11, wherein the sequence of the light chain variable domain and the amino acid sequence of SEQ ID NO: 9 have a Z score of from about 2 to about 8, or a Z score of at least 8, when aligned using distance matrix alignment.13. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of any of aspects 1 to 12, comprising the three CDR1, CDR2 and CDR3 complementarity determining regions (CDRs) of SEQ ID NO:8; or CDR1 amino acid (aa) residues 25-32, CDR2 aa residues 50-56, and / or CDR3 aa residues 94-104, of SEQ ID NO: 8.14. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of any of aspects 1 to 13, comprising the three CDR1, CDR2 and CDR3 complementarity determining regions (CDRs) of SEQ ID NO:9; or CDR1 amino acid (aa) residues 27-32, CDR2 aa residues 50-52, and / or CDR3 aa residues 89-100, of SEQ ID NO:9.15. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of any of aspects 1 to 14, wherein the antibody heavy chain is an IgM, IgG, IgA or IgE isotype heavy chain, and / or the light chain is a kappa or a lambda light chain.16. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of any of aspects 1 to 15, further comprising, or is bound to or covalently conjugated to, a detectable agent or a binding moiety.17. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of any of aspects 1 to 16, wherein the detectable agent comprises: an enzyme, a biotin, a fluorescent or chemiluminescent label, a fluorophore, sulfoindo-cyanine, nile red, rhodamine, perylene, fluorenyl, coumarin, 7- methoxycoumarin (Mca), dabcyl, [2-(4-nitro-2,l,3-benzoxadiazol-7-AFS Ref.: 039062.00181 yl)aminoethyl]trimethylammonium (NBD), Nile blue, Tamra, boron-dipyrromethene (BODIPY), or derivatives thereof), a dye, a radioisotope, a quantum dot or photoluminescent aqueous nanocrystal, a hapten or an antibody binding epitope or domain.18. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of aspect 17, wherein the enzyme is a peroxidase, an alkaline phosphatase, or a beta-galactosidase.19. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of aspect 17, wherein the peroxidase is a horse radish peroxidase (HRP).20. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen -binding protein, of aspect 17, wherein the hapten comprises a biotin, theophylline, digoxigenin, carborane, fluorescein or bromodeoxyuridine.21. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of aspect 17, wherein the dye comprises a cyanine dye; or Cy3 or Cy5.22. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of aspect 17, wherein the fluorophore comprises dansyl, fluorescein or carboxyfluorescein (FAM) or 6-FAM.23. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of aspect 16, wherein the binding moiety comprises: a glutathione S-transferase (GST) or ligandin tag, a polyhistidine (poly-his) tag, a chitin binding protein (CBP), a STREP-TAG™ (SEQ ID NO: 18) peptide tag, a FLAG tag (SEQ ID NO: 19) peptide tag, or a maltose binding protein.24. A recombinant nucleic acid molecule encoding a chimeric or a recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigenbinding protein according to any one of aspects 1 to 23.25. The recombinant nucleic acid molecule of aspect 24, wherein the recombinant nucleic acid further comprises and is operatively linked to a transcriptional regulatory element.26. The recombinant nucleic acid molecule of aspect 25, wherein the transcriptional regulatory element comprises a promoter.27. The recombinant nucleic acid molecule of aspect 26, wherein the promoter is an inducible promoter or a constitutive promoter.AFS Ref.: 039062.0018128. The recombinant nucleic acid molecule of any of aspects 20 to 27, wherein the recombinant nucleic acid further comprises sequence encoding an additional protein or peptide moiety or domain.29. The recombinant nucleic acid molecule of aspect 28, wherein the additional protein or peptide moiety or domain comprises a purification moiety or domain to aid in the purification or isolation of the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, encoded by the recombinant nucleic acid.30. The recombinant nucleic acid molecule of aspect 28 or aspect 29, wherein the additional protein or peptide moiety or domain comprises: a glutathione S-transferase (GST) or ligandin tag, a polyhistidine (poly-his) tag, a chitin binding protein (CBP), a STREP-TAG™ (SEQ ID NO: 18) peptide tag, a FLAG tag (SEQ ID NO: 19) peptide tag, or a maltose binding protein.31. The recombinant nucleic acid molecule of any of aspects 24 to 30, wherein the recombinant nucleic acid further comprises sequence encoding a protease cleavage site positioned between the purification moiety or domain and the sequence encoding the chimeric or a recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein.32. An expression cassette, vector, recombinant virus, artificial chromosome, cosmid or plasmid comprising a recombinant nucleic acid molecule of any of aspects 24 to 31.33. A cell comprising a chimeric or a recombinant antibody (Ab), or an antigenbinding fragment thereof, or a monomeric or dimeric antigen-binding protein of any of any one of aspects 1 to 23, a recombinant nucleic acid molecule of any of aspects 24 to 31, or an expression cassette, vector, recombinant virus, artificial chromosome, cosmid or plasmid of aspect 32.34. The cell of aspect 33, wherein the cell is a bacterial, fungal, mammalian, yeast, insect, avian or plant cell.35. A method for generating an polyclonal antibody, or for generating a polyclonal immune serum, that is specific for or specifically binds to a human T-cell immunoglobulin and mucin-domain containing-3 (TIM3) polypeptide, the method comprising administering to or immunizing a mammal or an avian species with a peptide or polypeptide, or an epitope, comprising the amino acid sequence of SEQ ID NO: 1, or a fragment thereof.AFS Ref.: 039062.0018136. A method for detecting the presence of a human TIM3 polypeptide in a cell, a tissue, an organ, or a portion thereof, comprising:(a) contacting the cell, tissue, organ, or portion thereof with a chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein of any of aspects 1 to 23, or encoded by a recombinant nucleic acid molecule of any of aspects 24 to 31, and(b) detecting the specific binding of the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, with a human TIM3 polypeptide having the amino acid sequence of SEQ ID NO:1, or a fragment thereof, in the cell, tissue, organ, or portion thereof.37. The method of aspect 36, wherein the contacting comprises use of an immunohistochemistry (IHC) assay.38. The method of aspect 36 or aspect 37, further comprising contacting the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein with a detectable agent to indicate or signal the specific binding of the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein to the human TIM3 polypeptide or the fragment thereof.39. The method of aspect 38, wherein the detectable agent specifically binds to the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein.40. The method of aspect 38 or 39, wherein the detectable agent is or comprises an antibody or an antigen-binding fragment or a secondary antibody thereof that specifically binds to the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein which is bound to the human TIM3 polypeptide.41. The method of any of aspects 38 to 40, wherein the detectable agent is or comprises an antibody or an antigen-binding fragment or a secondary antibody thereof that specifically binds to a hapten or tag attached or conjugated to the chimeric or recombinant antibody (Ab) or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein.AFS Ref.: 039062.0018142. The method of any of aspects 38 to 41, wherein the antibody or an antigenbinding fragment or the secondary antibody further comprises or has attached or conjugated thereto a second detectable agent or an enzyme.43. The method of aspect 42, wherein the enzyme is an alkaline phosphatase, a betagalactosidase or a peroxidase.44. The method of aspect 36 or 37, wherein the antibody or an antigen-binding fragment or the secondary antibody further comprises or has attached or conjugated thereto a biotin, a fluorescent or chemiluminescent label, a fluorophore, sulfoindo-cyanine, nile red, rhodamine, perylene, fluorenyl, coumarin, 7-m ethoxy coumarin (Mca), dabcyl, [2-(4-nitro- 2,l,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (NBD), Nile blue, Tamra, boron- dipyrromethene (BODIPY), or derivatives thereof), a dye, a radioisotope, a quantum dot or photoluminescent aqueous nanocrystal, a hapten.45. The method of aspect 44, wherein the dye comprises a cyanine dye, or Cy3 or Cy5.46. The method of aspect 44, wherein the hapten comprises a biotin, theophylline, digoxigenin, carborane, fluorescein or bromodeoxyuridine.47. A method for diagnosing a TIM3 protein-expressing cancer, comprising: detecting the expression or presence of a TIM3 polypeptide or fragment thereof, in a cell, tissue, or organ sample, or a portion thereof, by contacting the cell, tissue, organ sample, or portion thereof with a chimeric or recombinant antibody or antigen-binding fragment thereof of any one of aspects 1 to 23, or encoded by a recombinant nucleic acid molecule of any of aspects 24 to 31, and detecting whether or not the chimeric or recombinant antibody specifically binds to the human TIM3 polypeptide or fragment thereof in the cell, tissue, organ sample, or portion thereof, wherein the detecting of specific binding indicates the expression or presence of the human TIM3 polypeptide or fragment thereof, in the cell, tissue, or organ sample, or the portion thereof.48. The method of aspect 47, wherein the cell is an activated T cell, an activated T cell that has infiltrated a tumor, or a tumor infiltrating lymphocyte (TIL).49. The method of aspect 47 or aspect 48, wherein the detecting of specific binding indicates the expression or presence of the human TIM3 polypeptide or fragment thereof, in the cell, tissue or organ sample, or portion thereof, thereby diagnosing the cancer.AFS Ref.: 039062.0018150. The method of any of aspect 47 to 49, wherein the cancer is selected from the group consisting of: a Non-Small Cell Lung Cancer (NSCLC) and / or other types of lung cancer, Head and Neck Squamous Cell Carcinoma (HNSCC), Hepatocellular Carcinoma (HCC) or other types of liver cancer, Renal cell carcinoma, malignant melanoma, gastrointestinal cancer, colorectal cancer, urothelial carcinoma and other types of bladder cancer, mamma carcinoma and / or other types of breast cancer, ovarian cancer, cervical cancer, prostate cancer, pancreatic cancer, lymphoma / leukemia, malignant mesothelioma, or a cancer in another organ or cell type.51. The method of aspect 50, wherein the adenocarcinoma is a lung adenocarcinoma or a colon adenocarcinoma.52. The method of any of aspects 47 to 51, wherein the detection comprises using or conducting an immunohistochemistry (IHC) assay or a flow cytometry.53. The method of aspect 52, wherein the conducting or using of the flow cytometry comprises use of a fluorescence-activated cell sorter (FACS) or an impedance flow cytometer.54. The method of any of aspects 47 to 53, wherein the cell, tissue, organ sample, or portion thereof is or is derived from a biopsy from a patient.55. A method for treating, ameliorating or preventing a cancer comprising first characterizing the cancer in an individual in need thereof using a method of any of aspects 47 to 54, followed by treatment of the individual in need thereof.56. The method of aspect 55, wherein the cancer is selected from the group consisting of: a Non-Small Cell Lung Cancer (NSCLC) and / or other types of lung cancer, Head and Neck Squamous Cell Carcinoma (HNSCC), Hepatocellular Carcinoma (HCC) or other types of liver cancer, Renal cell carcinoma, malignant melanoma, gastro-intestinal cancer, colorectal cancer, urothelial carcinoma and other types of bladder cancer, mamma carcinoma and / or other types of breast cancer, ovarian cancer, cervical cancer, prostate cancer, pancreatic cancer, lymphoma / leukemia, malignant mesothelioma, or a cancer in another organ or cell type.57. Use of a chimeric or a recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein according to any one of aspects 1 to 23, or encoded by a recombinant nucleic acid molecule of any of aspects 24 to 31, for characterizing a cancer, or treating, ameliorating or preventing a cancer.58. A chimeric or a recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein according to any one of aspects 1AFS Ref.: 039062.00181 to 23, or encoded by a recombinant nucleic acid molecule of any of aspects 24 to 31, for use in characterizing a cancer, or treating, ameliorating or preventing a cancer.59. A kit comprising a chimeric or a recombinant antibody (Ab), or an antigenbinding fragment thereof, or a monomeric or dimeric antigen-binding protein according to any one of aspects 1 to 23, or encoded by a recombinant nucleic acid molecule of any of aspects 24 to 31.60. The kit of aspect 59, wherein comprising components needed for an immunohistochemistry (IHC) assay.61. The kit of aspect 59 or 60, comprising instructions for practicing a method of any of the preceding aspects.62. The kit of any of aspects 59 to 61, wherein the chimeric or the recombinant antibody (Ab), the antigen-binding fragment thereof, or the monomeric or dimeric antigenbinding protein, is substantially purified or isolated.63. A product of manufacture comprising a chimeric or a recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein according to any one of aspects 1 to 23, or encoded by a recombinant nucleic acid molecule of any of aspects 24 to 31.64. The product of manufacture of aspect 63, wherein the product of manufacture comprises or is fabricated as or manufactured as a slide, a well, a chip, a biochip, an array, a tray, a dish or a microtiter plate or dish.65. The product of manufacture of aspect 63 or aspect 64, wherein the chimeric or the recombinant antibody (Ab), the antigen-binding fragment thereof, or the monomeric or dimeric antigen-binding protein, is substantially purified or isolated, or is in the form of an unpurified or partially purified culture supernatant.66. A phage or a phagemid comprising or expressing on its surface a chimeric or a recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein according to any one of aspects 1 to 23, or encoded by a recombinant nucleic acid molecule of any of aspects 24 to 31.EXAMPLES

[0095] Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols, for example, as described in Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press,AFS Ref.: 039062.00181NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, 3rdEdition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR - Basics: From Background to Bench, 1stEdition, Springer Verlag, Germany.

[0096] Example 1 : Development of an exemplary anti-TIM3 antibody

[0097] This example describes the development of an exemplary rabbit anti-human TIM3 antibody. Three antigens were selected for study, including two peptide fragments of TIM3 and a recombinant TIM3 protein. Each antigen was used to immunize four rabbits. Post immunization bleed testing showed four rabbits from the recombinant track having TIM3 specific IHC staining, whereas in the two peptide tracks none of the rabbits showed TIM3 specific staining. The two rabbits from the recombinant track showing the best immune response against were chosen for B-cell selection using blood samples from the rabbits. Several B-cell clones were identified showing TIM3 specific staining in IHC. Cloning was initiated on chosen B-cell clones. Cloning resulting in functional antibody chains, heavy and light chains, respectively, were used for transient transfection in HEK cells and the resulting antibodies were tested for reactivity against the TIM3 antigen of BLItz. The transfected antibody T0234 (clone 1C12), recognising the antigen were further tested in IHC on different normal and clinical tissue samples including lung NSCLC, liver HCC and melanoma. This antibody was then purified and concentrated and tested in IHC on both normal and clinical tissues and compared to a TIM3 reference antibody. The staining obtained with antibodies produced from clone 1C12 was similar or superior to the staining obtained with the TIM3 reference antibody. FIGs. 1-5 illustrate the staining of various tissues using antibodies produced by clone 1C12 compared to the TIM3 reference and illustrates that TIM3, clone 1C12 is expressed in the same cells as the TIM3 reference. The results of exemplary IHC assays using these antibodies as a stain with various tissue types, are summarized below in Table 1.

[0098] The specificity of the new TIM3 antibody was verified by IHC double staining (DAB & magenta chromogenes) on the same slide with TIM3 and antibodies against PDL1 and CD8 respectively. These double staining showed that the new TIM3 antibody is expressed in a subpopulation of CD8+ activated T cells and in a subpopulation of PD-L1 positiveAFS Ref. : 039062.00181 activated T cells in tonsils and in tumor infiltrating lymphocytes (TILs). The specificity double staining is illustrated in FIGs. 6-11.

[0099] The results of exemplary IHC assays using these antibodies as a stain with various tissue types, are summarized below in Table 1.AFSRef.: 039062.00181AFSRef.: 039062.00181SEQUENCE LISTINGAFSRef.: 039062.00181

Claims

AFS Ref.: 039062.00181CLAIMSWe claim:

1. A chimeric or a recombinant antibody (“Ab”), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein, capable of specifically binding to a human T-cell immunoglobulin and mucin-domain containing-3 (“TIM3”) polypeptide having an amino acid sequence comprising or consisting of SEQ ID NO: 1, or a fragment thereof.

2. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of claim 1, fabricated as or in the form of: an antigen-binding fragment, a F(ab')2, a Fab', a single-chain variable fragment (“scFv”), a (SCFV)2, a minibody, a diabody, or a tetrabody.

3. The chimeric or recombinant antibody (Ab), or antigen-binding-fragment thereof, or monomeric or dimeric antigen-binding protein, of claim 1 or claim 2, wherein the sequence of the heavy chain variable domain is:QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYDMTWVRQAPGKGLEWIGIIASTGTTYY ASWAKGRFPISKTSSTTVDLMITSLTAADTATYFCARAHYSDGWYFDLWGQGTLVT VS S (SEQ ID NO: 8); or wherein the sequence of the light chain variable domain is: AYDMTQTPASVEVAVGGTVTIKCQASQSIYSYLAWYQKKPGQPPKLLIYGTSTLESG VPSRFSGSGSGTEFTLTISGVQCDDAATYYCQQGYSTRDVFKIFGGGTEVVVK (SEQ ID NOV); or wherein the sequence of the heavy chain variable domain is: QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYDMTWVRQAPGKGLEWIGIIASTGTTYY ASWAKGRFPISKTSSTTVDLMITSLTAADTATYFCARAHYSDGWYFDLWGQGTLVT VSS (SEQ ID NO:8), and the sequence of the light chain variable domain is: AYDMTQTPASVEVAVGGTVTIKCQASQSIYSYLAWYQKKPGQPPKLLIYGTSTLESG VPSRFSGSGSGTEFTLTISGVQCDDAATYYCQQGYSTRDVFKIFGGGTEVVVK (SEQ ID NOV).

4. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of any of claims 1 to 3, having an amino acid sequence further comprising:AFS Ref.: 039062.00181(a) a heavy chain variable domain and a constant domain having SEQ ID NO: 14;(b) a heavy chain signal peptide having SEQ ID NO: 12;(c) a heavy chain variable domain and a signal peptide sequence having SEQ ID NO: 16;(d) a heavy chain variable domain and a constant domain and a signal peptide sequence having SEQ ID NO: 10;(e) a light chain variable domain and a constant domain having SEQ ID NO: 15;(f) a light chain signal peptide having SEQ ID NO: 13;(g) a light chain variable domain and signal peptide sequence having SEQ ID NO: 17;(h) a light chain variable domain and a constant domain and a signal peptide sequence having SEQ ID NO: 11;(i) a heavy chain variable domain comprising one or more complementarity determining regions (CDRs) having an amino acid sequence selected from SEQ ID NOs:2, 3, or 4; or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions compared to any one of SEQ ID NOs: 2, 3, 4, optionally wherein the one or more substitutions are conservative substitutions; and / or(j) a light chain variable domain comprising one or more complementarity determining regions (CDRs) having an amino acid sequence selected from SEQ ID NOs:5, 6, or 7; or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions compared to any one of SEQ ID NOs: 5, 6, or 7, optionally wherein the one or more substitutions are conservative substitutions.

5. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of any of claims 1 to 4, wherein the sequence of the heavy chain variable domain comprises SEQ ID NO:8 having at least one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve conservative amino acid substitutions, wherein the heavy chain variable domain is capable of specifically binding to the human TIM3 polypeptide having the amino acid sequence of SEQ ID NO:1, or a fragment thereof, when either unpaired or paired with a light chain variable domain.

6. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of any of claims 1 to 5,AFS Ref. : 039062.00181 wherein the sequence of the light chain variable domain comprises SEQ ID NO: 9 having at least one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve conservative amino acid substitutions, wherein the light chain variable domain is capable of specifically binding to the human TIM3 polypeptide having the amino acid sequence of SEQ ID NO: 1, or a fragment thereof, when either unpaired or paired with a heavy chain variable domain; or wherein the sequence of the heavy chain variable domain has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity compared to SEQ ID NO:8; or wherein the sequence of the light chain variable domain has at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity compared to SEQ ID NO:9; or wherein the sequence of the heavy chain variable domain and the amino acid sequence of SEQ ID NO: 8 have a Z score of from about 2 to about 8, or a Z score of at least 8, when aligned using distance matrix alignment; or wherein the sequence of the light chain variable domain and the amino acid sequence of SEQ ID NO: 9 have a Z score of from about 2 to about 8, or a Z score of at least 8, when aligned using distance matrix alignment; or wherein the antibody heavy chain is an IgM, IgG, IgA or IgE isotype heavy chain, and / or the light chain is a kappa or a lambda light chain.

7. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of any of claims 1 to 6, comprising the three CDR1, CDR2 and CDR3 complementarity determining regions (CDRs) of SEQ ID NO:8; or CDR1 amino acid (aa) residues 25-32, CDR2 aa residues 50-56, and / or CDR3 aa residues 94-104, of SEQ ID NO:8; or comprising the three CDR1, CDR2 and CDR3 complementarity determining regions (CDRs) of SEQ ID NO:9; or CDR1 amino acid (aa) residues 27-32, CDR2 aa residues 50-52, and / or CDR3 aa residues 89-100, of SEQ ID NO:9.

8. The chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, of any of claims 1 to 7, further comprising, or is bound to or covalently conjugated to, a detectable agent or a binding moiety,AFS Ref. : 039062.00181 optionally, wherein the detectable agent comprises: an enzyme, a biotin, a fluorescent or chemiluminescent label, a fluorophore, sulfoindo-cyanine, nile red, rhodamine, perylene, fluorenyl, coumarin, 7-m ethoxy coumarin (Mca), dabcyl, [2-(4-nitro-2,l,3-benzoxadiazol-7- yl)aminoethyl]trimethylammonium (NBD), Nile blue, Tamra, boron-dipyrromethene (BODIPY), or derivatives thereof), a dye, a radioisotope, a quantum dot or photoluminescent aqueous nanocrystal, a hapten or an antibody binding epitope or domain; optionally, wherein the enzyme is a peroxidase, an alkaline phosphatase, or a betagalactosidase; optionally, wherein the peroxidase is a horse radish peroxidase (HRP); optionally, wherein the hapten comprises a biotin, theophylline, digoxigenin, carborane, fluorescein or bromodeoxyuridinel; optionally, wherein the dye comprises a cyanine dye; or Cy3 or Cy5; optionally, wherein the fluorophore comprises dansyl, fluorescein or carboxyfluorescein (FAM) or 6-FAM; optionally, wherein the binding moiety comprises: a glutathione S-transferase (GST) or ligandin tag, a polyhistidine (poly-his) tag, a chitin binding protein (CBP), a STREP-TAG™ (SEQ ID NO: 18) peptide tag, a FLAG tag (SEQ ID NO: 19) peptide tag, or a maltose binding protein.

9. A recombinant nucleic acid molecule encoding a chimeric or a recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigenbinding protein according to any one of claims 1 to 8, optionally, wherein the recombinant nucleic acid further comprises and is operatively linked to a transcriptional regulatory element; optionally, wherein the transcriptional regulatory element comprises a promoter; optionally, wherein the promoter is an inducible promoter or a constitutive promoter; optionally, wherein the recombinant nucleic acid further comprises sequence encoding an additional protein or peptide moiety or domain, optionally, wherein the additional protein or peptide moiety or domain comprises a purification moiety or domain to aid in the purification or isolation of the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, encoded by the recombinant nucleic acid; optionally, wherein the additional protein or peptide moiety or domain comprises: a glutathione S-transferase (GST) or ligandin tag, a polyhistidine (poly-his)AFS Ref.: 039062.00181 tag, a chitin binding protein (CBP), a STREP-TAG™ (SEQ ID NO: 18) peptide tag, a FLAG tag (SEQ ID NO: 19) peptide tag, or a maltose binding protein; optionally, wherein the recombinant nucleic acid further comprises sequence encoding a protease cleavage site positioned between the purification moiety or domain and the sequence encoding the chimeric or a recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein.

10. An expression cassette, vector, recombinant virus, artificial chromosome, cosmid or plasmid comprising a recombinant nucleic acid molecule of claim 9.

11. A cell comprising a chimeric or a recombinant antibody (Ab), or an antigenbinding fragment thereof, or a monomeric or dimeric antigen-binding protein of any of any one of claims 1 to 8, a recombinant nucleic acid molecule claim 9, or an expression cassette, vector, recombinant virus, artificial chromosome, cosmid or plasmid of claim 10, optionally, wherein the cell is a bacterial, fungal, mammalian, yeast, insect, avian or plant cell.

12. A method for generating an polyclonal antibody, or for generating a polyclonal immune serum, that is specific for or specifically binds to a human T-cell immunoglobulin and mucin-domain containing-3 (TIM3) polypeptide, the method comprising administering to or immunizing a mammal or an avian species with a peptide or polypeptide, or an epitope, comprising the amino acid sequence of SEQ ID NO: 1, or a fragment thereof.

13. A method for detecting the presence of a human TIM3 polypeptide in a cell, a tissue, an organ, or a portion thereof, comprising:(a) contacting the cell, tissue, organ, or portion thereof with a chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein of any of claims 1 to 8, or encoded by a recombinant nucleic acid molecule of claim 9, and(b) detecting the specific binding of the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein, with a human TIM3 polypeptide having the amino acid sequence of SEQ ID NO:1, or a fragment thereof, in the cell, tissue, organ, or portion thereof; optionally, wherein the contacting comprises use of an immunohistochemistry (IHC) assay; optionally, further comprising contacting the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein with aAFS Ref.: 039062.00181 detectable agent to indicate or signal the specific binding of the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein to the human TIM3 polypeptide or the fragment thereof; optionally, wherein the detectable agent specifically binds to the chimeric or recombinant antibody (Ab), or antigenbinding fragment thereof, or monomeric or dimeric antigen-binding protein; optionally, wherein the detectable agent is or comprises an antibody or an antigen-binding fragment or a secondary antibody thereof that specifically binds to the chimeric or recombinant antibody (Ab), or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein which is bound to the human TIM3 polypeptide; optionally, wherein the detectable agent is or comprises an antibody or an antigen-binding fragment or a secondary antibody thereof that specifically binds to a hapten or tag attached or conjugated to the chimeric or recombinant antibody (Ab) or antigen-binding fragment thereof, or monomeric or dimeric antigen-binding protein; optionally, wherein the antibody or an antigen-binding fragment or the secondary antibody further comprises or has attached or conjugated thereto a second detectable agent or an enzyme; optionally, wherein the enzyme is an alkaline phosphatase, a beta-galactosidase or a peroxidase; optionally, wherein the antibody or an antigen-binding fragment or the secondary antibody further comprises or has attached or conjugated thereto a biotin, a fluorescent or chemiluminescent label, a fluorophore, sulfoindo-cyanine, nile red, rhodamine, perylene, fluorenyl, coumarin, 7-m ethoxy coumarin (Mca), dabcyl, [2-(4-nitro-2,l,3-benzoxadiazol-7- yl)aminoethyl]trimethylammonium (NBD), Nile blue, Tamra, boron-dipyrromethene (BODIPY), or derivatives thereof), a dye, a radioisotope, a quantum dot or photoluminescent aqueous nanocrystal, a hapten; optionally, wherein the dye comprises a cyanine dye, or Cy3 or Cy5; optionally, wherein the hapten comprises a biotin, theophylline, digoxigenin, carborane, fluorescein or bromodeoxyuridine.

14. A method for diagnosing a TIM3 protein-expressing cancer, comprising: detecting the expression or presence of a TIM3 polypeptide or fragment thereof, in a cell, tissue, or organ sample, or a portion thereof, by contacting the cell, tissue, organ sample, or portion thereof with a chimeric or recombinant antibody or antigen-binding fragment thereof of any one of claims 1 to 8, or encoded by a recombinant nucleic acid molecule of claim 9, and detecting whether or not the chimeric or recombinant antibody specifically binds to the human TIM3 polypeptide or fragment thereof in the cell, tissue, organAFS Ref.: 039062.00181 sample, or portion thereof, wherein the detecting of specific binding indicates the expression or presence of the human TIM3 polypeptide or fragment thereof, in the cell, tissue, or organ sample, or the portion thereof.

15. The method of claim 14, wherein the cell is an activated T cell, an activated T cell that has infiltrated a tumor, or a tumor infiltrating lymphocyte (TIL); or wherein the detecting of specific binding indicates the expression or presence of the human TIM3 polypeptide or fragment thereof, in the cell, tissue or organ sample, or portion thereof, thereby diagnosing the cancer; or wherein the cancer is selected from the group consisting of: a Non-Small Cell Lung Cancer (NSCLC) and / or other types of lung cancer, Head and Neck Squamous Cell Carcinoma (HNSCC), Hepatocellular Carcinoma (HCC) or other types of liver cancer, Renal cell carcinoma, malignant melanoma, gastro-intestinal cancer, colorectal cancer, urothelial carcinoma and other types of bladder cancer, mamma carcinoma and / or other types of breast cancer, ovarian cancer, cervical cancer, prostate cancer, pancreatic cancer, lymphoma / leukemia, malignant mesothelioma, or a cancer in another organ or cell type; optionally, wherein the adenocarcinoma is a lung adenocarcinoma or a colon adenocarcinoma; or wherein the detection comprises using or conducting an immunohistochemistry (IHC) assay or a flow cytometry; optionally, wherein the conducting or using of the flow cytometry comprises use of a fluorescence-activated cell sorter (FACS) or an impedance flow cytometer; or wherein the cell, tissue, organ sample, or portion thereof is or is derived from a biopsy from a patient.

16. A method for treating, ameliorating or preventing a cancer comprising first characterizing the cancer in an individual in need thereof using a method of claim 14 or claim 15, followed by treatment of the individual in need thereof.

17. The method of claim 16, wherein the cancer is selected from the group consisting of: a Non-Small Cell Lung Cancer (NSCLC) and / or other types of lung cancer, Head and Neck Squamous Cell Carcinoma (HNSCC), Hepatocellular Carcinoma (HCC) or other types of liver cancer, Renal cell carcinoma, malignant melanoma, gastro-intestinal cancer, colorectal cancer, urothelial carcinoma and other types of bladder cancer, mamma carcinoma and / or other types of breast cancer, ovarian cancer, cervical cancer, prostate cancer, pancreaticAFS Ref.: 039062.00181 cancer, lymphoma / leukemia, malignant mesothelioma, or a cancer in another organ or cell type.

18. Use of a chimeric or a recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein according to any one of claims 1 to 8, or encoded by a recombinant nucleic acid molecule of claim 9, for characterizing a cancer, or treating, ameliorating or preventing a cancer.

19. A chimeric or a recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein according to any one of claims 1 to 8, or encoded by a recombinant nucleic acid molecule of claim 9, for use in characterizing a cancer, or treating, ameliorating or preventing a cancer.

20. A kit comprising a chimeric or a recombinant antibody (Ab), or an antigenbinding fragment thereof, or a monomeric or dimeric antigen-binding protein according to any one of claims 1 to 8, or encoded by a recombinant nucleic acid molecule of claim 9, optionally comprising components needed for an immunohistochemistry (IHC) assay; optionally comprising instructions for practicing a method of any of the preceding claims; optionally, wherein the chimeric or the recombinant antibody (Ab), the antigen-binding fragment thereof, or the monomeric or dimeric antigen-binding protein, is substantially purified or isolated.

21. A product of manufacture comprising a chimeric or a recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein according to any one of claims 1 to 8, or encoded by a recombinant nucleic acid molecule of claim 9, optionally, wherein the product of manufacture comprises or is fabricated as or manufactured as a slide, a well, a chip, a biochip, an array, a tray, a dish or a microtiter plate or dish; optionally, wherein the chimeric or the recombinant antibody (Ab), the antigen-binding fragment thereof, or the monomeric or dimeric antigen-binding protein, is substantially purified or isolated, or is in the form of an unpurified or partially purified culture supernatant.

22. A phage or a phagemid comprising or expressing on its surface a chimeric or a recombinant antibody (Ab), or an antigen-binding fragment thereof, or a monomeric or dimeric antigen-binding protein according to any one of claims 1 to 8, or encoded by a recombinant nucleic acid molecule of claim 9.

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