Cancer therapy by blocking the interaction between TIM-3 and its ligands

Abstract

To provide novel compositions and methods for curing cancer by blocking the interaction to activate immune response where TIM-3 interacts with a novel ligand galectin-3 (Gal-3) and the interaction inhibits T cell activation.SOLUTION: Disclosed is a method for activating the immune response of a patient and / or a method for treating cancer comprising administering to the patient a Gal3:TIM-3 inhibitor that interferes with the interaction between Gal3 and TIM-3, where the inhibitor is administered in an amount sufficient for activating immune response. Also provided are a humanized anti-Gal3 antibody capable of blocking the interaction between Gal3 and TIM-3, a method for treating cancer using an anti-Gal3 antibody, a method for determining whether a treatment using Gal3:TIM-3 inhibitor is suitable for the cancer of a patient or not, and a method for selecting compounds usable for the treatment.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 536,886, filed July 25, 2017, which is incorporated herein by reference in its entirety for all purposes. [Background technology]

[0002] Human cancers harbor numerous genetic and epigenetic alterations that result in neoantigens potentially recognizable by the immune system. While an innate immune response to cancer is observed in preclinical models and patients, the response is ineffective, and established cancers are often considered "self" and tolerated by the immune system. Additionally, tumors can utilize several different mechanisms to actively suppress the host's immune response. Among these mechanisms, immune checkpoints, which comprise various negative regulators of the immune system that normally shut off immune responses to mitigate collateral tissue damage, can be used by tumors to evade immune destruction.

[0003] T cell immunoglobulin and mucin domain-containing-3 (TIM-3) is known to be one such negative regulator of T cell activation, but the mechanism of TIM-3 suppression in T cells is largely unknown. Previous efforts have been made to identify the ligands of TIM-3 involved in this regulation, but data have been inconsistent and unreliable. For example, in 2005, it was reported that galectin 9 (Gal9) could bind to TIM-3 (Zhu et al., Nature Immunology 6, 1245); however, a later report indicated that the interaction between TIM-3 and Gal9 was nonspecific in nature (Leitner et al., PLoS Pathog 9(3):e1003253). CEACAM1 was reported as a TIM-3 ligand that regulates T cell tolerance and exhaustion (Huang et al., Nature 517, 386). However, Huang's results are inconsistent with our own data showing that TIM-3 does not bind to CEACAM1 (see below). Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention relates to the interaction of TIM-3 with a novel ligand, galectin-3 (Gal-3), which inhibits immune responses, such as T cell activation. The present invention provides novel compositions and methods for blocking the interaction, activating the immune response, and thus curing cancer. [Means for solving the problem]

[0005] In some embodiments, the present disclosure provides a method of activating an immune response in a patient, comprising administering to the patient a Gal3:TIM-3 inhibitor that interferes with the interaction between Gal3 and TIM-3 in the patient, wherein the inhibitor is administered in an amount sufficient to activate the immune response. In some embodiments, the patient is a host of cancer, and the interaction between Gal3 and TIM-3 occurs in the tumor microenvironment. In some embodiments, activating the immune response reduces cancer burden in the patient. In some embodiments, TIM-3 is present on immune cells. In some embodiments, the patient is a host of cancer, and Gal3 is overexpressed in the tumor microenvironment, and the Gal3:TIM-3 inhibitor is administered in an amount sufficient to reduce cancer burden in the patient. In some embodiments, the cancer comprises cancer cells that overexpress Gal3 on their surface. In some embodiments, the immune cells on which TIM-3 is expressed are T cells and / or NK cells.

[0006] In some embodiments, the present disclosure provides a method for the treatment of cancer in a tumor microenvironment where cells overexpress Gal3.

[0003] The present invention provides a method for activating T cells in a patient hosting a cancer comprising T cells, the method comprising administering to the patient a Gal3:TIM-3 inhibitor that interferes with the interaction between Gal3 and TIM-3 on the T cells, wherein the inhibitor is administered in an amount sufficient to activate the T cells and thereby reduce the cancer burden in the patient.

[0007] Optionally, the cells in the tumor microenvironment can include cancer cells. Optionally, the cells in the tumor microenvironment can include tumor-associated macrophages (TAMs), e.g., M2 TAMs.

[0008] In some embodiments, the Gal3:TIM-3 inhibitor binds to TIM-3. In some embodiments, the Gal3:TIM-3 inhibitor binds to Gal3.

[0009] In some embodiments, the disclosure provides a method for determining whether a patient's cancer is suitable for treatment with a Gal3:TIM-3 inhibitor, the method comprising: binding cells obtained from a tumor microenvironment of a known type from the patient with an antibody specific for Gal3; determining the level of Gal3 on the surface of primary cancer cells in the sample; comparing the level of Gal3 on the cell surface to a first activity threshold for Gal3; and determining that the patient's cancer is suitable for treatment with a Gal3:TIM-3 inhibitor if the level of Gal3 on the surface of the primary cancer cells is higher than the first activity threshold.

[0010] In some embodiments, the first Gal3 activity threshold is derived from a cohort of at least 100 test individuals suffering from the same type of cancer as the patient sample. In some embodiments, the first Gal3 activity threshold is based on the average, mean, or median Gal3 levels on the cell surface of similar tissue types from healthy individuals.

[0011] In some embodiments, the present disclosure provides a sterile solution capable of interfering with the interaction of Gal3 and TIM-3 on T cells in a cancer patient, the solution comprising 10 μg to 100 mg of an antibody per kilogram of patient body weight in 100 ml of solution suitable for intravenous delivery over 1 to 4 hours, the antibody being capable of interfering with the interaction of Gal3 and TIM-3 on T cells. In some embodiments, the sterile solution further comprises one or more other checkpoint inhibitor antibodies. In some embodiments, the one or more other checkpoint inhibitor antibodies are selected from the group consisting of anti-PD-1 and anti-CTLA-4 antibodies.

[0012] In some embodiments, the present disclosure provides a method for producing an anti-Gal3 antibody that can interfere with the interaction between Gal3 and TIM-3, the method comprising: introducing into an animal a peptide comprising any one of the sequences set forth in SEQ ID NOs: 5 to 8, and the animal producing the anti-Gal3 antibody.

[0013] In some embodiments, the disclosure provides a humanized anti-Gal3 antibody, the antibody comprising: (1) a light chain variable region comprising complementarity determining regions (CDRs) L1, CDR L2, and CDR L3; and (2) a heavy chain variable region comprising CDR H1, CDR H2, and CDR H3, wherein CDR L1 comprises the amino acid sequence of SEQ ID NO: 17, CDR L2 comprises the amino acid sequence of SEQ ID NO: 18, CDR L3 comprises the amino acid sequence of SEQ ID NO: 19, CDR H1 comprises the amino acid sequence of SEQ ID NO: 9, CDR H2 comprises the amino acid sequence of SEQ ID NO: 10, and CDR H3 comprises the amino acid sequence of SEQ ID NO: 11.

[0014] In some embodiments, the heavy chain variable region of the humanized antibody has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the light chain variable region of the humanized antibody has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 26.

[0015] In some embodiments, the humanized antibodies can block the interaction of Gal3 with TIM-3, thereby activating the immune response.

[0016] In some embodiments, the present disclosure provides a method for selecting a compound capable of blocking the interaction between Gal3 and TIM-3, a method for activating an immune response in a patient and / or treating cancer, comprising: (a) contacting a library of compounds with Gal3 and TIM-3; and (b) selecting one or more candidate compounds from the library capable of blocking the interaction between Gal3 and TIM-3. In some embodiments, the method further comprises: (c) contacting one or more candidate compounds selected from step (b) with a mixture containing T cells and allogeneic antigen-presenting cells to identify one or more compounds capable of stimulating T cells; and / or (d) administering one or more candidate compounds selected from (b) to a mammal hosting a tumor to identify one or more compounds capable of reducing tumor burden in the mammal; and, optionally, (e) administering to the patient an effective amount of a compound capable of stimulating T cells and / or reducing tumor burden in the mammal, thereby activating an immune response in the patient and / or treating cancer. In some embodiments, the compound is an antibody.

[0017] In some embodiments, the present disclosure provides a Gal3:TIM-3 inhibitor as disclosed in any of the above embodiments for use in a method of activating an immune response in a patient, comprising administering to the patient a Gal3:TIM-3 inhibitor capable of blocking the interaction between Gal3 and TIM-3, wherein the inhibitor is administered in an amount sufficient to activate the immune response. Optionally, the Gal3:TIM inhibitor is a humanized anti-Gal3 antibody described above.

[0018] In some embodiments, the present disclosure provides use of a Gal3:TIM-3 inhibitor disclosed in any of the above embodiments in the manufacture of a medicament (i.e., a pharmaceutical composition) for activating an immune response and / or treating cancer. Optionally, the Gal3:TIM inhibitor is a humanized anti-Gal3 antibody described above. [Brief explanation of the drawings]

[0019] [Figure 1] Figure 1 shows the results of a co-immunoprecipitation assay demonstrating that human Gal3 (hGal3) specifically pulls down human TIM-3 (hTIM-3). Figure 1A shows TIM-3 expression in 293T cells co-transfected with a plasmid encoding HA-tagged hTIM-3 and a plasmid encoding hGal3, hGal9, or hCEACAM1. Figure 1B shows the expression of hGal9, hGal3, or hCEACAM1. Figure 1C shows that hGal3, but not CEACAM1, pulled down HA-tagged hTIM-3 in co-transfected 293T cells. The results also show that human Gal9 (hGal9) pulled down hTIM-3, but this pull-down was accompanied by protein aggregation (Figure 1B), indicating that the binding between hGal9 and hTIM-3 may be nonspecific. [Figure 2] This figure shows the results of a pull-down assay using a fusion protein consisting of the hTIM-3 extracellular domain fused to the Fc portion of hIgG (hTIM-3 Fc). The results demonstrate that the binding between Gal3 and TIM-3 is specific. As shown in this figure, hTIM-3 Fc, but not hFc or hPD1, pulled down Flag-tagged hGal3 protein overexpressed from 293T cells. [Figure 3]Figure 1 shows the results of a cell adhesion assay demonstrating the specific interaction between hGal3 and hTIM-3. As shown in the figure, significantly more A20 cells expressing hGal3 (A20 Gal3 cells) were able to adhere to plates coated with hTIM-3 Fc than to plates coated with hVISTA Fc or hPD1 Fc. The results also show that more A20 PDL1 cells were able to adhere to plates coated with hPD1 Fc than to plates coated with human VISTA Fc (hVISTA Fc) or hTIM-3 Fc. [Figure 4] Figure 4A shows live (left peak) and dead (right peak) A20 cells by flow cytometry analysis. Figures 4B and 4C show live (Figure 4B) and dead (Figure 4C) cells stained with anti-hFc APC antibody. In group 1, A20 Gal3 cells were incubated without mTIM-3 Fc protein as a control; in group 2, A20 Gal3 cells were incubated with mTIM-3 Fc protein; and in groups 3, 4, and 5, in addition to mTIM-3 Fc protein, anti-mouse TIM-3 polyclonal antibody (R&D System, Minneapolis, MN, USA) (group 3), monoclonal antibody RMT3-23 (Bio X cell, West Lebanon, NH, USA) (group 4), and monoclonal antibody 215015 (R&D System) (group 5) were also added to test whether these antibodies could block Gal3 and Tim3 binding. [Figure 5]Figures 5A-5C show ELISA results demonstrating the specific binding of Gal3 to TIM-3. In Figure 5A, plates were coated with 10 μg / ml mGal3, and the mGal3 polyclonal antibody (mGal3 pAb) and monoclonal antibody IMT001, but not monoclonal antibody M3 / 38, were found to block the interaction between Gal3 and TIM-3. Figure 5B shows that lactose blocked Gal9 binding to TIM-3 but not Gal3 binding, indicating that the binding between Gal3 and Tim3 is sugar-independent. Figure 5C shows that antibody RMT3-23 blocked phosphatidylserine (PS) binding to TIM-3 but not Gal3 binding, indicating that the epitope on TIM-3 that binds Gal3 is distinct from that that binds PS. [Figure 6] Figures 6A and 6B show that overexpressed Gal3 suppressed T cell activation. Figure 6A shows that mouse A20 cell clones #41, #31, and #15 overexpress Gal3. Figure 6B shows that when these cells were mixed with mouse DO11.10 T cells, much less IL-2 was produced compared to parental A20 cells (Figure 6B). [Figure 7-1]Figures 7A-7E show that Gal3 antibodies have antitumor effects in a lung metastasis model. Figure 7A shows high expression of Gal3 on B16F10 tumor cells. Figure 7B shows representative images of whole lungs from three treatment groups. Figure 7C shows numerous metastatic colonies on the surface of the left lung lobe (mean ± SEM (standard error of the mean)). Figures 7D and 7E show lung and body weights (mean ± SEM) for the various treatment groups. Compared with animals treated with an isotype control, animals treated with a monoclonal anti-human Gal3 antibody showed a significant reduction in tumor number (p < 0.01) (Figure 7B) and a much lower tumor burden, as indicated by lung weight (p < 0.05) (Figure 7D). However, animals treated with a PD1 antibody did not show a significant reduction in tumor number or tumor burden in this lung metastasis model (p > 0.05). FIG. 7E shows that animals treated with either PD1 or Gal3 antibodies had similar body weights to the control group, indicating that there were no adverse effects associated with administration of either antibody. [Figure 7-2] Continued from Figure 7-1. [Figure 8] Figures 8A-8C show the antitumor effect of Gal3 antibody on 4T1 orthotopic tumor-induced lung metastasis. Figure 8A shows images of metastatic tumor colonies on the lungs of mice implanted with 4T1 cells and then treated with a control antibody ("isotype") or IMT001. The antibodies were administered intraperitoneally on days 0, 3, 7, 10, and 14 over a 30-day period. Images were taken on day 30 when the mice were sacrificed. Figure 8B shows the weight measurements of these mice over the same period. Figure 8C shows the number of metastatic tumor colonies on the surface of the left lobe of these mice on day 30. [Figure 9] Figure 1 shows tumor growth in mice implanted with Renca tumor cells and treated with Gal3 antibody. Compared to mice implanted with Renca tumor cells and treated with an isotype control antibody ("Iso"), mice treated with Gal3 antibody ("IMT001") showed significantly reduced tumor size (p<0.05), whereas the anti-mouse PD-1 antibody 29F had no effect (p>0.05). [Figure 10]Figure 1 shows tumor growth in mice implanted with MC38 colon cancer cells and treated with anti-Gal3 antibody. Compared to mice implanted with MC38 tumor cells and treated with an isotype control antibody ("Iso"), mice treated with Gal3 antibody ("IMT001") showed significantly reduced tumor size (p<0.05). [Figure 11-1] Figures 11A-11D show the results of epitope mapping. A peptide array derived from the hGal3 protein sequence was synthesized (Figure 11A) and dot-blotted with the anti-Gal3 antibody IMT001 (Figure 11B). Peptides 5 and 6 showed good signals, indicating that the anti-Gal3 monoclonal antibody, IMT001, could bind to these peptides. To further map the binding epitopes of IMT001 to these peptides, several shorter peptides derived from these peptide sequences were synthesized (Figure 11C), and their binding to IMT001 was measured by ELISA (Figure 11D). The peptide with the sequence GQAPPGAYPG (SEQ ID NO: 8) produced the highest signal. [Figure 11-2] Continued from Figure 11-1. [Figure 12] Immune cell counts are summarized from mice transplanted with B16F10 cells expressing various lymphocyte markers: CD3, CD4, CD8, CD19, or DX5. These mice were treated with an isotype control antibody or IMT001. [Figure 13] Figures 13A and 13B show Gal3 expression on tumor-associated macrophages of human lung cancer in an immunohistochemistry (IHC) assay. IMT001 was used to stain human lung cancer frozen slides to detect Gal3 expression on tumor-associated macrophages. Figure 13A shows the staining results for squamous cell carcinoma, and Figure 13B shows the staining results for adenocarcinoma. [Figure 14-1] Figures 14A-14C show that Gal3 expression was detected on human M2 macrophages (Figure 14C), but not on dendritic cells (DCs) (Figure 14A) or M1 macrophages (Figure 14B). [Figure 14-2] Continued from Figure 14-1. [Figure 15] Figure 15 shows the immunological effect of Gal3 antibody ("IMT001") on mouse macrophage / T cell reactions. Figure 15B shows the detection of Gal3 expression by IHC on the mouse macrophage cell line RAW264.7 compared to the control (Figure 15A). Figure 15C shows Gal3 expression on mouse macrophage cell lines by flow cytometry using cells stained with IMT001. The anti-Gal3 antibody IMT001 promoted IL-2 production in the RAW macrophage / DO11.10 T cell mixed reaction, but the anti-mouse PD-1 antibody 29F did not (Figure 15D). DETAILED DESCRIPTION OF THE INVENTION

[0020] definition As used herein, the terms "a," "an," or "the" not only include embodiments of one member, but also encompass embodiments of more than one member. For example, the singular forms "a," "an," or "tha" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, reference to "the agent" includes reference to one or more agents known to those of skill in the art, and so forth.

[0021] The term "comprise" means that the composition includes the recited elements, but does not exclude others. Thus, comprising also means that the composition includes only the recited elements. For example, "the light chain comprises SEQ ID NO:24" includes the scenario in which the light chain has the sequence set forth in SEQ ID NO:24.

[0022] The terms "subject," "patient," or "individual" are used interchangeably herein and refer to a human or an animal. For example, an animal subject can be a mammal, a primate (e.g., a monkey), a livestock animal (e.g., a mare, cow, sheep, pig, or goat), a companion animal (e.g., a dog, cat), a laboratory animal (e.g., a mouse, rat, guinea pig, bird), an animal of veterinary importance, or an animal of economic importance.

[0023] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein and include polymers of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding natural amino acids, as well as natural and unnatural amino acid polymers. As used herein, the terms include amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked by covalent peptide bonds.

[0024] The term "amino acid" encompasses natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to natural amino acids. Natural amino acids are those encoded by the genetic code, as well as amino acids that have been modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs include compounds that have the same basic chemical structure as natural amino acids, i.e., an α-carbon bonded to a hydrogen, a carboxylic acid group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as natural amino acids. "Amino acid mimetics" include chemical compounds that have a structure different from the general chemical structure of amino acids but function similarly to natural amino acids.

[0025] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes.

[0026] The term "therapeutically effective amount" or "effective amount" includes an amount or quantity effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0027] The term "administering" includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration, or implantation of a sustained-release device, such as a mini-osmotic pump, to a subject. Administration can be by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, intravaginal, rectal, or transdermal). Parenteral administration can include, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other delivery methods include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like. Those skilled in the art will be aware of additional methods for administering a therapeutically effective amount of a Gal3:TIM-3 inhibitor described herein that interferes with the interaction between Gal3 and TIM-3 on T cells to reduce cancer burden in patients. "Co-administering" means administering a first compound described herein simultaneously with, immediately before, or immediately after the administration of a second compound described herein.

[0028] The terms "tumor" and "cancer" are used interchangeably and both refer to the abnormal growth of tissue resulting in excessive cell division.

[0029] The term "tumor microenvironment" refers to the tumor cells and surrounding blood vessels, immune cells, fibroblasts, and bone marrow-derived cells. It represents the cellular environment in which the tumor resides, including inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix.

[0030] The term "immune cells" refers to cells of hematopoietic origin that are involved in the specific recognition of antigens. Immune cells include antigen-presenting cells (APCs), such as dendritic cells or macrophages, B cells, T cells, natural killer cells, and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0031] The term "immune response" refers to a T cell-mediated immune response and / or a B cell-mediated immune response. Exemplary immune responses include a B cell response (e.g., antibody production), a T cell response (e.g., cytokine production and cytotoxic activity), and activation of cytokine-responsive cells, such as macrophage activation. The term "activation of an immune response" refers to enhancing the level of a T cell-mediated immune response and / or a B cell-mediated immune response using methods known to those skilled in the art. In one embodiment, the level of enhancement is at least 20-50%, alternatively at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 150%, or at least 200%.

[0032] The term "recognize" refers to the phenomenon whereby a molecule can specifically and selectively bind to a second molecule. Typically, specific or selective binding will be at least 2 times the background signal or noise, and more typically greater than 10 to 100 times the background.

[0033] The term "Gal3:TIM-3 inhibitor" refers to a molecule that inhibits the interaction between Gal3 and TIM-3, which results in T cell activation.

[0034] The term "TIM-3:Gal3" or "Gal3:TIM-3" pathway refers to a signaling pathway in which TIM-3 binds to Gal3, an interaction that inhibits T cell activation.

[0035] The term "T cell activation" refers to the phenomenon in which T cells become activated and engage in signal transduction pathways that promote an immune response. T cell activation is typically accompanied by T cell proliferation and / or release of cytokines, such as interferon-γ, IL-2, IL-5, IL-10, IL-12, or transforming growth factor (TGF)-β.

[0036] The term "Gal3-overexpressing cancer" refers to a cancer that expresses higher levels of Gal3 on the cell surface compared to control cells. In some cases, the control cells are cells from a similar tissue in a healthy individual. In some cases, the control cells are non-cancerous cells from the same individual that is the host of the cancer.

[0037] The terms "cancer burden," "tumor burden," or "tumor burden" generally refer to the number of cancer cells, tumor size, or amount of cancer in a subject's body at any given time. Tumor burden can be detected, for example, by measuring the expression of tumor-specific gene markers and measuring tumor size by a number of well-known biochemical or imaging methods disclosed herein below.

[0038] The term "activity threshold" refers to an expression level or activity level used for comparison, which may help determine whether a diagnosis can be made or a treatment can be prescribed. In some embodiments, the activity threshold is the median Gal3 expression level on cancer cells from a heterogeneous population with the same type of cancer as the patient being treated. In some embodiments, the activity threshold is the Gal3 level on non-cancerous tissues of a patient hosting a cancer. In some embodiments, the activity threshold is the Gal3 expression level on cells of a similar tissue type from a healthy individual. or activity level.

[0039] The term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies, e.g., bispecific antibodies, chimeric antibodies, humanized antibodies, fully synthetic antibodies, and antibody fragments, so long as they exhibit the desired biological action, i.e., binding specificity. Antibodies are monomeric or multimeric proteins comprising one or more polypeptide chains. Antibodies can specifically bind to an antigen and can modulate the biological action of the antigen. The term "antibody" also encompasses antibody fragments. Specific antibody fragments include (i) Fab fragments consisting of the VL, VH, CL, and CH1 domains; (ii) Fd fragments consisting of the VH and CH1 domains; (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of a single variable region (Ward et al., 1989, Nature 341:544-546); (v) isolated CDR regions; (vi) F(ab')2 fragments, bivalent fragments comprising two linked Fab fragments; and (vii) single-chain Fv molecules (scFv) in which the VH and VL domains are linked by a peptide linker that allows the two domains to participate in forming the antigen-binding site (Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA). 85:5879-5883), (viii) bispecific single-chain Fv dimers (PCT Application No. US92 / 09965), and (ix) "diabodies" or "triabodies," multivalent or multispecific fragments constructed by gene fusion (Tomlinson et al., 2000, Methods Enzymol. 326:461-479; WO 94 / 13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448). In certain embodiments, antibodies are produced by recombinant DNA technology.Other examples of antibody formats and architectures are described in Holliger & Hudson, 2006, Nature Biotechnology 23(9):1126-1136, and Carter 2006, Nature Reviews Immunology 6:343-357, and the references therein, which are expressly incorporated by reference in their entireties. In additional embodiments, antibodies are produced by enzymatic or chemical cleavage of native antibodies.

[0040] The term "humanized antibody" refers to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Framework region modifications may be made within the human framework sequences.

[0041] The term "framework" refers to variable domain residues other than hypervariable region residues. The "framework regions" or "FRs" of different light or heavy chains are relatively conserved within a species. The framework of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4, and framework region modifications may be made within human framework sequences. The framework region of an antibody, which is the combined framework regions of the constituent light and heavy chains, serves to position and arrange the CDRs in three-dimensional space. Framework sequences can be obtained from public DNA databases or published references containing germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBASE2" germline variable gene sequence database of human and mouse sequences.

[0042] As used herein, the terms "variable region" and "variable domain" refer to the portions of the light and heavy chains of an antibody that contain the amino acid sequences of the complementarity-determining regions (CDRs, e.g., CDR H1, CDR H2, CDR H3, CDR L1, CDR L2, and CDR L3) and framework regions (FRs). The amino acid positions designated as CDRs and FRs are: These sequences can be defined by Chothia and Kabat (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), or the International Immunogenetics (IMGT) database. The variable regions in antibody heavy or light chains are derived from germline variable (V), diversity (D), or joining (J) genes (not from constant (Cμ and Cδ) gene segments) and confer their specificity for binding to antigens. Typically, antibody variable regions contain four conserved "framework" regions interspersed with three hypervariable "complementarity-determining regions."

[0043] As used herein, the terms "complementarity determining region" and "CDR" refer to the regions of an antibody variable region that are hypervariable in the sequence and / or configuration of structurally defined loops. CDRs are also known as hypervariable regions. The light chain and heavy chain variable regions each have three CDRs. The light chain variable region contains CDR L1, CDR L2, and CDR L3. The heavy chain variable region contains CDR H1, CDR H2, and CDR H3. Each CDR is defined as a CDR sequence as described in Chothia, Kabat (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health The amino acid residues may be derived from the complementarity determining regions defined by the International Immunogenetics and Glycosciences (IMGT) database, or by the National Institutes of Health, Bethesda, MD (1991).

[0044] The term "human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or an antibody derived from a non-human source that utilizes the human antibody repertoire or human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0045] The term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0046] The term "checkpoint inhibitor therapy" refers to a therapy that suppresses checkpoint pathways.Non-limiting examples of checkpoint inhibitor therapy include therapy that inhibits PD1 signaling pathways and therapy that inhibits CTLA4 signaling pathways.Checkpoint inhibitor therapy can be a peptide, a nucleoside analog (e.g., aptamer), a small molecule compound, or a combination thereof.

[0047] The term "primary cancer" refers to the location in the body or tissue where a particular cancer begins. Primary cancer is often called the first cancer or original cancer. Primary cancer is the opposite of metastasis, which refers to the migration of cancer cells from the original tumor site to produce cancer in other tissues.

[0048] The term "metastatic cancer" refers to cancer that has spread from its original site (where it started) to a different area of ​​the body.

[0049] The term "primary cancer cells" refers to cancer cells that have been isolated from a cancer patient, e.g., a cancer biopsy, and have not been cultured in vitro.

[0050] The phrase, a cancer is "suitable for treatment with a Gal3:TIM-3 inhibitor" refers to a cancer that is likely to respond to treatment with a Gal3:TIM-3 inhibitor, e.g., patients receiving a Gal3:TIM-3 inhibitor have an overall survival, time to progression, disease-free survival, progression-free survival, tumor burden reduction, or any of the other beneficial clinical outcomes disclosed below or according to RECIST criteria. This means that the drug is likely to have beneficial clinical outcomes, such as

[0051] overview The present invention is based on the surprising discovery that TIM-3 specifically binds to the Gal3 protein, and that this interaction results in the suppression of T cell activation. The present disclosure provides methods for treating patients hosting cancer, particularly cancer types that overexpress Gal3, by administering inhibitors that interfere with the interaction between Gal3 and TIM-3 to restore T cell activation. The present disclosure additionally provides methods for determining whether a cancer is suitable for treatment using Gal3:TIM-3 therapy by determining Gal3 levels on the surface of cells in the tumor microenvironment, e.g., cancer cells and tumor-associated macrophages, and comparing the Gal3 levels to an activation threshold.

[0052] 1. Patient population selection Gal3, also known as galectin 3, is expressed in several cell types and is involved in a wide range of physiological and pathological processes, including cell adhesion, cell activation and chemoattraction, the cell cycle, apoptosis, cell proliferation and differentiation, and tumor progression and metastasis. Gal3 is expressed on tumor cells and cells in the tumor microenvironment, such as tumor-associated macrophages, particularly M2 macrophages, as described below.

[0053] TIM-3 is a molecule expressed on immune cells, particularly T cells, and can suppress immune responses, e.g., T cell signaling, through its interaction with Gal3. The Gal3:TIM-3 inhibitors disclosed herein can prevent the interaction between Gal3 and TIM-3 and activate the immune response. The Gal3:TIM-3 inhibitors disclosed herein can be used to treat cancer or other diseases that may benefit from an activated immune response.

[0054] Cancer cells in solid tumors create a tumor microenvironment in their surroundings that can support their proliferation and metastasis. The tumor microenvironment is the cellular milieu in which the tumor resides, including surrounding cells, immune cells, fibroblasts, other cells, soluble factors, signaling molecules, extracellular matrix, and mechanistic cues that can promote malignant transformation. It supports tumor growth and invasion, protects the tumor from host immunity, fosters therapeutic resistance, and provides a niche for dormant metastases to become active. The tumor and its surrounding microenvironment are intimately intertwined and constantly interact. The tumor can influence its microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing immune tolerance in the surrounding area, while immune cells in the microenvironment can influence the proliferation and evolution of cancerous cells. See et al. “Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy,” Cancer Res, vol., 72, pp. 2473-2480, 2012.

[0055] Tumors are often associated with immune infiltrates as part of a macrophage-rich reactive stroma. Tumor-associated macrophages (TAMs) play a key role in promoting tumor growth by promoting angiogenesis and matrix degradation. When associated with tumors, macrophages exhibit functional polarization toward one of two phenotypically distinct subsets of macrophages: M1 macrophages (also known as TH1) or M2 macrophages (also known as TH2). M1 macrophages are known to produce proinflammatory cytokines and play an active role in cell destruction, while M2 macrophages primarily capture debris and promote angiogenesis and repair. As a result, many tumors with large numbers of TAMs have increased tumor growth rates, local proliferation, and distant metastasis. The M2 macrophage population is phenotypically similar to the TAM population, which promotes tumor proliferation and growth. In addition to expressing Gal3, M2 macrophages may also express one or more cell surface markers selected from the group consisting of CD206, IL-4r, IL-1ra, decoy IL-1rll, IL-10r, CD23, macrophage capture receptors A and B, Ym-1, Ym-2, low density receptor-related protein 1 (LRP1), IL-6r, CXCR1 / 2, CD136, CD14, CD1a, CD1b, CD93, CD226, (FcyR), and PD-L1.

[0056] The Gal3:TIM-3 inhibitors disclosed herein can be used to treat cancers that overexpress Gal3 in the tumor microenvironment. In some cases, the cancer includes cancer cells that overexpress Gal3 on their surface. In some cases, the cancer includes other types of cells contained in the tumor microenvironment, such as tumor-associated macrophages, blood vessels, stromal cells, and fibroblasts, that overexpress Gal3 on their surface. In some cases, the cancer overexpresses Gal3, and Gal3 is present in the tumor microenvironment as a soluble protein. Unless otherwise specified, the term "overexpress" refers to an expression level that is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher than the expression level in a control, e.g., a similar cell, tissue, or region of the body of a healthy individual.

[0057] In some embodiments, the Gal3:TIM-3 inhibitors disclosed herein are useful for treating various types of cancers in which cells in the tumor microenvironment, such as cancer cells or tumor-associated macrophages, have higher levels of Gal3 on their surface compared to control cells. The expression level of Gal3 on the cell surface can be measured by methods well known in the art, including, but not limited to, flow cytometry and immunohistochemistry. Typically, detecting the expression level of Gal3 in the tumor microenvironment involves combining a sample containing cells from the tumor microenvironment, including cancer cells and / or tumor-associated macrophages (e.g., M2 TAMs), with an anti-Gal3 antibody, and the level of Gal3 on the cell surface is indicated by the amount of Gal3 antibody that can bind to the cell surface. In some embodiments, the Gal3 level is determined by measuring a detectable label bound to the Gal3 antibody. In some embodiments, a labeled secondary antibody that binds to the Gal3 antibody is used, and the Gal3 expression level is determined by measuring the signal from the label of the secondary antibody. Alternatively, the antibody can be conjugated with biotin, and detectably labeled avidin (a polypeptide that binds biotin) can be used to detect the presence of the biotinylated antibody. Suitable detectable labels that can be used include radionuclides (e.g., 125 I, 131 I, 35 S, 3 H, or 32 P), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or β-galactosidase), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., Qdot™ nanoparticles supplied by Quantum Dot Corporation, Palo Alto, Calif., USA).

[0058] In some embodiments, the determined cancer Gal3 expression level is compared to an activity threshold to determine whether the cancer is suitable for treatment with a Gal3:TIM-3 inhibitor disclosed herein. In some embodiments, the activity threshold is the Gal3 expression or activity level in cells of non-cancerous tissue from a patient hosting the cancer. In some embodiments, the activity threshold is the Gal3 expression or activity level in cells of a similar tissue type from a healthy individual. In some embodiments, the activity threshold is derived from the median of individual Gal3 expression levels in a cohort of patients suffering from the same type of cancer, where the patient cohort is heterogeneous with respect to Gal3 expression levels. The test cohort preferably includes at least 25, 50, 100, 200, 1000, or more individuals, including all values ​​and ranges thereof. In some embodiments, the patient Gal3 expression levels and activity threshold are normalized before comparison.

[0059] Thus, in some embodiments, the disclosure provides a method for determining whether a patient's cancer is suitable for treatment with a Gal3:TIM-3 inhibitor, the method comprising obtaining a sample containing cancer cells from the patient, determining the level of Gal3 on the cell surface in the sample, comparing the Gal3 level on the cells to an activity threshold, and determining that the patient's cancer is suitable for treatment with a Gal3:TIM-3 inhibitor if Gal3 surface expression on the patient's cancer cells is at least 15%, at least 25%, at least 50%, at least 75%, at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold, at least 1000-fold, or at least 10,000-fold higher than the activity threshold. In some embodiments, the activity threshold used for comparison can be based on the average, mean, or median Gal3 levels on the surface of cancer cells of the same cancer type from at least 100, 200, 300, or 500 cancer patients. In some embodiments, the activity threshold is based on the average, mean, or median Gal3 levels on the cell surface of similar tissue types from healthy individuals. In some embodiments, the cancer cells in the sample used to determine whether a cancer is suitable for treatment with Gal3:TIM-3 are primary cancer cells.

[0060] Some cancer types, including those that metastasize, overexpress Gal3 on their surface and therefore may be suitable for treatment using the methods disclosed herein. These cancer types include, but are not limited to, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, head / neck cancer, melanoma, sarcoma, renal cell tumor, stem cell tumor, glioblastoma, neuroendocrine tumor, bladder cancer, pancreatic cancer, gallbladder cancer, stomach cancer, prostate cancer, endometrial cancer, thyroid cancer, and mesothelioma. Therefore, in some cases, cancers suitable for treatment using the methods disclosed herein are metastatic cancers derived from the above tumors, such as metastatic lung cancer.

[0061] 2. Gal3:TIM-3 inhibitor The present disclosure provides methods for treating cancer by administering to a patient a therapeutically effective amount of at least one Gal3:TIM-3 inhibitor. A Gal3:TIM-3 inhibitor can be any molecule that inhibits the interaction between Gal3 and TIM-3, resulting in T cell activation. In some embodiments, a Gal3:TIM-3 inhibitor binds to a TIM-3 protein, and such inhibitors are referred to in this disclosure as TIM-3 inhibitors. In some embodiments, a Gal3:TIM-3 inhibitor binds to a Gal3 protein, and such inhibitors are referred to as Gal3 inhibitors. A Gal3:TIM-3 inhibitor can be a protein (e.g., an antibody) or a small molecule. Antibodies that are Gal3:TIM-3 inhibitors are referred to in this disclosure as GIAs.

[0062] i.Gal3:TIM-3 Inhibitory Antibody ("GIA") In one embodiment, a method for treating cancer comprises administering a Gal3:TIM-3 inhibitory antibody. Such an antibody can inhibit the interaction between Gal3 and TIM-3 and activate T cells. In some embodiments, the Gal3:TIM-3 inhibitory antibody is a Gal3 inhibitory antibody. In some embodiments, the Gal3:TIM-3 inhibitory antibody is a TIM-3 inhibitory antibody.

[0063] GIA Creation GIA can be expressed using methods well known in the art. See, for example, Kohler and Milstein, Nature 256:495 (1975), and Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pp. 2.5.1-2.6.7 (John Wiley & Sons 1991). Antigens, such as Gal3 or its epitopes, can be expressed. Monoclonal antibodies can be obtained by injecting a composition containing the antigen into a mouse, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies against the antigen, culturing the clones that produce antibodies against the antigen, and isolating the antibodies from the hybridoma culture medium. In some embodiments, the Gal3 epitopes used to produce Gal3 inhibitory antibodies are: SEQ ID NO:5 (PGAYPGQAPPGAYPGQAPPG), SEQ ID NO:6 (GAYPGQAPPGAYPGAPGAYP), SEQ ID NO:7 (PGAYPGQAPPGAYPGQAPPGAYPGAPGAYP), or SEQ ID NO:8 (GQAPPGAYPG).

[0064] The monoclonal antibodies produced can be isolated and purified from the hybridoma culture medium by a variety of established, well-known techniques. Such isolation techniques include affinity chromatography using protein A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, e.g., Coligan, pp. 2.7.1-2.7.12 and 2.9.1-2.9.3. See also Baines et al., "Purification of Immunoglobulin G (IgG)," in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pp. 79-104 (The Humana Press, Inc. 1992). After initial incubation of the antibody with the target protein, the antibody can be sequenced and subsequently produced by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art. See, e.g., Leung et al. Hybridoma 13:469 (1994); U.S. Patent Application Publication No. 2014 / 0099254(A1).

[0065] Human antibodies can be produced using genetically engineered transgenic mice that produce specific human antibodies in response to antigen challenge using target proteins. See Green et al., Nature Genet. 7:13 (1994); Lonberg et al., Nature 368:856 (1994). Human antibodies against target proteins can also be constructed by gene or chromosome transfection, phage display technology, or in vitro activated B cells. See, for example, McCafferty et al., 1990, Nature 348:552-553; U.S. Patent Nos. 5,567,610 and 5,229,275.

[0066] In some embodiments, the GIA is an anti-Gal3 antibody. In some embodiments, the GIA binds to a peptide having the sequence of SEQ ID NO: 5, 6, 7, or 8. In some embodiments, the GIA is an antibody that binds to Gal3 and can interfere with the interaction between TIM-3 and Gal3. In some embodiments, the GIA is an antibody that binds to a peptide comprising a sequence selected from any of SEQ ID NOs: 5-8 and can interfere with the interaction between TIM-3 and Gal3. In some embodiments, the GIA is an antibody that blocks the binding of known GIA to Gal3 and can interfere with the interaction between TIM-3 and Gal3. In some cases, administration of a Gal3:TIM-3 inhibitor disclosed herein, e.g., a Gal3 inhibitory antibody, can reduce tumor burden by at least 20%, e.g., at least 30%, at least 40%, or at least 46%, in a mouse model over a treatment period, e.g., a 3-12 week period.

[0067] In some embodiments, the GIA is an anti-Gal3 antibody. In some embodiments, the anti-Gal3 antibody is an IgG4 isotype. In some embodiments, the anti-Gal3 antibody comprises heavy chain variable region complementarity determining regions CDR1, 2, and 3 (CDR H1, CDR H2, and CDR H3), where CDR H1 comprises the amino acid sequence of SEQ ID NO: 9, CDR H2 comprises the amino acid sequence of SEQ ID NO: 10, and / or CDR H3 comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the heavy chain variable region of the anti-Gal3 antibody comprises framework regions 1 to 4 (FR H1, FR H2, FR H3, and FR H4), where FR H1 comprises the amino acid sequence of SEQ ID NO: 12, FR H2 comprises the amino acid sequence of SEQ ID NO: 13, FR H3 comprises the amino acid sequence of SEQ ID NO: 14, and / or FR H4 comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the heavy chain of the anti-Gal3 antibody comprises the amino acid sequence of SEQ ID NO: 16.

[0068] In some embodiments, the anti-Gal3 antibody comprises light chain variable region complementarity determining regions CDR1, 2, and 3 (CDR L1, CDR L2, and CDR L3), where CDR L1 comprises the amino acid sequence of SEQ ID NO: 17, CDR L2 comprises the amino acid sequence of SEQ ID NO: 18, and / or CDR L3 comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the heavy chain variable region of the anti-Gal3 antibody comprises frame regions 1-4 (FR L1, FR L2, FR L3, and FR L4), where FR L1 comprises the amino acid sequence of SEQ ID NO: 20, FR L2 comprises the amino acid sequence of SEQ ID NO: 21, FR L3 comprises the amino acid sequence of SEQ ID NO: 22, and / or FR L4 comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the light chain of the anti-Gal3 antibody comprises the amino acid sequence of SEQ ID NO: 24.

[0069] In some embodiments, the anti-Gal3 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the anti-Gal3 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26.

[0070] GIA Modifications GIAs may be produced by introducing conservative modifications into existing GIAs. For example, the modified GIAs contain heavy and light chain variable regions and / or Fc regions that are homologous to the corresponding antibodies produced above. Modified GIAs that can be used in the methods disclosed herein must retain the desired functional property of being able to block the Gal3:TIM-3 signaling pathway.

[0071] The GIA described herein can be conjugated with another functional molecule, such as another peptide or protein (such as albumin, another antibody), a toxin, a radioisotope, a cytotoxic drug, or a cytostatic agent. For example, the antibody can be conjugated by chemical crosslinking or recombinant methods. The antibody can also be conjugated with one of various non-protein polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, by the methods described in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337. The antibody can be chemically modified by covalently binding to a polymer, for example, to increase its circulating half-life. Exemplary polymers and methods of binding thereto are also set forth in US Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546.

[0072] GIAs may be produced by modifying protein modification sites. For example, glycosylation sites of antibodies can be modified to produce non-glycosylated antibodies, and such modified GIAs typically increase the affinity of the antibody for the antigen. Antibodies can also be PEGylated by reaction with polyethylene glycol (PEG), under conditions in which one or more PEG groups are attached to the antibody. PEGylation can increase the biological half-life of the antibody. Such modified antibodies can also be used to treat tumors that overexpress Gal3, as long as they retain the desired functional property of blocking the TIM-3-Gal3 pathway.

[0073] The antibody may be tagged with a detectable or functional label. Detectable labels include: 131 I or 99 Detectable labels include radiolabels such as Tc, which can be bound to antibodies using conventional chemistry. Detectable labels also include enzyme labels such as horseradish peroxidase or alkaline phosphatase. Detectable labels also include chemical moieties such as biotin, which can be detected by binding with a specific similar detectable moiety, such as labeled avidin.

[0074] In another aspect, the present invention features bispecific molecules comprising an anti-Gal3 or anti-TIM-3 antibody of the invention, or a fragment thereof. An antibody of the invention, or an antigen-binding portion thereof, can be derivatized or conjugated to another functional molecule, such as another peptide or protein (e.g., a ligand for another antibody or receptor), to create a bispecific molecule that binds to at least two different binding sites or target molecules. Indeed, an antibody of the invention may be derivatized or conjugated to two or more other functional molecules to create a multispecific molecule that binds to three or more different binding sites and / or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule of the invention, an antibody of the invention can be functionally conjugated (e.g., by chemical linkage, genetic fusion, noncovalent association, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimic, to generate a bispecific molecule. In one illustrative embodiment, bispecific antibodies can be generated using knobs-into-holes technology. The technique typically involves first generating a first half of an antibody that recognizes a first antigen, e.g., Gal3, and a second half of an antibody that recognizes a second antigen, and then combining the two halves to generate a bispecific antibody.

[0075] Thus, the present invention includes dual-specificity molecules that have at least one first binding specificity for Gal3 or TIM-3 and a second binding specificity for a second target. In some embodiments, the second target is a known cancer target, e.g., PD-L1. In some embodiments, the second target epitope is TIM-3 or Gal3, and the dual-specificity molecule is capable of simultaneously binding to TIM-3 and Gal3. In some embodiments, the second target is an Fc receptor, e.g., human Fc.gamma.RI (CD64) or human Fc.alpha. receptor (CD89). Thus, the present invention includes dual-specificity molecules that can bind to both effector cells (e.g., monocytes, macrophages, or polymorphonuclear cells (PMNs)) that express Fc.gamma.R or Fc.alpha.R, as well as target cells that express Gal3. These dual-specific molecules target effector cells to Gal3-expressing cells and trigger Fc receptor-mediated effector cell actions such as phagocytosis of PD-1-expressing cells, antibody-dependent cellular cytotoxicity (ADCC), cytokine release, or superoxide anion generation.

[0076] ii. Other Gal3:TIM-3 inhibitor molecules In another embodiment, the Gal3:TIM-3 inhibitors disclosed herein are non-proteinaceous compounds that interfere with the interaction of small molecules, Gal3, with TIM-3, thus antagonizing the immunosuppressive function of TIM-3. These small molecules are typically organic molecules with molecular weights between 50 and 2500 daltons. The compounds can also be identified using any of the numerous techniques in combinatorial library methods known in the art and disclosed, for example, in EP 2360254. Combinatorial libraries include biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; "one-bead one-compound" library methods; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer, or small molecule libraries of compounds (Lam, KS (1997) Anticancer Drug Des. 12:145).

[0077] iii. Evaluation of Gal3:TIM-3 inhibitor candidates Several well-known assays can be used to evaluate whether candidates, such as antibodies generated by immunizing animals with an antigen containing a Gal3 protein or a test compound from a combinatorial library, can block the interaction between Gal3 and TIM-3. Typically, this involves evaluating the candidates using one or more of the following types of assays: i) binding assays to test whether the candidate binds to a target protein, i.e., Gal3 or TIM-3; ii) blocking assays to test whether the candidate can block the interaction between Gal3 and TIM-3; iii) cell-based functional assays to test whether the candidate can activate T cells by blocking the interaction between Gal3 and TIM-3; and iv) in vivo efficacy assays to test whether the candidate can reduce tumor burden.

[0078] Binding assay Any assay used to evaluate the interaction of two molecules can be used to determine whether a candidate can bind to a target protein. Non-limiting exemplary assays include binding assays, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS) analysis. In some cases, the target protein, i.e., Gal3 or TIM-3 protein, can be conjugated with a radioisotope or enzyme label, so that the binding of the target protein and the candidate can be determined by detecting the labeled target protein in the complex. For example, the target protein can be directly or indirectly conjugated to the target protein. 125 I, 35 S, 14 C, or 3 H, and the radioisotope can be detected by direct counting of radioactive emissions or by scintillation counting. Alternatively, target protein molecules can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and binding of the candidate to the target protein determined by conversion of an appropriate substrate to product.

[0079] In some embodiments, immunoassays such as enzyme-linked immunosorbent assays (ELISAs) can be used to assess the binding specificity of candidate Gal3:TIM-3 inhibitors to their target proteins. In some embodiments, a sample containing the candidate is added to a plate pre-coated with the target protein and incubated for a period of time. A labeled secondary antibody that recognizes the candidate can be added, and the signal from the labeled secondary antibody is detected. In some cases, the secondary antibody is conjugated to an enzyme, and binding is assessed by adding a substrate specific for the enzyme and reading at an appropriate wavelength according to the manufacturer's instructions. Non-limiting examples of enzymes that can be used include horseradish peroxidase and alkaline phosphatase. For horseradish peroxidase, an ABTS substrate can be used, and readings at 415-490 nm can be taken to assess the candidate's ability to bind to Gal3 or TIM-3. Alternatively, ELISAs can be performed by coating the candidate on a plate, adding the target protein to the plate, and detecting said binding.

[0080] The binding rate (e.g., binding affinity) of the candidate can also be evaluated by standard assays known in the art, such as Biacore analysis (Biacore AB, Uppsala, Sweden). In one exemplary assay, the target protein is covalently coupled to a chip, e.g., a carboxymethyldextran-coated chip, using standard amine coupling chemistry and a kit provided by Biacore. Binding is measured by flowing the candidate in a buffer solution (provided by Biacore AB) at the appropriate concentration and flow rate recommended by the manufacturer. Association and dissociation rates are recorded, and the association and dissociation curves are fitted to a binding model using BIA evaluation software (Biacore AB). The K of the interaction D value, K on value and K off A preferred Gal3:TIM-3 inhibitor is a 1×10 -7 M or less, e.g., 5 x 10-7 M or less or 1 x 10 -8 It can bind to its target protein at M or less.

[0081] Blocking assay Candidates that exhibit target protein binding ability are then evaluated for their ability to block the interaction between Gal3 and TIM-3 in a blocking assay. In some embodiments, the blocking assay is an immunoassay, e.g., ELISA. In one embodiment, a method for determining whether a candidate blocks the interaction between TIM-3 and Gal3 includes coating a plate with one of the target proteins, TIM-3 or Gal3, and adding a mixture of the candidate and the other target protein, i.e., Gal3 or TIM-3, to the coated plate and detecting a signal corresponding to the binding of TIM-3 to Gal3. A decrease in signal compared to a control reaction in which no candidate is added indicates that the candidate can block the interaction between Gal3 and TIM-3.

[0082] In some embodiments, the blocking assay is a flow cytometry assay. Generally, the candidate is mixed with one of the target proteins, TIM-3 or Gal3, and the mixture is added to cells overexpressing the other target protein, Gal3 or TIM-3. The binding of TIM-3 to Gal3 on the cell surface can be detected by a fluorescently labeled antibody. A decrease in the signal in the reaction containing the candidate compared to the control indicates that the candidate can block the interaction between Gal3 and TIM-3. An exemplary blocking assay that can be used to determine whether a candidate can block the interaction between Gal3 and TIM-3 is described in Example 2.

[0083] Functional assays In some cases, candidates that exhibit binding to the target protein are further evaluated for their ability to activate T cells using a mixed lymphocyte reaction (MLR) assay. One exemplary assay is described in U.S. Patent No. 8,008,449, the relevant disclosure of which is incorporated herein by reference in its entirety. The MLR assay can be used to measure T cell proliferation, IL-2 and / or IFN-γ production. In one exemplary assay, the candidate is added to several purified T cells cultured with antigen-presenting cells (APCs) at various concentrations. The cells are then cultured in the presence of the candidate at 37°C for a period of 4 to 7 days. A specific volume of culture medium is then collected for cytokine measurement. The levels of IFN-γ and other cytokines can be measured. Methods for measuring cytokine production are well known, and commercially available kits are readily available, such as the OptEIA ELISA kit (BD Biosciences). In some embodiments, T cell proliferation is measured for a period of 12 to 24 hours, e.g., 18 hours. 3 Cells were cultured in the presence of H-thymidine, and intracellular H-thymidine levels were determined, which positively correlated with cell proliferation. 3 The amount of H-thymidine incorporation is analyzed. Results showing that cultures containing the candidate exhibit increased T cell proliferation and increased production of IL-2 and / or IFN-γ compared to controls indicate that the candidate is effective in activating T cells by blocking the interaction of TIM-3 and Gal3. One exemplary MLR assay that can be used to evaluate the ability of a candidate to activate T cells is disclosed in Example 11.

[0084] In vivo assay In another embodiment, an in vivo assay is used to determine whether a candidate is effective in treating cancer. In vivo assays can be performed in tumor models, such as mouse tumor models, according to established, known procedures. Briefly, animals, e.g., mice, are implanted subcutaneously with a human tumor cell line. Tumors are allowed to grow and reach a specific size, e.g., 100-300 mm. 3When the tumor size reaches 1500 mm, the candidate is administered to the mice at an appropriate dose and at a predetermined frequency. The candidate can be administered by several routes, such as intraperitoneal or intravenous injection. The tumor growth is typically monitored once or twice weekly for 4-8 weeks. The tumor is measured three-dimensionally (height x width x length) and the tumor volume is calculated. When the mice reach a tumor endpoint, e.g., 1500 mm, the candidate is administered to the mice at an appropriate dose and at a predetermined frequency. The candidate can be administered by several routes, such as intraperitoneal or intravenous injection. The tumor growth is typically monitored once or twice weekly for 4-8 weeks. The tumor is measured three-dimensionally (height x width x length) and the tumor volume is calculated. 3 At the end of the experiment, mice are usually euthanized when the weight loss reaches 100%, or when the mice show significant weight loss, for example, more than 15%, more than 20%, or more than 25%. Results showing slower tumor growth or a longer mean time to reach tumor endpoint volume in the candidate treatment group compared to the control indicate that the candidate has the effect of inhibiting cancer growth. One exemplary in vivo efficacy assay that can be used to evaluate the ability of a candidate in tumor treatment is disclosed in Example 4.

[0085] 4. Evaluation of the efficacy of Gal3:TIM-3 inhibitor therapy Gal3:TIM-3 inhibitor therapy as disclosed herein can reduce tumor burden and provide beneficial clinical outcomes to cancer patients, particularly those afflicted with cancers that overexpress Gal3. Methods for measuring these responses are well known to those skilled in the art of cancer therapy, for example, as described in the Response Evaluation Criteria in Solid Tumors ("RECIST") guidelines, available at ctep.cancer.gov / protocolDevelopment / docs / recist_guideline.pdf.

[0086] One approach is to measure tumor burden by assessing the expression of tumor-specific biomarkers. This approach is particularly useful for metastatic tumors. Tumor-specific biomarkers are proteins or other molecules that are unique to cancer cells or that are much more abundant in cancer cells than non-cancerous cells. Useful biomarkers for various cancers are known, and non-limiting examples of tumor-specific gene markers include alpha-fetal protein (AFP) for liver cancer, beta-2-microglobulin (B2M) for multiple myeloma, beta-human chorionic gonadotropin (β-hCG) for choriocarcinoma and germ cell tumors, CA19-9 for pancreatic cancer, gallbladder cancer, bile duct cancer, and gastric cancer, CA-125 and HE4 for ovarian cancer, carcinoembryonic antigen (CEA) for colorectal cancer, chromogranin A (CgA) for neuroendocrine tumors, fibrin / fibrinogen for bladder cancer, prostate-specific antigen (PSA) for prostate cancer, and thyroglobulin for thyroid cancer. See www.cancer.gov / about-cancer / diagnosis-staging / diagnosis / tumor-markers-fact-sheet.

[0087] Methods for measuring the expression level of tumor-specific gene markers are well known. In some embodiments, real-time reverse transcriptase polymerase chain reaction (RT-PCR) is performed to isolate the mRNA of the gene marker from blood samples or tumor tissues and quantify the expression of the gene marker. In some embodiments, Western blot, immunohistochemistry, or flow cytometry analysis is performed to evaluate the protein expression of the tumor-specific gene marker. Typically, the level of tumor-specific gene markers is measured in multiple samples collected over the course of the treatment of the present invention, and a decrease in the level correlates with a decrease in tumor burden.

[0088] In another approach, reduction in tumor burden with Gal3:TIM-3 inhibitor therapy as disclosed herein is indicated by a decrease in tumor size or cancer burden in the body. Measurement of tumor size is typically performed by imaging techniques. For example, computed tomography (CT) scans can be used to characterize either the growth of existing lesions or the development of new lesions or tumor metastases. These measurements can provide accurate and reliable anatomical information on disease progression as well as tumor shrinkage or growth.

[0089] In yet another approach, tumor burden reduction can be assessed by functional and metabolic imaging techniques. These techniques can provide early assessment of therapeutic response by observing changes in perfusion, oxygenation, and metabolism. For example, 18 F-FDG PET assesses tissue metabolism using radiolabeled glucose analogue molecules. Tumors typically increase glucose consumption, and changes in glucose corresponding to decreased tumor tissue metabolism indicate decreased tumor burden. A similar imaging technique is disclosed in Kang et al., Korean J.Radiol. (2012) 13(4) 371-390.

[0090] Patients receiving the treatments disclosed herein may experience varying degrees of tumor burden reduction. In some cases, patients may experience a complete response (CR), also known as "disease-free survival (NED)." CR means that all detectable tumor has disappeared, as evidenced by tests, physical examination, and scans. In some cases, patients receiving the combination therapy disclosed herein experience a partial response (PR), which roughly corresponds to a reduction in total tumor volume of at least 50%, but with evidence that some residual disease still remains. In some cases, the residual disease in a deep partial response may actually be dead tumor or scar, and as a result, a small number of patients classified as having a PR may actually be in CR. Many patients who experience a reduction during treatment may continue treatment and experience further reduction, resulting in a CR. In some cases, patients receiving treatment may experience a minor response (MR), which roughly refers to a small reduction in total tumor volume of more than 25% but less than the 50% that constitutes a PR. In some cases, treated patients may have stable disease (SD), which means that the tumor remains roughly the same size but may include either a small amount of growth (usually less than 20% or 25%) or a small amount of shrinkage (less than PR unless there is some benefit, in which case SD is usually defined as less than 25%).

[0091] The desired effect or desired clinical outcome of treatment may include, for example, a reduction in cancer cell infiltration into surrounding organs (i.e., delay and / or stop to some extent); inhibition of tumor metastasis (i.e., delay and / or stop to some extent); an increase in response rate (RR); an increase in the duration of response; a partial alleviation of one or more cancer-related symptoms; a reduction in the dosage of other drugs required to treat the disease; a delay in disease progression; and / or an increase in the patient's survival time and / or quality of life. Methods for assessing these effects are well known and / or are disclosed, for example, at cancerguide.org / endpoints.html and in the above-mentioned RECIST guidelines.

[0092] In some cases, administration of a Gal3:TIM-3 inhibitor disclosed herein may reduce tumor burden by at least 20%, at least 30%, at least 40%, or at least 46% within the treatment period.

[0093] 4. Combination with other treatments In some embodiments, a combination of a Gal3:TIM-3 inhibitor and one or more second anti-cancer agents ("second agents") may be used to reduce tumor burden in a patient. "Combination therapy" or "in combination" does not imply that the therapeutic agents must be administered simultaneously and / or formulated for delivery together, although these delivery methods are within the scope described herein. The Gal3:TIM-3 inhibitor and second agent may be administered according to the same or different dosing regimens. In some embodiments, the Gal3:TIM-3 inhibitor and second agent are administered sequentially, in any order, during the entire treatment period or a portion of the treatment period. In some embodiments, the Gal3:TIM-3 inhibitor and second anti-cancer agent are administered simultaneously or nearly simultaneously (e.g., within about 1, 5, 10, 15, 20, or 30 minutes of each other). Non-limiting examples of combination therapy include the following: for example, administration of a Gal3 and a second anti-cancer agent, where the Gal3:TIM-3 inhibitor is "A" and the second anti-cancer agent or compound is "B":

[0094] A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B

[0095] B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / A B / B / A / A

[0096] B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / A A / A / B / A

[0097] Administration of a second anti-cancer agent to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the treatment. We next disclose several preferred second agents that can be used in combination with Gal3:TIM-3 inhibitors to treat cancer.

[0098] i.Targeted therapy In some embodiments, the second anti-cancer agent is a targeted therapeutic agent, i.e., comprises an agent directed against a specific molecular or genetic target, such as one associated with a receptor tyrosine kinase.

[0099] ii. Chemotherapy and radiation therapy Chemotherapeutic agents suitable for use in combination with the Gal3:TIM-3 inhibitor of the present invention include agents that have the properties of killing cancer cells or inhibiting cancer cell growth. Compared to the above-mentioned targeted therapies, chemotherapy generally excludes agents that function nonspecifically, for example, inhibit the cell division process known as mitosis, and more selectively block extracellular growth signals (i.e., signal transduction blockers). These agents include, but are not limited to, microtubule inhibitors (e.g., taxanes and vinca alkaloids), topoisomerase inhibitors and antimetabolites (e.g., nucleoside analogs that act like gemcitabine), mitotic inhibitors, alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, anthracyclines, intercalators, agents that can interfere with signal transduction pathways, agents that promote apoptosis, and proteosome inhibitors.

[0100] Alkylating agents work best in the quiescent phase of cells. These classes of agents are cell cycle non-specific. Exemplary alkylating agents that can be used in combination with the Gal3:TIM-3 inhibitors of the present invention include nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes: uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard, etc.). Mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™), ifosfamide (Mitoxana®), melphalan (Alkeran®), chlorambucil (Leukeran®), pipobroman (Amedel®, Vercyte®), These include, but are not limited to, triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and dacarbazine (DTIC-Dome®).Additional exemplary alkylating agents include oxaliplatin (Eloxatin®); temozolomide (Temodar® and Temodal®); dactinomycin (also known as actinomycin-D, Cosmegen®); melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran®); altretamine (also known as hexamethylmelamine (HMM), Hexalen®); carmustine (BiCNU®); bendamustine (Treanda®); busulfan (Busulfex® and Myleran®); carboplatin (Paraplatin®); lomustine (also known as CCNU, CeeNU®); cisplatin (also known as CDDP, Platinol® and Platinol®-AQ); chlorambucil (Leuk eran®); cyclophosphamide (Cytoxan® and Neosar®); dacarbazine (also known as DTIC, DIC, and imidazole carboxamide, DTIC-Dome®); altretamine (also known as hexamethylmelamine (HMM), Hexalen®); ifosfamide (Ifex®); prednummustine; procarbazine (Matulane®); mechlorethamine (also known as nitrogen mustard, mustine, and mechlorethamine hydrochloride, Mustargen®); streptozocin (Zanosar®); thiotepa (also known as thiophosphamide, TESPA, and TSPA, Thioplex®); cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®); and bendamustine HCl These include, but are not limited to, Treanda®.

[0101] Antitumor antibiotics are chemical agents derived from natural products produced by the soil fungus Streptomyces. These drugs act in multiple phases of the cell cycle and are considered to be cell cycle specific. There are several types of antitumor antibiotics, including, but not limited to, anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, mitoxantrone, and idarubicin), chromomycins (e.g., dactinomycin and plicamycin), mitomycin, and bleomycin.

[0102] Antimetabolites are a type of chemotherapy treatment that is cell cycle specific. When cells incorporate these antimetabolites into their metabolism, the cells cannot divide. These classes of chemotherapy drugs include folate antagonists such as methotrexate; pyrimidine antagonists such as 5-fluorouracil, floxuridine, cytarabine, capecitabine, and gemcitabine; purine antagonists such as 6-mercaptopurine and 6-thioguanine; and adenosine deaminase inhibitors such as cladribine, fludarabine, nelarabine, and pentostatin.

[0103] Exemplary anthracyclines that can be used in combination with the Gal3:TIM-3 inhibitors of the invention include, for example, doxorubicin (Adriamycin® and Rubex®); bleomycin (Lenoxane®); daunorubicin (daunorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, Cerubidine®); daunorubicin liposomal formulation (daunorubicin citrate liposomal formulation, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence®); idarubicin (Idamycin®, Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; ravidomycin; and desacetylravidomycin.

[0104] Microtubule inhibitors include vinca alkaloids and taxanes. Exemplary vinca alkaloids that can be used in combination with the Gal3:TIM-3 inhibitors of the present invention include, but are not limited to: vinorelbine tartrate (Navelbine®), vincristine (Oncovin®), and vindesine (Eldisine®); vinblastine (also known as vinblastine sulfate, vincaleukoblastine and VLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®). Exemplary taxanes that can be used in combination with the Gal3:TIM-3 inhibitors of the present invention include, but are not limited to: paclitaxel and docetaxel. Non-limiting examples of paclitaxel-based drugs include nanoparticle albumin-bound paclitaxel (ABRAXANE, marketed by Abraxis Bioscience), docosahexaenoic acid-bound paclitaxel (DHA-paclitaxel, Taxoprexin, marketed by Protarga), polyglutamic acid-bound paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX marketed by Cell Therapeutic), tumor-activated prodrug (TAP), ANG105 (Angiopep-2 conjugated with 3 molecules of paclitaxel, marketed by ImmunoGen), paclitaxel-EC-1 (paclitaxel conjugated with the erbB2-recognizing peptide EC-1; Li et al. al., Biopolymers (2007) 87:225-230), and glucose-conjugated paclitaxel (e.g., 2'-paclitaxel methyl 2-glucopyranosylsuccinate, see Liu et al., Bioorganic & Medicinal Chemistry Letters (2007) 17:617-620).

[0105] Exemplary proteosome inhibitors that can be used in combination with the Gal3:TIM-3 inhibitors of the present invention include bortezomib (Velcade.RTM.); carfilzomib (PX-171-007, (S)-4-methyl-N--((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4- ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O-methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(1S)-2-[(-2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912).

[0106] In some embodiments, the chemotherapeutic agent is chlorambucil, cyclophosphamide, ifosfamide, melphalan, streptozocin, carmustine, lomustine, bendamustine, uramustine, estramustine, carmustine, nimustine, ranimustine, mannosulfan, busulfan, dacarbazine, temozolomide, thiotepa, altretamine, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, daunorubicin, doxorubicin, epirubicin, idarubicin, SN-38, ARC, NPC, campothecin, topotecan, 9-nitrocamptothecin, 9-aminocamptothecin, leuprolide ... Bifen, gimatecan, diflomotecan, BN80927, DX-895If, MAG-CPT, amsacrine, etoposide, etoposide phosphate, teniposide, doxorubicin, paclitaxel, docetaxel, gemcitabine, accatin III, 10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol, Selected from the group consisting of cephalomannine, 10-deacetyl-7-epitaxol, 7-epitaxol, 10-deacetylbaccatin III, 10-deacetylcephalomannine, gemcitabine, irinotecan, albumin-bound paclitaxel, oxaliplatin, capecitabine, cisplatin, docetaxel, liposomal irinotecan, and etoposide, and combinations thereof.

[0107] In certain embodiments, chemotherapeutic agents are administered at dosages and schedules that may be guided by dosages and schedules approved by the U.S. Food and Drug Administration (FDA) or other regulatory agencies following empirical optimization.

[0108] In still further embodiments, two or more chemotherapeutic agents may be administered simultaneously or sequentially in any order during all or part of the treatment period. The two agents may be administered according to the same or different dosing regimens.

[0109] Radiation therapy requires maximal exposure of the affected tissue while sparing surrounding normal tissue. Interstitial therapy, in which needles containing radioactive sources are implanted into the tumor, has become an important novel approach. This method allows for the local delivery of high doses of radiation while avoiding surrounding normal tissue. Intraoperative radiotherapy, in which the beam is placed directly on the tumor during surgery while normal structures are safely moved away from the beam, is another specialized radiation technique. This also achieves effective irradiation of the tumor while limiting exposure to surrounding structures. Despite the clear advantages of approaches based on local control of irradiation, patient survival rates are still very low.

[0110] iii. Other treatments The present methods involving Gal3:TIM-3 inhibitors can be combined with other therapeutic approaches, such as surgery, radiation, and / or hormone therapy, which can block growth-promoting signals derived from classical endocrine hormones, e.g., primarily estrogens in breast cancer and androgens in prostate cancer.

[0111] 5. Pharmaceutical Compositions The Gal3:TIM-3 inhibitors disclosed herein are useful for preparing pharmaceutical compositions or medicaments for treating the above-mentioned inflammatory diseases. Pharmaceutical compositions or medicaments for use in the present invention can be formulated by standard techniques using one or more physiologically acceptable carriers or excipients. Suitable pharmaceutical carriers are described herein and, for example, in "Remington's Pharmaceutical Sciences" by E.W. Martin. The Gal3:TIM-3 inhibitors of the present invention and their physiologically acceptable salts and solvates can be prepared for administration by any suitable route, including, but not limited to, oral, topical, intranasal, rectal, parenteral (e.g., intravenous, subcutaneous, intramuscular, etc.), and combinations thereof. In some embodiments, the therapeutic agent is dissolved in a liquid, such as water.

[0112] For oral administration, the pharmaceutical compositions or medicaments disclosed herein can be in the form of, for example, tablets or capsules formulated by conventional methods. (a) Diluents or fillers, such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose (e.g., ethyl cellulose, crystalline cellulose), glycine, pectin, Preferred are tablets and gelatin capsules containing the active ingredient together with (b) lubricants such as silica, anhydrous colloidal silica, talc, stearic acid, its magnesium or calcium salts (e.g., magnesium stearate or calcium stearate), metal stearates, colloidal silicon dioxide, hydrogenated vegetable oil, corn starch, sodium benzoate, sodium acetate and / or polyethylene glycol; for tablets, (c) binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone and / or hydroxypropylmethylcellulose; and optionally (d) disintegrants such as starches (e.g., potato starch or sodium starch), glycolic acid, agar, alginic acid or its sodium salts, or effervescent mixtures; (e) wetting agents such as sodium lauryl sulfate and / or (f) absorbents, flavorings, and sweeteners. In some embodiments, the tablets contain a mixture of hydroxypropyl methylcellulose, polyethylene glycol 6000, and titanium dioxide. Tablets may be either thin- or enteric-coated according to methods known in the art.

[0113] Liquid preparations for oral administration can be in the form of, for example, solution, syrup, or suspension, or can be provided as a dry product, which can be prepared with water or other suitable medium before use.Such liquid preparations can be prepared by conventional methods with pharmaceutically acceptable additives, such as suspending agents, for example, sorbitol syrup, cellulose derivatives, or edible hydrogenated fats; emulsifiers, for example, lecithin or acacia; non-aqueous medium, for example, almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils; and preservatives, for example, methyl or propyl p-hydroxybenzoate or sorbic acid.Preparations can also contain buffer salts, light, coloring agents, and / or sweeteners as needed.If necessary, preparations for oral administration can be appropriately formulated to control the release of active compounds.

[0114] For topical administration, the compositions of the present invention may be in the form of emulsions, lotions, gels, creams, jellies, solutions, suspensions, ointments, and transdermal patches. For inhalation delivery, the compositions may be delivered as dry powders or liquids via nebulizers. For parenteral administration, the compositions may be in the form of sterile injectable solutions and sterile packed powders. Preferably, the injectable solutions are formulated at a pH of about 4.5 to about 7.5.

[0115] The compositions of the present invention can also be provided in a lyophilized form. Such compositions can contain a buffer, such as bicarbonate, for preparation before administration. For example, a buffer can be included in the lyophilized composition for preparation for administration with water. The lyophilized composition can further contain a suitable vasoconstrictor, such as epinephrine. If necessary, the lyophilized composition can be provided in a syringe packaged together with a buffer for preparation for administration, so that the prepared composition can be immediately administered to a patient.

[0116] The compound can be encapsulated in a controlled drug delivery system, such as a pressure-controlled delivery capsule (see, e.g., Takaya et al., J. Control Rel., 50:111-122 (1998)), a colon-targeted delivery system, an osmotic drug delivery system, and the like. The pressure-controlled delivery capsule can comprise an ethylcellulose membrane. The colon-targeted delivery system can comprise a lactose-containing tablet core overcoated with an acid-soluble substance, e.g., Eudragit E®, and then overcoated with an enteric substance. The osmotic drug delivery system can be a single or multiple osmotic units encapsulated in a hard gelatin capsule (e.g., a capsule osmotic pump; e.g., commercially available from Alzet, Inc., Cupertino, California, USA). Typically, the osmotic unit comprises an osmotic push layer and a drug layer, the drug being surrounded by a semipermeable membrane.

[0117] 6. Dosage The pharmaceutical composition or drug can be administered to a subject in a therapeutically effective dose to treat a cancer described herein. In some embodiments, the pharmaceutical composition or drug is administered to a subject in an amount sufficient to elicit an effective therapeutic response in the subject.

[0118] The administered dose will vary depending on several factors, including, but not limited to, the subject's weight, age, individual condition, the volume of the surface area or region to be treated, and / or the mode of administration. The dose size will be determined by the existence, nature, and extent of side effects associated with the administration of a particular compound in a particular subject. Preferably, the lowest dose and concentration required to achieve the desired results should be used. Dosages should be appropriately adjusted for children, the elderly, debilitated patients, and patients with cardiac and / or hepatic disease. Further guidance can be obtained from studies known in the art using experimental animal models to evaluate dosages.

[0119] Dosing regimens are adjusted to obtain the optimal desired response, e.g., a therapeutic response or minimal side effects. For administration of Gal3:TIM-3 inhibitory antibodies, the dosage ranges from about 0.0001 to about 100 mg / kg of the subject's body weight, usually from about 0.001 to about 20 mg / kg, or from about 0.01 to about 40 mg / kg, more usually from about 0.01 to about 10 mg / kg. Preferably, the dosage is within the range of 0.1 to 10 mg / kg of body weight. For example, the dosage can be 0.1, 0.3, 1, 3, 5, or 10 mg / kg of body weight, more preferably 0.3, 1, 3, or 10 mg / kg of body weight.

[0120] The dosing schedule is usually designed to achieve an exposure that results in sustained receptor occupancy (RO) based on the typical pharmacokinetic properties of the Ab. Exemplary treatment regimens involve administration once weekly, once every other week, once every three weeks, once every four weeks, once monthly, once every three months, or once every three to six months. The dosing and schedule may be modified during treatment. For example, dosing schedules may include: (i) every other week in a six-week cycle; (ii) six doses every four weeks, followed by every three months; (iii) every three weeks; or (iv) one dose of 3-10 mg / kg of body weight, followed by 1 mg / kg of body weight every two to three weeks. Considering that IgG4 Abs typically have a half-life of 2-3 weeks, a preferred dosing regimen for the Gal3:TIM-3 inhibitors of the present invention comprises 0.3-10 mg / kg of body weight, preferably 3-10 mg / kg of body weight, more preferably 3 mg / kg of body weight, administered intravenously, with the Ab given every 14 days in cycles of 6 or 12 weeks or less until disease-free survival or progression is confirmed.

[0121] In some cases, two or more antibodies with different binding specificities are administered simultaneously, with the dosage of each Ab administered falling within a specified range. Typically, the antibodies are administered multiple times. The interval between single doses can be, for example, weekly, biweekly, every three weeks, monthly, quarterly, or annually. The intervals can also be irregular, as indicated by measuring the patient's blood levels of Abs against the target antigen. In some methods, the dosage is adjusted to achieve a plasma Ab concentration of about 1-1000 mg / ml, and in some methods, a plasma Ab concentration of about 25-300 mg / ml.

[0122] In some cases, the Gal3:TIM-3 inhibitor is a compound that may be administered at a therapeutically effective daily dose for multiple days, and treatment may be continued for periods ranging from 3 days to 2 weeks or more. While continuous daily dosing is the preferred means of achieving a therapeutically effective dose, it is also possible to administer the agent daily, even if the agent is administered daily, as long as the administration is repeated frequently enough to maintain a therapeutically effective concentration of the agent in the subject. A beneficial therapeutic effect can be achieved without administration of the agent daily, every other day, or, if a higher dose range is used and tolerated by the subject, twice weekly.

[0123] In some embodiments, the present disclosure provides that a unit dosage for oral administration to an individual weighing approximately 50-70 kg may contain approximately 20-300 mg of active ingredient. Typically, the dosage of Gal3:TIM-3 is sufficient to achieve the desired effect. Optimal administration schedules can be calculated from measurements of drug accumulation in the subject's body. Generally, dosages may be given one or more times daily, weekly, or monthly. One of ordinary skill in the art can readily determine optimal dosages, administration methods, and repetition rates.

[0124] Thus, in some embodiments, the pharmaceutical compositions provided herein are sterile solutions containing an antibody capable of interfering with the interaction between Gal3 and TIM-3 on T cells in a cancer patient, the solution containing 10 μg to 100 mg, e.g., 10 μg to 40 mg, 100 μg to 40 mg, or 1 mg to 10 mg of antibody per kilogram of patient body weight in 100 ml of solution suitable for intravenous delivery over a period of time, e.g., 1 to 4 hours. The antibody in the sterile solution can be an anti-Gal3 antibody or an anti-TIM-3 antibody. In some embodiments, the sterile solution further comprises one or more targeted therapeutic agents, e.g., one or more checkpoint inhibitor therapeutic agents described above. In some embodiments, the sterile solution further comprises one or more nanoparticles having a diameter of 10 to 100 nm, e.g., 40 to 100 nm, or 50 to 80 nm.

[0125] In some embodiments, the compositions of the invention are administered for one week or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 weeks or more. In still other embodiments, the compounds are administered for one month or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more.

[0126] Alternatively, Abs can be administered as sustained-release formulations, in which case less frequent administration is required. The dosage and frequency will vary depending on the half-life of the Ab in the patient. Generally, human Abs exhibit the longest half-life, followed by humanized Abs, chimeric Abs, and non-human Abs. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low dosages are administered at relatively infrequent intervals over an extended period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high dosages are required at relatively short intervals until the progression of the disease is reduced or stopped, preferably until the patient shows partial or complete remission of the disease symptoms. Thereafter, the patient can be administered in a prophylactic form.

[0127] The dosage of the compositions of the present invention can be monitored and adjusted throughout treatment depending on the severity of symptoms, the frequency of recurrence, and / or the physiological response to the treatment regimen. Such adjustments of the treatment regimen are routinely performed by those skilled in the art.

[0128] Non-limiting exemplary embodiments The present invention is illustrated by the following non-limiting exemplary embodiments. 1. A method for activating an immune response in a patient, comprising administering to the patient a Gal3:TIM-3 inhibitor that interferes with the interaction between Gal3 and TIM-3 in the patient, wherein the inhibitor is administered in an amount sufficient to activate the immune response. 2. The method of embodiment 1, wherein said TIM-3 is expressed on immune cells of said patient. 3. The method of embodiment 1 or 2, wherein the patient is a host of cancer, the interaction between Gal3 and TIM-3 occurs in the tumor microenvironment, and the Gal3:TIM-3 inhibitor is administered in an amount sufficient to reduce the cancer burden in the patient. 4. The method of embodiment 3, wherein said cancer comprises cells in a tumor microenvironment, said cells overexpressing Gal3 on their surface. 5. A method of activating an immune response in a patient hosting a cancer comprising cells in a tumor microenvironment, the cells overexpressing Gal3 on their surface, the method comprising administering to the patient a Gal3:TIM-3 inhibitor that interferes with the interaction of Gal3 with TIM-3 on the immune cells in the tumor microenvironment, the inhibitor being administered in an amount sufficient to reduce the cancer burden in the patient by activating the immune response. 6. The method of embodiment 2 or 5, wherein the immune cells are T cells and / or NK cells. 7. The method of any one of embodiments 3 to 5, wherein the cancer is a metastatic cancer or a primary cancer. 8. The method of any one of embodiments 1 to 7, wherein the inhibitor binds to TIM-3. 9. The method of any one of embodiments 1 to 8, wherein the inhibitor binds to Gal3. 10. The method of any one of embodiments 1 to 9, wherein said Gal3:TIM-3 inhibitor is an antibody. 11. The method of embodiment 5, wherein the antibody recognizes a peptide comprising a sequence selected from the group consisting of SEQ ID NOs: 5-8. 12. The method of embodiment 5, wherein the antibody is a single chain antibody or a Fab. 13. The method of embodiment 5, wherein the antibody is a humanized or human antibody. 14. The method of any one of embodiments 1-13, wherein said administering said Gal3:TIM-3 inhibitor is by intravenous infusion. 15. The method of any one of embodiments 1-14, wherein said Gal3:TIM-3 inhibitor is administered in combination with one or more other treatments. 16. The method of embodiment 15, wherein the one or more other treatments are selected from the group consisting of chemotherapy, radiation therapy, and checkpoint inhibitor therapy. 17. The method of embodiment 15 or 16, wherein said checkpoint inhibitor therapy is selected from the group consisting of anti-PD-1 therapy and anti-CTLA4 therapy. 18. The method of any one of embodiments 1-17, wherein said administration of said inhibitor is administered every other week at a dosage of 100 μg / kg body weight to 40 mg / kg body weight. 19. A method for determining whether a patient's cancer is suitable for treatment with a Gal3:TIM-3 inhibitor, said method comprising: combining cells obtained from a known type of tumor microenvironment from a patient with an antibody specific for Gal3; determining the level of Gal3 on the cells; comparing the Gal3 level on the cell surface to a first activation threshold of Gal3; and determining that the patient's cancer is suitable for treatment with a Gal3:TIM-3 inhibitor if the level of Gal3 on the cell surface is greater than the first activity threshold; A method comprising: 20. The method of embodiment 19, wherein said first activity threshold of Gal3 is derived from a cohort of at least 100 test individuals suffering from the same type of cancer as said patient. 21. The method of embodiment 20, wherein determining that the patient's cancer is suitable for a treatment step further comprises determining whether the Gal3 level on the cell surface obtained from the tumor microenvironment is 25% or greater compared to a second activity threshold of Gal3, wherein the second activity threshold is derived from a sample containing corresponding cells from a healthy patient. 22. The method of any one of embodiments 19 to 21, wherein the cells obtained from the tumor microenvironment comprise at least cancer cells and / or tumor-associated macrophages. 23. Determining that the patient's cancer is suitable for a treatment step includes determining whether the tumor microcirculation 22. The method of embodiment 21, further comprising determining whether the level of Gal3 on the cell surface obtained from the border is 75% or greater compared to said second activity threshold. 24. A sterile solution capable of interfering with the interaction of Gal3 and TIM-3 on T cells in a cancer patient, said solution comprising 10 μg to 100 mg of an antibody per kilogram of patient body weight in 100 ml of solution suitable for intravenous delivery over 1 to 4 hours, said antibody being capable of interfering with the interaction of Gal3 and TIM-3 on T cells. 25. The sterile solution of embodiment 23, wherein the sterile solution further comprises one or more other checkpoint inhibitor antibodies. 26. The sterile solution of embodiment 23, wherein the one or more other checkpoint inhibitor antibodies are selected from the group consisting of anti-PD-1 and anti-CTLA-4 antibodies. 27. The sterile solution of any one of embodiments 23 to 25, wherein the sterile solution further comprises nanoparticles having a diameter of 10 to 100 nm. 28. The sterile solution of embodiment 23, wherein the antibody is an anti-Gal3 antibody. 29. The sterile solution of embodiment 23, wherein the antibody is an anti-TIM-3 antibody. 30. A method for producing an anti-Gal3 antibody capable of interfering with the interaction between Gal3 and TIM-3, the method comprising: introducing into an animal a peptide comprising an array selected from the group consisting of SEQ ID NOs: 5 to 8, and the animal producing a Gal3 antibody. 31. A humanized or chimeric anti-Gal3 antibody, the antibody comprising: (1) a light chain variable region comprising complementarity-determining regions (CDRs) L1, L2, and L3; and (2) a heavy chain variable region comprising CDRs H1, H2, and H3; the CDR L1 comprises the amino acid sequence of SEQ ID NO: 17; the CDR L2 comprises the amino acid sequence of SEQ ID NO: 18; the CDR L3 comprises the amino acid sequence of SEQ ID NO: 19; the CDR H1 comprises the amino acid sequence of SEQ ID NO: 9; the CDR H2 comprises the amino acid sequence of SEQ ID NO: 10, and the CDR H3 comprises the amino acid sequence of SEQ ID NO: 11; Humanized or chimeric anti-Gal3 antibodies. 32. The humanized or chimeric anti-Gal3 antibody of embodiment 31, wherein the heavy chain variable region has a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 25. 33. The humanized or chimeric anti-Gal3 antibody of embodiment 31 or 32, wherein the light chain variable region has a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 26. 34. A method for selecting a compound that blocks the interaction between Gal3 and TIM-3 and is capable of activating an immune response and / or treating cancer in a patient, said method comprising: (a) contacting a library of compounds with Gal3 and TIM-3; and (b) selecting one or more candidate compounds from the library that are capable of blocking the interaction between Gal3 and TIM-3; A method comprising: 35. (c) contacting one or more candidate compounds selected from step (b) with a mixture containing T cells and allogeneic antigen-presenting cells to identify one or more compounds capable of stimulating T cells; and / or (d) administering to a mammal hosting a tumor one or more candidate compounds selected from (b) above to identify one or more compounds capable of reducing tumor burden in said mammal, and optionally (e) administering to said patient an effective amount of a compound capable of stimulating said T cells and / or reducing tumor burden in said mammal, thereby activating an immune response and / or treating cancer in said patient; 35. The method of embodiment 34, further comprising: 36. The method of embodiment 35, wherein the compound is an antibody. 37. A method for activating an immune response in a patient, comprising administering to the patient a Gal3:TIM-3 inhibitor that interferes with the interaction between Gal3 and TIM-3, wherein the inhibitor is administered in an amount sufficient to activate the immune response, and the inhibitor comprises a humanized antibody described in any one of embodiments 31 to 33. 38. A method of activating an immune response in a patient, comprising administering to the patient an antibody, wherein the antibody comprises a means for inhibiting the interaction between Gal3 and TIM-3. 39. The method of embodiment 38, wherein the antibody further comprises means for binding to Gal3 or TIM-3. [Example]

[0129] The present invention will now be described with reference to the following examples, which are provided by way of illustration and are not intended to be limiting in any way. Unless otherwise indicated, standard techniques well known in the art or those detailed below were employed.

[0130] Example 1. Construction of Gal3-overexpressing cell lines A20, a murine B lymphoma cell line obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), was transfected with a nucleic acid construct encoding either Flag-tagged human Gal3 protein or Flag-tagged human PDL1 protein. Additionally, the construct contained an antibiotic resistance marker. Transformed cells were selected based on antibiotic resistance to generate A20 cells stably expressing Flag-tagged human Gal3 protein (A20 Gal3 cells) or Flag-tagged human PDL1 protein (A20 hPDL1 cells).

[0131] Example 2. Gal3 specifically binds to TIM-3 This example describes various assays performed to assess the interaction between Gal3 and TIM-3.

[0132] Binding assay - co-immunoprecipitation To test whether TIM-3 specifically interacts with Gal3, we performed coimmunoprecipitation experiments. 293T cells were cotransfected with a plasmid encoding HA-tagged TIM-3 and a plasmid encoding Flag-tagged Gal3, Flag-tagged Gal9, or Flag-tagged CEACAM1. Transfections were performed using Lipofectamine 3000 (Waltham, MA, USA) according to the manufacturer's protocol. Transfected cells were grown overnight, washed, and lysed in 1 ml of lysis buffer. Lysed cells were centrifuged, and the supernatant (lysate) was collected. Lysates were prepared, separated by SDS-PAGE, and probed with anti-HA (Figure 1A) and anti-Flag antibodies (Figure 1B), respectively. Both anti-Flag and anti-HA antibodies were purchased from Sigma. Arrows in Figures 1A and 1B indicate the presence of various proteins.

[0133] For immunoprecipitation, anti-Flag agarose beads (Abcam, Cambridge, MA, USA) were added to the supernatant (lysate) obtained above. The beads and lysate were incubated overnight at 4°C with rotation to allow binding of Flag-tagged proteins. The beads were then washed three times with lysis buffer, mixed with 1x SDS PAGE sample buffer, boiled, and separated by SDS-PAGE. The SDS-PAGE gel was transferred to a membrane probed with anti-HA antibody (Figure 1C). In Figures 1A-1C, lanes 1-3 represent cells cotransfected with a plasmid encoding HA-tagged TIM-3 and a plasmid encoding Flag-tagged Gal3, cells cotransfected with a plasmid encoding HA-tagged TIM-3 and a plasmid encoding Flag-tagged Gal9, or cells cotransfected with a plasmid encoding HA-tagged TIM-3 and a plasmid encoding Flag-tagged Gal9, respectively. Results from lysates obtained from cells cotransfected with a plasmid encoding EACAM1 are shown.

[0134] As shown in Figure 1, the results indicate that human Gal3 specifically pulled down human TIM-3, whereas human CEACAM1 was unable to pull down HA-tagged human TIM-3. Human Gal9 also appeared to pull down human TIM-3 (Fig. 1C, lane 2), but this appeared to be nonspecific due to Gal9 protein aggregation—the molecular weight of Gal9 is likely much larger than its actual size of 40 kD. The conclusion that the interaction between Gal9 and TIM-3 is nonspecific in nature is also supported by evidence shown below in Figure 5B.

[0135] To test whether Gal3 specifically interacts with TIM-3, we performed further co-immunoprecipitation experiments. Flag-human Gal3 plasmid (OriGene, Rockville, MD, USA) was transfected into 80% confluent 293T cells. Transfections were performed in 10 cm plates using Lipofectamine 3000 as described above. After overnight transfection, cells were plated on 10 cm plates coated with human Fc, human PD1-Fc, or human TIM-3Fc for 3 hours. Cells were washed once with 1x PBS and then lysed in 1 ml lysis buffer. The cell lysate was collected and centrifuged. Protein G beads were added to the supernatant obtained after centrifugation and incubated with rotation at 4°C for 4 hours. The beads were then washed three times with lysis buffer, followed by the addition of 1x SDS-PAGE sample buffer. The samples containing the beads were boiled, separated by SDS-PAGE, and transferred to a membrane. The membrane was then probed with anti-Flag antibody. As shown in Figure 2, human TIM-3 specifically pulled down Flag-tagged Gal3. In contrast, neither human Fc nor human PD1 Fc was able to pull down TIM-3. This indicates that Gal3 does not bind to PD1 Fc and that the binding between Gal3 and TIM-3 is specific.

[0136] Binding assay - cell adhesion assay Next, a cell adhesion assay was performed to confirm the binding between Gal3 and TIM-3. In this experiment, a 96-well plate was coated with human Fc, human PD1-Fc, human VISTA-Fc, or human TIM-3-Fc overnight at 4°C and then blocked with 2% BSA in PBS for 2 hours at 37°C. A20, A20 cells overexpressing human Gal3 (A20 Gal3), or A20 cells overexpressing human PDL1 (A20 PDL1) were seeded into the wells coated with the various Fc proteins. The plate was then centrifuged at 720 rpm and stopped. The plate was incubated at 37°C for 30 minutes and then submerged in PBS. The plate was slowly inverted 180 degrees and held in the inverted position for 30 minutes. After uninverting, the plate was removed from the PBS, and 200 μl of solution from each well was removed and discarded. Approximately 100 μl of the remaining solution was transferred to the 96-well plate. Cells were counted by flow cytometry analysis.

[0137] The results show that the number of A20 cells expressing human Gal3 (A20 Gal3) that adhered to human TIM-3 Fc-coated plates was significantly greater than the number of cells that adhered to plates coated with human VISTA Fc or human PD1 Fc. As expected, the number of A20 PDL1 cells, which are known to adhere to hPD1 Fc because PDL1 is a known ligand for PD1, was significantly greater than the number that adhered to plates coated with human VISTA Fc or human TIM-3 Fc. These results further confirmed that the interaction between Gal3 and TIM-3 is specific.

[0138] Blocking assay - flow cytometry Flow cytometry analysis was performed to determine the intercellular interactions between TIM-3 and Gal3 using A20 cells. The binding of mTIM-3 Fc to A20 Gal3 cells was assessed. A20 Gal3 cells were incubated with 10% FBS HBSS solution with or without mouse TIM-3 Fc for 20 minutes on ice. There were five experimental groups: in group 1, A20 Gal3 cells were incubated without mTIM-3 Fc protein as a control; in group 2, A20 Gal3 cells were incubated with mTIM-3 Fc protein. In groups 3, 4, and 5, in addition to the mTIM-3 Fc protein, anti-mouse TIM-3 polyclonal antibody (R&D System, Minneapolis, MN, USA) (group 3), monoclonal antibody RMT3-23 (Bio X) Monoclonal antibody 215015 (R&D Systems, West Lebanon, NH, USA) (group 4) and monoclonal antibody 215015 (R&D Systems, West Lebanon, NH, USA) (group 5) were also added to test whether these antibodies could block Gal3 and Tim3 binding. For blocking, cells were incubated with the indicated antibodies in 10% FBS HBSS, then added with mTIM-3 Fc in 10% FBS HBSS for 20 minutes. Samples were centrifuged, and the pellet was added to 10% FBS HBSS containing APC-conjugated anti-hFc antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 20 minutes. After centrifugation, live / dead cells were analyzed using a violet dead cell staining kit (Life Violet Dead Cell Staining Kit). The stained cells were subjected to flow analysis.

[0139] Figure 4 shows that mTIM-3 can bind to Gal3 protein on both dead and live cells, and that Gal3 and dead cells bind to different epitopes on TIM-3. In this assay, TIM-3 Fc binds to both dead cells (Figure 4C, column 2) and Gal3 expressed on live cells (Figure 4B, column 2). However, the mTIM-3 monoclonal antibody RMT3-23 blocked the binding of TIM-3 to dead cells (Figure 4C, column 4) but not to Gal3 expressed on live cells (Figure 4B, column 4). This indicates that Gal3 and dead cells bind to different epitopes on TIM-3. As a control, neither the mTIM-3 polyclonal antibody nor the monoclonal antibody 215015 (R&D System, Minneapolis, MN, USA) had any effect on Tim3 Gal3 (Fig. 4B, lanes 3 and 5) or dead cells (Fig. 4C, lanes 3 and 5), respectively.

[0140] Blocking assay - ELISA ELISA was also performed to test the interaction between Gal3 and TIM-3. A 96-well ELISA plate (ThermoFisher Scientific) was coated with mouse Gal3 protein (BioLegend, San Diego, CA, USA) in PBS, human Gal9 protein (R&D Systems, Inc.) in PBS, or phosphatidylserine (PS) (Sigma) in ethanol and incubated overnight at 4°C. The plate was washed three times with TBST and then blocked with PBS buffer containing 2% BSA for 1 hour at room temperature. In Figure 5A, various anti-Gal3 antibodies, namely, mGal3 polyclonal antibody (R&D Systems), mAb IMT001, and mAb M3 / 38 (ThermoFisher Scientific) (Figure 5A), were added to the Gal3-coated wells. The antibodies were then incubated for 10 minutes, after which TIM-3 Fc was added to the plate and incubated for an additional hour. The plates were then washed three times and then incubated with anti-human IgG-HRP (Jackson ImmunoResearch) for 1 hour at room temperature. After washing three times with TBST, color was developed with TMB substrate (GeneTex, Irvine, CA, USA), and the reaction was stopped with 1N HCl. Optical density (OD) was read at 450 nm. Results are expressed as the mean OD ± SD of duplicate experiments. Figure 5A shows that, among all antibodies tested, the mouse Gal3 polyclonal antibody and the monoclonal antibody IMT001 blocked the interaction between Gal3 and TIM-3 (Figure 5A).

[0141] In Figure 5B, mouse Gal3 protein (BioLegend) in PBS (groups 1 and Plates were coated with mTIM-3 (groups 2 and 2) or PS (Sigma-Aldrich, St. Louis, MO, USA) in ethanol (groups 3 and 4) and incubated overnight at 4°C. Anti-mTIM-3 mouse antibody, mAb RMT3-23 (Bio X Cell) was added only to the coated plates of groups 2 and 4. Secondary anti-human IgG-HRP antibody and substrate were added as described above to detect binding of mTIM-3 to mGal3 or PS. The results showed a significant decrease in signal in group 4 compared with group 3, indicating that RMT3-23 blocked PS binding to TIM-3; whereas, the results showed no significant decrease in signal in group 2 compared with group 1, indicating that RMT3-23 did not block Gal3 binding to TIM-3. Because TIM-3 binds to dead cells through its interaction with PS externalized and exposed on the surface of dead cells, these experiments substantiated the observations in Figures 4A-4C that Gal3 and PS bind to different epitopes on TIM-3.

[0142] For the sugar-dependent assay, ELISA plates were coated with either mGal3 (groups 1 and 2) or hGal9 (groups 3 and 4). Mouse TIM-3 Fc protein (R&D Systems) was added to the coated ELISA plates with (groups 2 and 4) or without (groups 1 and 3) 25 mM α-lactose (Sigma-Aldrich) for 1 hour at room temperature. Secondary anti-human IgG-HRP antibody and substrate were added as described above to detect binding of mTIM-3-Fc to mGal3 or hGal9. Figure 5C shows that lactose blocked Gal9 binding to TIM-3, as indicated by a significant, >10-fold decrease in signal in group 4 (with lactose) compared to group 3 (without lactose), indicating sugar-dependent binding between Gal9 and TIM-3. In contrast, although the blocking effect of lactose on the binding of Gal3 to TIM-3 was minimal, there was no significant difference in the signal resulting from the binding of TIM-3 to Gal3 between Group 2 (with lactose) and Group 1 (without lactose), indicating that the interaction of Gal3 with TIM-3 was not affected by the presence of sugar, i.e., the interaction was sugar-independent.

[0143] Example 3. Overexpression of Gal3 suppresses T cell activation This example describes experiments performed to evaluate the functional properties of Gal3 overexpression in A20 cells.

[0144] A20 clones, #41, #31, and #15, stably overexpressing hGal3, were generated as described above. Figure 6A shows the results of flow cytometry analysis demonstrating the hGal3 expression levels in these clones. A20 cells or A20 Gal3 clones were mixed with mouse DO11.10 T cells. The mixture was placed in each well of a flat 96-well plate, and then OVA323-339 peptide (Invivogen, San Diego, CA, USA) was added to the plate. After overnight incubation, the supernatant was used to measure IL-2 production by T cells by ELISA (Thermo Fisher Scientific). As shown in Figure 6B, IL-2 production by mouse DO11.10 T cells was significantly reduced when the T cells were mixed with any of the three mouse A20 cell clones compared to when the T cells were mixed with parental A20 cells (Figure 6B).

[0145] Example 4. Anti-Gal3 antibody exhibits antitumor activity in a mouse lung metastasis model The experiment in this example was conducted to evaluate the antitumor efficacy of Gal3:TIM-3 inhibitors in vivo. Animal experiments were conducted in accordance with protocols approved by the Institute for Molecular Medicine Animal Care and Use Committee. C57BL / 6 mice were housed in an Association for Accreditation of Laboratory Animal Care-accredited facility upon arrival. Thirty-six 7-week-old female mice were randomly assigned to three groups (n=12). On day 0, B16F10 cells (2×10 in 0.1 mL of PBS) were inoculated into 100% PBS-treated mice. 5 ) was washed, resuspended in PBS, and then injected into the tail vein of mice using a syringe with a 27-gauge needle. After injection of B16F10 cells, animals were intraperitoneally administered 10 mg / kg of mouse IgG2b (Bio X Cell, West Lebanon, NH, USA) on days 0, 3, 7, and 10, and mPD1 antibody (Bio X Cell, West Lebanon, NH, USA) on days 0, 3, and 7, or Gal3 antibody IMT001 on days 0, 3, 7, 10, and 15. The Gal3 antibody clone IMT001 used in this experiment recognizes an epitope on Gal3 (SEQ ID NO: 5). On day 21, animals were humanely sacrificed, and lung tissue was removed and fixed in 10% buffered formaldehyde solution. The number of black metastatic colonies on the surface of one of the left lung lobes was counted (Figure 7B). Results are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA in comparison with the IgG control group.

[0146] Figure 7A shows that the mean fluorescence intensity (MFI) of B16F10 cells stained with anti-mGAL3 antibody was almost 10-fold higher than that of cells stained with an isotype control antibody. Specifically, B16F10 cells were incubated on ice for 20 minutes in 10% FBS containing control rat IgG PE or rat anti-mouse Gal3 PE antibody (Thermo Fisher Scientific, Waltham, MA, USA). The cells were incubated with HBSS solution. After centrifugation, live / dead cells were stained with a violet dead cell staining kit (Thermo Fisher Scientific, Waltham, MA, USA). The stained cells were subjected to flow analysis. Figure 7B shows representative images of whole lungs from the three treatment groups. Figure 7C shows numerous metastatic colonies on the surface of the left lung lobe (mean ± SEM). Figures 7D and 7E show lung and body weights for the various treatment groups (mean ± SEM). Compared with the isotype control group, the Gal3 antibody-treated group showed a significant (approximately 46%) reduction in tumor number, as indicated by the number of black metastatic colonies (p<0.01). However, compared with the isotype control group, the anti-mouse PD1 antibody 29F did not show significant antitumor effects in this lung metastasis model (p>0.05).

[0147] Example 5. Anti-Gal3 antibody exhibits anti-tumor activity in the 4T1 orthotopic lung metastasis model Animal experiments were performed according to a protocol approved by the Institute for Molecular Medicine Animal Care and Use Committee. Seven-week-old female Balb / c mice were housed in an Association for Accreditation of Laboratory Animal Care-accredited facility upon arrival. On the day of tumor implantation, 4T1 cells were collected, washed, and resuspended in PBS. Mice were anesthetized with inhalation anesthetic (3-5% isoflurane in medical-grade air). 2 × 10 cells were added to 0.1 mL of PBS. 5 Cells were injected subcutaneously into the mammary gland using a syringe with a 25-gauge needle. Mice were randomly assigned to two groups (n = 10). After 4T1 cell injection, mice were intraperitoneally administered 10 mg / kg of mouse IgG2b (Bio X Cell) on days 0, 3, and 7, or Gal3 antibody IMT001 on days 0, 3, 7, 10, and 14. Tumor volume and body weight were monitored twice weekly. On day 30, mice were humanely sacrificed, and lung tissue was inflated with 30% sucrose, removed, and fixed in Boynes' solution (Sigma-Aldrich). The number of metastatic colonies on the surface of one of the left lung lobes was counted. Results are expressed as mean ± SEM. Statistical analysis was performed using an unpaired t-test compared with the IgG control group.

[0148] Figure 8A shows representative images of whole lungs from treatment groups. Figure 8B shows the body weights of the different treatment groups (mean ± SEM). Figure 8C shows numerous metastatic colonies on the surface of one of the left lung lobes (mean ± SEM). Compared to mice treated with an isotype control antibody, animals treated with monoclonal anti-human Gal3 antibody showed a significant reduction in the number of lung metastases (p<0.05).

[0149] Example 6. Anti-Gal3 antibody exhibits anti-tumor activity in a primary murine RENCA kidney tumor model To evaluate the antitumor efficacy of Gal3:TIM-3 inhibitors in primary tumor models Experiments were performed (Figure 9). Animal experiments were conducted in accordance with a protocol approved by the Institute for Molecular Medicine Animal Care and Use Committee. Balb / c mice were placed in a facility accredited by the Association for Accreditation of Laboratory Animal Care upon arrival. Seven-week-old female mice were randomly assigned to three groups (n = 15). On the day of tumor implantation, mice were anesthetized with an inhalation anesthetic (3-5% isoflurane in medical-grade air). Renca cells were washed and resuspended in PBS, and then 2 x 10 cells were implanted in 0.1 mL of PBS using a syringe with a 25-gauge needle. 5 After Renca cell injection, mice were intraperitoneally administered 10 mg / kg mouse IgG2b (Bio X Cell) on days 0, 3, and 7, mPD1 antibody (Bio X Cell) on days 0, 3, and 7, or Gal3 antibody IMT001 antibody on days 0, 3, 7, 10, and 14. Tumor volumes in the control group were 2000–2500 mm. 3 Animals were humanely sacrificed when the IgG2b concentration reached 0.05. Results are expressed as mean ± SEM. Statistical analysis was performed in comparison with the IgG2b control group using an unpaired t-test.

[0150] The results demonstrate the antitumor effect of the Gal3 antibody (IMT001) in a renal cancer model. Compared with the isotype control group, the anti-Gal3 antibody-treated group showed a significant (approximately 35%) reduction in tumor growth (p<0.05), whereas the anti-PD-1 antibody had no effect (Figure 9).

[0151] Example 7. Anti-Gal3 antibody exhibits anti-tumor activity in a primary murine MC38 colon cancer model Animal experiments were conducted according to a protocol approved by the Institute for Molecular Medicine Animal Care and Use Committee. Seven-week-old female C57BL / 6 mice were housed in an Association for Accreditation of Laboratory Animal Care-accredited facility upon arrival. On the day of tumor implantation, MC38 mouse colon adenocarcinoma cells were collected, washed, and resuspended in PBS. Mice were anesthetized with inhalation anesthetic (3-5% isoflurane in medical-grade air). 5 × 10 cells were placed in 0.1 mL of PBS. 5Cells were injected subcutaneously into the right side of the mice using a syringe with a 25-gauge needle. On day 7, tumor volume was measured, and mice were randomly assigned to two groups (n=8). Mice were intraperitoneally administered 10 mg / Kg of mouse IgG2b (BioXCell) or Gal3 antibody IMT001 on days 7, 10, 14, 17, and 22. Tumor volume and body weight were monitored twice weekly. When tumor volume reached 3000 mm 3 Animals were humanely sacrificed when the IgG antibody titer reached 100%. Results are expressed as mean ± SEM. Statistical analysis was performed using an unpaired t-test in comparison with the IgG control group.

[0152] The results in Figure 10 demonstrate that the IMT001 antibody has antitumor activity in the MC38 colon cancer model. Compared to mice treated with an isotype control antibody, mice treated with the IMT001 antibody showed a significant reduction in tumor burden (approximately 33%) at day 24 (p<0.05).

[0153] Example 8. Epitope binding of Gal3 antibody clone IMT001 A peptide array containing 24 20-amino acid peptides overlapping by 10 amino acids and covering the entire human Gal3 protein sequence was synthesized (Genscript, Piscataway, NJ, USA) (Figure 11A). 20 μg of each peptide was dot-blotted onto a membrane. After blocking with 5% milk in PBS, the membrane was incubated with 1 μg / ml of IMT001 antibody overnight at 4°C. After three washes, the membrane was incubated with a 1:8000 dilution of anti-mIgG HRP antibody (Southern Biotech, Birmingham, AL, USA) for 1 hour. After three washes, the membrane was incubated with Western ECL blotting substrate (Bio-Rad, Hercules, CA, USA) and reacted (Figure 11B). Peptides 5 and 6 showed good signals, indicating that the epitope on hGal3 to which IMT001 binds is PGAYPGQAPPGAYPGQAPPGAYPGAPGAYP (SEQ ID NO: 7).

[0154] To further define the binding epitope of IMT001 on the above peptide, eight shorter peptides derived from it were synthesized (Genscript, Piscataway, NJ, USA) (Figure 11C), and their binding by IMT001 was determined by ELISA (Figure 11D). A 96-well ELISA plate (Thermo Scientific) was coated with these peptides in PBS buffer and incubated overnight at 4°C. The plate was washed three times with TBST and then blocked with PBST buffer containing 2% BSA for 1 hour at room temperature. 10 μg / mL of IMT001 was incubated in the coated ELISA plate for 1 hour at room temperature. The plate was washed three times and then incubated with a 1:8000 dilution of anti-mouse IgG-HRP for 1 hour at room temperature. After washing three times with TBST, the plate was developed with 100 μL of TMB substrate (GeneTex) and stopped with 50 μL of 1N HCl. Optical density (OD) was read at 450 nm. Results were expressed as the mean OD±SD of duplicates. Pep-2 showed a good signal, indicating that the binding epitope of IMT001 on human Gal3 is GQAPPGAYPG (SEQ ID NO: 8).

[0155] Example 9. Immune profiling of B16F10 lung metastatic mouse tumors Mice were intravenously implanted with one million B16F10 cells. They were then treated with IMT001 or an isotype control (10 mg / kg, i.p.) on days 0, 1, 3, and 7, and sacrificed on day 8 to isolate and phenotype lung immune cells. Cells were isolated from the lungs, stained with fluorescently labeled antibodies against lymphocyte markers CD3, CD4, CD8, CD19, and DX5, and analyzed by flow cytometry. The results in Figure 12 show that, compared with isotype control antibody treatment, anti-Gal3 antibody IMT001 treatment increased the number of various immune effector cells, including CD3 T lymphocytes, CD4 T helper cells, CD8 cytotoxic T cells, CD19 B cells, and DX5 natural killer cells, in the tumor-host lungs. This indicates that the anti-Gal3 antibody was able to activate immune cells.

[0156] Example 10. Gal3 expression detected on human lung cancer-associated macrophages Immunohistochemistry (IHC) experiments were performed to detect Gal3 expression in human lung cancer. Frozen human lung cancer tissue slides (US Biomax Inc.) were fixed in 10% neutral buffered formalin (Fisher Scientific) for 10 minutes at room temperature and washed twice in PBS for 5 minutes. Endogenous peroxidase was blocked by immersing the slides in 3% H2O2 for 10 minutes at room temperature. After washing twice in PBS for 5 minutes, the slides were incubated in streptavidin reagent (Molecukar Probes) for 15 minutes at room temperature, thoroughly washed with PBS, incubated in biotin reagent (Molecukar Probes) for 15 minutes, and further rinsed in PBS to block endogenous biotin background. Slides were blocked with 10% FBS, 200 μg / mL mIgG, and 200 μg / mL hIgG for 1 hour, incubated with primary antibody IMT001-biotin (5 μg / mL) overnight at 4°C, washed three times, and then incubated with secondary antibody HRP-avidin (BioLegend) at 1:100 for 1 hour and washed three times. Staining was performed by incubation with DAB substrate (Vector Laboratories) and stopped by immersing the slides in distilled water. Human lung cancer slides were then counterstained in hematoxylin QS (Vector Laboratories), washed in distilled water, dehydrated in a graded series of ethanol and xylene solutions, and mounted with VectaMount™ mounting medium (Vector Laboratories).

[0157] The results in Figure 13 show that canopy-shaped tumor-associated macrophages in these human lung cancer slides (squamous cell carcinoma and adenocarcinoma) express Gal3, as evidenced by their positive staining with IMT001.

[0158] Example 11. Gal3 expression on human M2 macrophages Human CD14 monocytes were isolated from peripheral blood mononuclear cells (PBMCs) using a CD14 cell positive selection kit (Miltenyi, Auburn, CA, USA) and differentiated into dendritic cells (DCs), M1 macrophages, or M2 macrophages in the presence of GM-CSF + IL-4 or GM-CSF or M-CSF (Rocky Hill, NJ, USA), respectively. Flow cytometry analysis was then performed to detect Gal3 expression on human dendritic cells (DCs), M1, and M2 macrophages. Specifically, 100,000 DCs, M1, or M2 cells were incubated with 100 μl of 10% FBS HBSS solution containing control mIgG-biotin (BioLegend) or IMT001-biotin at 10 μg / ml for 20 minutes on ice. The cells were then washed and incubated with PE-streptavidin (BioLegend) at a 1:1000 dilution on ice for 20 minutes. After centrifugation, live / dead cells were stained with a Violet Dead Cell Stain Kit (Life Technologies). The stained cells were subjected to flow analysis. The results in Figure 14 show that the mean fluorescence intensity (MFI) of M2 cells stained with IMT001 was much higher than that of cells stained with the isotype control antibody, indicating that IMT001 specifically binds to M2 cells but fails to stain dendritic cells (Figure 14A) and M1 macrophages (Figure 14B).

[0159] Example 12. Anti-Gal3 antibody promotes murine T cell activation in macrophage / T cell reactions Gal3 expression on mouse macrophages was detected by IHC and flow cytometry analysis. For IHC, 100,000 cells were seeded per well overnight. On day 2, cells were washed once with PBS, fixed with 3% formaldehyde for 10 minutes at room temperature, washed twice with PBS, and blocked in PBS containing 10% FBS and 200 μg / mL formaldehyde for 1 hour at room temperature. After blocking, cells were incubated with 10 μg / mL primary antibody mIgG-biotin (BioLegend) or IMT001-biotin overnight at 40°C, washed three times with PBST, stained with avidin-HRP (1:1000) for 1 hour at room temperature, and then washed three times with PBST. Staining was developed using a peroxidase substrate and counterstained with hematoxylin QS (Vector Laboratories). The results show that IMT001 clearly detected Gal3 expression on macrophages (FIG. 15B) compared to the mIgG control (FIG. 15A).

[0160] For flow cytometry experiments, 100,000 RAW cells were blocked with 10% FBS + 200 μg / mL hIgG for 20 minutes on ice and then incubated with 100 μl of 10% FBS HBSS solution containing control mIgG (BD Biosciences) or IMT001 at 10 μg / mL for 20 minutes on ice. The cells were then washed and incubated with APC-conjugated anti-mFc antibody (Jackson ImmunoResearch) at a 1:100 ratio for 20 minutes on ice. After centrifugation, live / dead cells were stained with a Violet Dead Cell Stain Kit (Life Technologies). The stained cells were subjected to flow analysis. Figure 15C shows that the mean fluorescence intensity (MFI) of RAW cells stained with IMT001 was more than 10-fold higher than that of cells stained with an isotype control antibody.

[0161] The ability of IMT001 to activate T cells was demonstrated by a mixed lymphocyte reaction (MLR) assay. RAW mouse macrophage cells were mixed with DO11 mouse T cells at a 1:1 ratio, treated with OVA peptide, and cultured overnight at 37°C in the presence of mIgG (BD Biosciences), anti-mPD1 antibody 29F (BioXCell), or IMT001 at 10 μg / ml. 50 μl of culture medium was collected for mIL-2 measurement. mIL-2 production was measured according to the commercial kit Mouse IL-2 Elisa Ready-SET-Go from eBioscience.

[0162] Figure 15D shows that compared to mIgG or mPD1 antibody-treated cells, IMT001 antibody, but not murine PD-1 antibody 29F, promoted IL-2 production, indicating reversal of macrophage-induced T cell inactivation.

[0163] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present invention that certain variations, changes, modifications, and substitutions of equivalents may be made without necessarily departing from the spirit and scope of the present invention. Consequently, the embodiments described herein are subject to various modifications and alterations, and the scope of the present invention is determined solely by reference to the embodiments attached hereto. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed, altered, or modified to achieve essentially similar results. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention is limited only by the attached embodiments. In addition, each reference provided herein is incorporated herein by reference in its entirety to the same extent as if each reference were individually incorporated by reference. In the event of a conflict between the present application and a reference provided herein, the present application shall control.

[0164] Sequence Listing Information Array No. 1 Mouse Gal3 nucleic acid (cDNA) sequence (the underlined parts are the start codon and the stop codon). GGGAGGGCGG GCCCGGGGAA AAGAGTACTA GAAGCGGCCG AGCCACCGCC CAGCTCTGAC AGCTAGCGGA GCGGCGGGTG GAGCACTAAT CAGGTGAGCG GCACAGAGAG CACTACCCAG GAAA ATG GCA GACAGCTTTT CGCTTAACGA TGCCTTAGCT GGCTCTGGAA ACCCAAACCC TCAAGGATAT CCGGGTGCAT GGGGGAACCA GCCTGGGGCA GGGGGCTACC CAGGGGCTGC TTATCCTGGG GCCTACCCAG GACAAGCTCC TCCAGGGGCC TACCCAGGAC AGGCTCCTCC[[ID=!3]] AGGGGCCTAC CCAGGACAGG CTCCTCCTAG TGCCTACCCC GGCCCAACTG CCCCTGGAGC TTATCCTGGC CCAACTGCCC CTGGAGCTTA TCCTGGCTCA ACTGCCCCTG GAGCCTTCCC AGGGCAACCT GGGGCACCTG GGGCCTACCC CAGTGCTCCT GGAGGCTATC CTGCTGCTGG CCCTTATGGT GTCCCCGCTG GACCACTGAC GGTGCCCTAT GACCTGCCCT TGCCTGGAGG AGTCATGCCC CGCATGCTGA TCACAATCAT GGGCACAGTG AAACCCAACG CAAACAGGAT TGTTCTAGAT TTCAGGAGAG GGAATGATGT TGCCTTCCAC TTTAACCCCC GCTTCAATGA GAACAACAGG AGAGTCATTG TGTGTAACAC GAAGCAGGAC AATAACTGGG GAAAGGAAGA AAGACAGTCA GCCTTCCCCT TTGAGAGTGG CAAACCATTC AAAATACAAG TCCTGGTTGA AGCTGACCAC TTCAAGGTTG CGGTCAACGA TGCTCACCTA CTGCAGTACA ACCATCGGAT GAAGAACCTC CGGGAAATCA GCCAACTGGG GATCAGTGGT GACATAACCC TCACCAGCGC TAACCACGCC ATGATC TAA G CCAGAAGGGG CGGCACCGAA ACCGGCCCTG TGTGCCTTAG GAGTGGGAAA CTTTGCATTT CTCTCTCCTT ATCCTTCTTG TAAGACATCC ATTTAATAAA GTCTCATGCT GAGAGATACC CATCGCTTTG GGGGTTTTTA TGATACTGGA TGTCAAATCT TAGGACTGCT CGTGACTGCT AGGCAAGTGT TCTCTCACTG AGCTACACAT CCCTAGCCTT TTAAACTTTG TGTGTTGTGT GTCTGTGCAC ATGGGTACAG GTGCCTGCTC ACTTGAGAGG CACCAGGCCT CCTGGAGCTG GAGTTACAGG TGGTTGTAAG TAAGCTGTGT GACCAGGTTG CTGGGAACCA GTCTCAGATC CTCCTGAGAC AGGTCAGGTC CACTGATGCC TCCAGCTGCC TGTCTTTATA TGCCCTTTGA TTTGGTGCAG TTTTATATAA AGGGAACTAT GTAATTATCA ATAAACCATC CTGATTTTTA CAAAGG SEQ ID NO:2: Mouse Gal3 polypeptide sequence MADSFSLNDALAGSGNPNPQGYPGAWGNQPGAGGYPGAAYPGAY PGQAPPGAYPGQAPPGAYPGQAPPSAYPGPTAPGAYPGPTAPGAYPGSTAPGAFPGQP GAPGAYPSAPGGYPAAGPYGVPAGPLTVPYDLPLPGGVMPRMLITIMGTVKPNANRIV LDFRRGNDVAFHFNPRFNENNRRVIVCNTKQDNNWGKEERQSAFPFESGKPFKIQVLV EADHFKVAVNDAHLLQYNHRMKNLREISQLGISGDITLTSANHAMI SEQ ID NO: 3: Human Gal3 nucleic acid (cDNA) sequence (the underlined parts are the start and stop codons.) GAGTATTTGA GGCTCGGAGC CACCGCCCCG CCGGCGCCCG CAGCACCTCC TCGCCAGCAG CCGTCCGGAG CCAGCCAACG AGCGGAAA AT G GCAGACAAT TTTTCGCTCC ATGATGCGTT ATCTGGGTCT GGAAACCCAA ACCCTCAAGG ATGGCCTGGC GCATGGGGGA ACCAGCCTGC TGGGGCAGGG GGCTACCCAG GGGCTTCCTA TCCTGGGGCC TACCCCGGGC AGGCACCCCC AGGGGCTTAT CCTGGACAGG CACCTCCAGG CGCCTACCCT GGAGCACCTG GAGCTTATCC CGGAGCACCT GCACCTGGAG TCTACCCAGG GCCACCCAGC GGCCCTGGGG CCTACCCATC TTCTGGACAG CCAAGTGCCA CCGGAGCCTA CCCTGCCACT GGCCCCTATG GCGCCCCTGC TGGGCCACTG ATTGTGCCTT ATAACCTGCC TTTGCCTGGG GGAGTGGTGC CTCGCATGCT GATAACAATT CTGGGCACGG TGAAGCCCAA TGCAAACAGA ATTGCTTTAG ATTTCCAAAG AGGGAATGAT GTTGCCTTCC ACTTTAACCC ACGCTTCAAT GAGAACAACA GGAGAGTCAT TGTTTGCAAT ACAAAGCTGG ATAATAACTG GGGAAGGGAA GAAAGACAGT CGGTTTTCCC ATTTGAAAGT GGGAAACCAT TCAAAATACA AGTACTGGTT GAACCTGACC ACTTCAAGGT TGCAGTGAAT GATGCTCACT TGTTGCAGTA CAATCATCGG GTTAAAAAAC TCAATGAAAT CAGCAAACTG GGAATTTCTG GTGACATAGA CCTCACCAGT GCTTCATATA CCATGATA TA A TCTGAAAGG GGCAGATTAA AAAAAAAAA AGAATCTAAA CCTTACATGT GTAAAGGTTT CATGTTCACT GTGAGTGAAA ATTTTTACAT TCATCAATAT CCCTCTTGTA AGTCATCTAC TTAATAAATA TTACAGTGAA TTACCTGTCT CAATATGTCA AAAAAAAAA AAAAAAA SEQ ID NO:4: ヒトGal3 polypeptide sequence MADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGA YPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYP ATGPYGAPAGPLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFN PRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHL LQYNHRVKKLNEISKLGISGDIDLTSASYTMI SEQ ID NO: 5: hGal3 epitope, corresponding to peptide_5 in Figure 11A PGAYPGQAPPGAYPGQAPPG SEQ ID NO: 6: hGal3 epitope, corresponding to peptide_6 in Figure 11A GAYPGQAPPGAYPGAPGAYP SEQ ID NO: 7: hGal3 epitope PGAYPGQAPPGAYPGQAPPGAYPGAPGAYP SEQ ID NO: 8: hGal3 epitope, corresponding to Pep-2 in FIG. 11C GQAPPGAYPG Humanized IMT001 in hIgG4 isotype SEQ ID NO: 9: Heavy chain CDR1 GYTFTNY SEQ ID NO: 10: Heavy chain CDR2 NTNTGE SEQ ID NO: 11: Heavy chain CDR3 YDNFFAY SEQ ID NO: 12: Heavy chain FR1 QVQLVQSGSELKKPGASVKVSCKAS SEQ ID NO: 13: Heavy chain FR2 GMNWVRQAPGQGLKWMGWI SEQ ID NO: 14: Heavy chain FR3 PTYAQEFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCAP SEQ ID NO: 15: Heavy chain FR4 WGQGTTVTVS SEQ ID NO: 16: Heavy chain QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTNTGEPTYAQEFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCAPYDNFFAYWGQGTT VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG** SEQ ID NO: 17: Light chain CDR1 RSSKSLLYKDGKTYLN SEQ ID NO: 18: Light chain CDR2 LMSTHAS SEQ ID NO: 19: Light chain CDR3 QQLVDYPLT SEQ ID NO: 20: Light chain FR1 DIVLTQSPLSLPVTPGEPASISC SEQ ID NO: 21: Light chain FR2 WFLQKPGQSPQLLIY SEQ ID NO: 22: Light chain FR3 GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC SEQ ID NO: 23: Light chain FR4 FGGGTKLEIK SEQ ID NO: 24: Light chain DIVLTQSPLSLPVTPGEPASISCRSSKSLLYKDGKTYLNWFLQKPGQSPQLLIYLMSTHASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQLVDYPLTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 25: Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTNTGEPTYAQEFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCAPYDNFFAYWGQGTTVTVS SEQ ID NO: 26: Light chain variable region DIVLTQSPLSLPVTPGEPASISCRSSKSLLYKDGKTYLNWFLQKPGQSPQLLIYLMSTHASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQLVDYPLTFGGGTKLEIK SEQ ID NO: 27: Peptide_1, disclosed in Figure 11A ADNFSLHDALSGSGNPNPQG SEQ ID NO: 28: Peptide_2, disclosed in Figure 11A SGSGNPNPQGWPGAWGNQPA SEQ ID NO: 29: Peptide_3, disclosed in Figure 11A WPGAWGNQPAGAGGYPGASY SEQ ID NO: 30: Peptide_4, disclosed in Figure 11A GAGGYPGASYPGAYPGQAPP SEQ ID NO: 31: Peptide_7, disclosed in Figure 11A AYPGAPGAYPGAPAPGVYPG SEQ ID NO: 32: Peptide_8, disclosed in Figure 11A GAPAPGVYPGPPSGPGAYPS SEQ ID NO: 33: Peptide_9, disclosed in Figure 11A PPSGPGAYPSSGQPSATGAY SEQ ID NO: 34: Peptide_10, disclosed in Figure 11A SGQPSATGAYPATGPYGAPA SEQ ID NO: 35: Peptide_11, disclosed in Figure 11A PATGPYGAPAGPLIVPYNLP SEQ ID NO: 36: Peptide_12, disclosed in Figure 11A GPLIVPYNLPLPGGVVPRML SEQ ID NO: 37: Peptide_13, disclosed in Figure 11A LPGGVVPRMLITILGTVKPN SEQ ID NO: 38: Peptide_14, disclosed in Figure 11A ITILGTVKPNANRIALDFQR SEQ ID NO: 39: Peptide_15, disclosed in Figure 11A ANRIALDFQRGNDVAFHFNP SEQ ID NO: 40: Peptide_16, disclosed in Figure 11A GNDVAFHFNPRFNENNRRVI SEQ ID NO: 41: Peptide_17, disclosed in Figure 11A RFNENNRRVIVCNTKLDNNW SEQ ID NO: 42: Peptide_18, disclosed in Figure 11A VCNTKLDNNWGREERQSVFP SEQ ID NO: 43: Peptide_19, disclosed in Figure 11A GREERQSVFPFESGKPFKIQ SEQ ID NO: 44: Peptide_20, disclosed in Figure 11A FESGKPFKIQVLVEPDHFKV SEQ ID NO: 45: Peptide_21, disclosed in Figure 11A VLVEPDHFKVAVNDAHLLQY SEQ ID NO: 46: Peptide_22, disclosed in Figure 11A AVNDAHLLQYNHRVKKLNEI SEQ ID NO: 47: Peptide_23, disclosed in Figure 11A NHRVKKLNEISKLGISGDID SEQ ID NO: 48: Peptide_24, disclosed in Figure 11A SKLGISGDIDLTSASYTMI SEQ ID NO: 49: Pep-1, disclosed in FIG. 11C PGAYPGQAPP SEQ ID NO: 50: Pep-3, disclosed in Figure 11C GAYPGQAPPGA SEQ ID NO: 51: Pep-4, disclosed in Figure 11C APPGAYPGAP SEQ ID NO: 52: Pep-5, disclosed in Figure 11C YPGAPGAYP SEQ ID NO: 53: Pep-6, disclosed in Figure 11C APPGAY SEQ ID NO: 54: Pep-7, disclosed in Figure 11C GAYPGQ SEQ ID NO: 55: Pep-8, disclosed in Figure 11C PGQAPP

Claims

1. A pharmaceutical composition for use in treating cancer in a patient, the pharmaceutical composition comprising an anti-Gal3 antibody, wherein the anti-Gal3 antibody interferes with the interaction between Gal3 and TIM-3, the anti-Gal3 antibody enhances the level of a T cell-mediated immune response and / or a B cell-mediated immune response in the patient, and the anti-Gal3 antibody recognizes a peptide consisting of a sequence selected from the group consisting of SEQ ID NOs: 5-8.

2. The pharmaceutical composition of claim 1, wherein the TIM-3 is expressed on an immune cell.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the interaction between Gal3 and TIM-3 occurs in the tumor microenvironment, and wherein enhancing the level of the T cell-mediated immune response and / or B cell-mediated immune response reduces the cancer burden in the patient.

4. The pharmaceutical composition of claim 3, wherein the Gal3 is overexpressed in the tumor microenvironment.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the cancer is a metastatic cancer or a primary cancer.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the anti-Gal3 antibody is a single-chain antibody or a Fab.

7. The pharmaceutical composition according to any one of claims 1 to 5, wherein the anti-Gal3 antibody is a humanized antibody or a human antibody.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the pharmaceutical composition is formulated for administration to the patient by intravenous infusion.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein the pharmaceutical composition is formulated for use in combination with one or more other treatments.

10. 10. The pharmaceutical composition of claim 9, wherein the one or more other treatments are selected from the group consisting of chemotherapy, radiation therapy, and checkpoint inhibitor therapy.

11. 11. The pharmaceutical composition of claim 10, wherein the checkpoint inhibitor therapy is selected from the group consisting of an anti-PD-1 therapy and an anti-CTLA4 therapy.

12. 12. The pharmaceutical composition of any one of claims 1 to 11, wherein the pharmaceutical composition is formulated for administration to the patient at a dosage of 10 μg / kg to 100 mg / kg of body weight every two weeks.

13. 1. An in vitro method for determining whether an anti-Gal3 antibody that interferes with the interaction between Gal3 and TIM-3 is suitable for treating cancer, wherein the anti-Gal3 antibody recognizes a peptide consisting of a sequence selected from the group consisting of SEQ ID NOs: 5-8, the method comprising: combining cells obtained from the tumor microenvironment of a known type of cancer with an anti-Gal3 antibody; determining the level of Gal3 on the surface of said cells; comparing the Gal3 level on the surface of the cells with Gal3 levels on the surface of cells obtained from a population of subjects with the same type of cancer; and determining that the anti-Gal3 antibody is suitable for treating cancer if the Gal3 level on the surface of cells obtained from the tumor microenvironment is higher than the Gal3 level on the surface of cells obtained from a population of subjects having the same type of cancer.

14. 14. The method of claim 13, wherein the Gal3 levels on the surface of cells obtained from the population of subjects are derived from a cohort of at least 100 test individuals suffering from the same type of cancer as the patient.

15. The method of claim 14, wherein determining whether the anti-Gal3 antibody that interferes with the interaction between Gal3 and TIM-3 is suitable for treating cancer further comprises determining whether the Gal3 level on the surface of cells obtained from the tumor microenvironment is 25% or greater compared to the Gal3 level on the surface of corresponding cells obtained from a healthy subject.

16. The method of any one of claims 13 to 15, wherein the cells obtained from the tumor microenvironment comprise at least cancer cells and / or tumor-associated macrophages.

17. The method of claim 15, wherein determining whether the anti-Gal3 antibody that interferes with the interaction between Gal3 and TIM-3 is suitable for treating cancer further comprises determining whether the Gal3 level on the surface of cells obtained from the tumor microenvironment is 75% or greater compared to the Gal3 level on the surface of corresponding cells obtained from a healthy subject.

18. A sterile solution comprising an anti-Gal3 antibody, wherein the antibody interferes with the interaction between Gal3 on cancer cells and TIM-3 on T cells in a cancer patient, the anti-Gal3 antibody recognizing a peptide consisting of a sequence selected from the group consisting of SEQ ID NOs: 5-8, the solution comprising 10 μg to 100 mg of anti-Gal3 antibody per kilogram of patient body weight in 100 ml of solution suitable for intravenous delivery over 1 to 4 hours.

19. 20. The sterile solution of claim 18, wherein the sterile solution further comprises one or more other checkpoint inhibitor antibodies.

20. The one or more other checkpoint inhibitor antibodies may be anti-PD-1 and anti-CTLA-4 antibodies.

20. The sterile solution of claim 19, wherein the sterile solution is selected from the group consisting of:

21. The sterile solution according to any one of claims 18 to 20, further comprising one or more nanoparticles having a diameter of 10 to 100 nm.

22. A composition for producing an anti-Gal3 antibody that interferes with the interaction between Gal3 and TIM-3, the composition comprising a peptide having a sequence selected from the group consisting of SEQ ID NOs: 5 to 8.

23. 1. An in vitro method for selecting a pharmaceutical composition for use in treating cancer in a patient, the pharmaceutical composition comprising an anti-Gal3 antibody that interferes with the interaction of Gal3 with TIM-3; The method comprises: (a) contacting a library of anti-Gal3 antibodies with Gal3 and TIM-3; and (b) selecting from the library one or more candidate antibodies capable of blocking the interaction between Gal3 and TIM-3; A method comprising:

24. (c) contacting one or more candidate anti-Gal3 antibodies selected from step (b) with a mixture containing T cells and allogeneic antigen-presenting cells to identify one or more anti-Gal3 antibodies capable of stimulating T cells.

24. The method of claim 23, further comprising:

Images