Anti-galectin-9 antibodies and uses thereof

Abstract

The present invention provides a humanized monoclonal antibody against Gal9 and a method of using the antibody to treat cancer, including combination therapy with an antagonist of the PD-1 / PD-L1 immune checkpoint. One embodiment of the invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, wherein the antibody of the present invention is widely used to treat hematological cancers, such as AML and DLBCL, and solid cancers, such as breast cancer, head and neck cancer, lung cancer, melanoma (including uveal melanoma), colon cancer, renal cancer, ovarian cancer, liver cancer, and prostate cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 957,910, filed January 7, 2020, the entire contents of which are incorporated herein by reference, including all sequences and figures. [Background technology]

[0002] Background of the Invention Galectin-9 (or Gal9) is a member of the galectin (or S-type lectin) protein family, which has at least 15 members in vertebrates, including 10 in humans. Galectin-9 is a soluble 34-39 kDa protein lacking a leader peptide, but is secreted via a non-canonical mechanism. Galectin-9 preferentially interacts with beta-galactoside residues on glycoproteins and glycolipids. In humans, galectin-9 exists in three isoforms: long, medium, and short.

[0003] Galectin-9 is one of the most studied ligands for HAVCR2 (TIM-3) and is expressed in a variety of hematological malignancies, including CLL, MDS, Hodgkin's and non-Hodgkin's lymphoma, and AML, as well as solid tumors, such as lung cancer, breast cancer, and hepatocellular carcinoma.

[0004] HAVCR2 / galectin-9 interaction has been shown to attenuate T cell proliferation and effector function in the tumor microenvironment and in chronic infections. Furthermore, galectin-9 contributes to tumorigenesis through tumor cell transformation, cell cycle regulation, angiogenesis, and cell adhesion.

[0005] Galectin-9 is also expressed directly by regulatory T lymphocytes (or Tregs), and its expression increases during Treg activation. On the other hand, galectin-9 is expressed by effector T lymphocytes (CD8 +Galectin-9 is expressed very weakly by effector T lymphocytes (e.g., CTLs), and this expression disappears during effector T lymphocyte activation. Inhibition of galectin-9 by anti-Gal9 antibodies has been found to inhibit the suppressive activity of Tregs.

[0006] Wu et al. (Immunity 41(2):270-282, 2014) reported that Gal-9 is important for regulating immune responses. Gal-9 is highly expressed by induced regulatory T cells (iTregs). Gal-9 was important for the generation and function of iTreg cells, but not natural regulatory T (nTreg) cells. Gal-9 expression in iTreg cells was driven by the transcription factor Smad3, forming a feed-forward loop that further promoted Foxp3 expression. Gal-9 directly bound to the iTreg receptor CD44, increasing its stability and function, forming a complex with transforming growth factor-β (TGF-β) receptor I (TGF-βRI) and activated Smad3. Gal-9 signaling was further found to regulate iTreg cell induction by acting predominantly through the CNS1 region of the Foxp3 locus. In addition to being an effector molecule for Treg cells, exogenous Gal-9 acts synergistically with TGF-β to enhance the differentiation and maintenance of iTreg cells.

[0007] Various T lymphocyte types typically develop into, for example, "effector" cells or effector T lymphocytes, which perform specialized immune functions to defend the host organism. + T lymphocytes or accessory T lymphocytes support, in particular, B lymphocytes in humoral function (production of specific antibodies) and CD8 + They secrete key cytokines that help T lymphocytes.

[0008] CD4 + Another population of T lymphocytes consists of natural regulatory T lymphocytes, or "regulatory T lymphocytes (Treg)." Regulatory T lymphocytes bind to the CD25 molecule (hence, "CD4 + CD25+ Constitutively overexpress the Foxp3 transcription factor (also called "Foxp3"). A small percentage of CD4 + CD25 + T lymphocytes have the special feature of negatively regulating the immune response factors that recognize various self-antigens through their TCR. Regulatory T lymphocytes also play a key role in the physiology of the immune system, especially in protecting the organism from the emergence of autoimmune diseases. In other words, Tregs are a subpopulation of natural regulatory T lymphocytes (or "nTregs") that are characterized by the expression of CD25, CTLA-4, and GITR, and the specific expression of the transcription factor Foxp3.

[0009] Tregs exert immunosuppressive activity against effector T lymphocytes. When activated in pathological conditions such as tumors, this activity promotes tumor growth. Therefore, the suppressive activity of Tregs can be understood as an activity that reduces antitumor immune responses by inhibiting the function of effector T lymphocytes.

[0010] Galectin-9 is primarily associated with tumor immunosuppression due to its interaction with different immune receptors. For example, Gal9 inhibits Th1 responses and induces peripheral tolerance, as evidenced by reduced Th1 apoptosis upon Gal9 blockade, increased susceptibility of Gal9 knockout mice to collagen-induced arthritis (CIA), and long-term graft survival and AID suppression upon Gal9 administration. Gal9 also regulates peripheral NK cell function, promotes maternal-fetal tolerance, promotes MDSC proliferation, and synergizes with TGF-β to promote Treg proliferation.

[0011] Circulating levels of Gal9 are significantly higher in patients with certain cancers compared to healthy controls. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] WU et al. (Immunity 41(2):270-282,2014) Summary of the Invention [Means for solving the problem]

[0013] Summary of the Invention The invention described herein relates to humanized antibodies directed against galectin-9 and their use for the treatment of diseases associated with the suppressor activity of regulatory T lymphocytes (Tregs).

[0014] In particular, the invention described herein provides anti-Gal9 neutralizing antibodies that relieve immunosuppression in the TME (tumor microenvironment), resulting in anti-tumor activity and clinical responses in cancer patients.

[0015] The anti-Gal9 neutralizing antibodies of the present invention are derivative antibodies based on two anti-Gal9 neutralizing antibodies (Ab1 and Ab2, respectively) disclosed in U.S. Patent Application Publication No. 2017 / 0283499A1 (filed June 5, 2015, and incorporated herein by reference), both of which bind to recombinant human Gal9 with sub-nM EC 50 Combined with the value, CD4 + They block human Gal9-induced apoptosis of T cells or proliferation of Tregs derived from peripheral blood of healthy donors. However, these two antibodies differ in that Ab2 blocks recombinant human Gal9 interaction with two immune receptors (R1 and R2), whereas Ab1 does not.

[0016] The invention described herein provides multiple humanized monoclonal antibodies based on Ab1 and Ab2. These humanized monoclonal antibodies bind to recombinant human and / or mouse Gal9, block Gal9-induced Th1 apoptosis, and block Gal9-induced Treg proliferation. More importantly, the humanized monoclonal antibodies of the present invention act synergistically with antibodies targeting the PD-1 / PD-L1 immune checkpoint, providing a therapeutic advantage for overcoming resistance encountered in immunotherapy (e.g., ineffective resistance using PD-1 and PD-L1 antagonists).

[0017] The antibodies of the present invention are widely used to treat hematological cancers such as AML and DLBCL, as well as solid cancers such as breast cancer, head and neck cancer, lung cancer, melanoma (including uveal melanoma), colon cancer, renal cancer, ovarian cancer, liver cancer, and prostate cancer.

[0018] Thus, one aspect of the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof is specific for galectin-9, and the monoclonal antibody comprises: (1a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 2, the HCVR CDR2 sequence of SEQ ID NO: 4, and the HCVR CDR3 sequence of SEQ ID NO: 6; and (1b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 10, the LCVR CDR2 sequence of SEQ ID NO: 12, and the LCVR CDR3 sequence of SEQ ID NO: 14; or (2a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 18, the HCVR CDR2 sequence of SEQ ID NO: 20, and the HCVR CDR3 sequence of SEQ ID NO: 22; and (2b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 26, the LCVR CDR2 sequence of SEQ ID NO: 28, and the LCVR CDR3 sequence of SEQ ID NO: 30; or (3a) an HCVR CDR1 sequence of SEQ ID NO: 34. a heavy chain variable region (HCVR) comprising the CDR1 sequence of SEQ ID NO: 36, the HCVR CDR2 sequence of SEQ ID NO: 36, and the HCVR CDR3 sequence of SEQ ID NO: 38; and (3b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 42, the LCVR CDR2 sequence of SEQ ID NO: 44, and the LCVR CDR3 sequence of SEQ ID NO: 46; or (4a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 50, the HCVR CDR2 sequence of SEQ ID NO: 52, and the HCVR CDR3 sequence of SEQ ID NO: 54; and (4b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 58, the LCVR CDR2 sequence of SEQ ID NO: 60, and the LCVR CDR3 sequence of SEQ ID NO: 62; or (5a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 66, the HCVR CDR2 sequence of SEQ ID NO: 68, and the HCVR CDR3 sequence of SEQ ID NO: 70; and (5b) a LCVR CDR1 sequence of SEQ ID NO: 74, the LCVR CDR2 sequence of SEQ ID NO: 76. (6a) a light chain variable region (LCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 82, the HCVR CDR2 sequence of SEQ ID NO: 84, and the HCVR CDR3 sequence of SEQ ID NO: 86; or (6b) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 82, the HCVR CDR2 sequence of SEQ ID NO: 84, and the HCVR CDR3 sequence of SEQ ID NO: 86;and (6b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 90, the LCVR CDR2 sequence of SEQ ID NO: 92, and the LCVR CDR3 sequence of SEQ ID NO: 94; or (7a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 98, the HCVR CDR2 sequence of SEQ ID NO: 100, and the HCVR CDR3 sequence of SEQ ID NO: 102; and (7b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 106, the LCVR CDR2 sequence of SEQ ID NO: 108, and the LCVR CDR3 sequence of SEQ ID NO: 110; or (8a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 114, the HCVR CDR2 sequence of SEQ ID NO: 116, and the HCVR CDR3 sequence of SEQ ID NO: 118; and (8b) a LCVR CDR1 sequence of SEQ ID NO: 122, the LCVR CDR2 sequence of SEQ ID NO: 124, and the LCVR of SEQ ID NO: 128. Contains a light chain variable region (LCVR) containing CDR3 sequences;

[0019] In certain embodiments, in the isolated monoclonal antibody or antigen-binding fragment thereof, (1c) the antibody or antigen-binding fragment thereof of (1a) and (1b) further comprises the HFR3 sequence of SEQ ID NO: 5, and optionally further comprises the HFR1 sequence of SEQ ID NO: 1; or (2c) the antibody or antigen-binding fragment thereof of (2a) and (2b) further comprises the HFR3 sequence of SEQ ID NO: 21, and optionally further comprises the HFR1 sequence of SEQ ID NO: 17; or (3c) the antibody or antigen-binding fragment thereof of (3a) and (3b) further comprises the HFR3 sequence of SEQ ID NO: 37, and optionally further comprises the HFR1 sequence of SEQ ID NO: 33; or (4c) the antibody or antigen-binding fragment thereof of (4a) and (4b) further comprises the HFR3 sequence of SEQ ID NO: 53, and optionally further comprises the HFR1 sequence of SEQ ID NO: or (5c) the antibodies or antigen-binding fragments thereof of (5a) and (5b) further comprise the HFR3 sequence of SEQ ID NO: 69, and optionally further comprise the HFR1 sequence of SEQ ID NO: 65; or (6c) the antibodies or antigen-binding fragments thereof of (6a) and (6b) further comprise the HFR3 sequence of SEQ ID NO: 85, and optionally further comprise the HFR1 sequence of SEQ ID NO: 81; or (7c) the antibodies or antigen-binding fragments thereof of (7a) and (7b) further comprise the HFR3 sequence of SEQ ID NO: 101, and optionally further comprise the HFR1 sequence of SEQ ID NO: 97; or (8c) the antibodies or antigen-binding fragments thereof of (8a) and (8b) further comprise the HFR3 sequence of SEQ ID NO: 117, and optionally further comprise the HFR1 sequence of SEQ ID NO: 113.

[0020] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof, (1A) the HCVR sequence is SEQ ID NO: 8; and / or (1B) the LCVR sequence is SEQ ID NO: 16, or (2A) the HCVR sequence is SEQ ID NO: 24; and / or (2B) the LCVR sequence is SEQ ID NO: 32, or (3A) the HCVR sequence is SEQ ID NO: 40; and / or (3B) the LCVR sequence is SEQ ID NO: 48, or (4A) the HCVR sequence is SEQ ID NO: 56; and / or (4B) the LCVR sequence is SEQ ID NO: 58. The CVR sequence is SEQ ID NO: 64, or (5A) the HCVR sequence is SEQ ID NO: 72; and / or (5B) the LCVR sequence is SEQ ID NO: 80, or (6A) the HCVR sequence is SEQ ID NO: 88; and / or (6B) the LCVR sequence is SEQ ID NO: 96, or (7A) the HCVR sequence is SEQ ID NO: 104; and / or (7B) the LCVR sequence is SEQ ID NO: 112, or (8A) the HCVR sequence is SEQ ID NO: 120; and / or (8B) the LCVR sequence is SEQ ID NO: 128.

[0021] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is a humanized antibody and comprises: (1) the HCVR sequence of SEQ ID NO: 8 and the LCVR sequence of SEQ ID NO: 16; or (2) the HCVR sequence of SEQ ID NO: 72 and the LCVR sequence of SEQ ID NO: 80.

[0022] In certain embodiments, the antigen-binding fragment is a Fab, Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-linked Fv, V-NAR domain, Ignar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0023] In some embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the present invention have an engineered Fc region that abolishes immune effector function. For example, the engineered Fc region of the subject antibodies may have a "LALA" double mutation (Leu234Ala together with Leu235Ala), thereby reducing effector function. Such an antibody may have the designation G1AA, for the LALA double mutation on IgG1.

[0024] Other recombinant human IgG antibodies (hIgG) with abolished immune effector functions by partially or completely lacking binding to Fcγ receptors (FcγRs) and the complement protein C1q are known in the art and are useful in various therapeutic applications to reduce FcγR activation and Fc-mediated toxicity. Certain such Fc-engineered antibodies / fragments partially achieve this goal, while others completely abolish FcγR activation and Fc-mediated toxicity. In a specific embodiment, the antibodies / fragments of the present invention have engineered hIgG Fc domains containing the hIgG1-P329G LALA or hIgG4-P329G SPLE (human IgG4 S228P / L235E mutant of IgG4) mutations, completely abolishing FcγR and C1q interactions and leaving FcRn interaction and Fc stability unaffected. The P329G Fc mutation disrupts the formation of a proline sandwich motif with FcγRs. Because this motif is present at the interface of all IgG Fc / FcγR complexes, its disruption can be effector-silent across all human and most other mammalian IgG subclasses, creating effector-silent IgG molecules. Thus, in certain embodiments, the subject antibodies / fragments possess any one IgG subclass that possesses such effector-silent Fc mutations.

[0025] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof cross-reacts with mouse Gal9.

[0026] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof binds to human Gal9 with an EC50 of about 0.1-0.2 nM and / or binds to mouse Gal9 with an EC50 of about 0.5-1.0 nM.

[0027] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof binds to human Gal9 with a KD of less than about 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 2 nM, or 1 nM.

[0028] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present invention contains one or more point mutations in its amino acid sequence designed to improve the developability of the antibody. For example, in certain embodiments, the one or more point mutations make the antibody more stable during its expression in a host cell, its purification during the manufacturing and / or formulation process, and / or its administration to a subject patient. In certain embodiments, the one or more point mutations make the antibody less prone to aggregation during the manufacturing and / or formulation process.

[0029] In certain embodiments, the present invention provides therapeutic antibodies in which developability issues are minimized or reduced, such as by substituting one or more amino acids in the sequence (e.g., in one or more CDRs) to eliminate or reduce hydrophobicity and / or optimize charge.

[0030] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof binds to Gal9 and inhibits binding of Gal9 to a Gal9 receptor (eg, TIM3 or CD44).

[0031] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof neutralizes Gal9-induced Th1 apoptosis of T cells (such as CD4+ T cells).

[0032] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof inhibits Gal9-induced Treg expansion.

[0033] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof synergizes with an immune checkpoint antagonist to inhibit tumor growth in vivo and / or prolong survival in mice bearing xenograft (XENOGRAPH) tumors.

[0034] In certain embodiments, the immune checkpoint antagonist is an antibody or antigen-binding fragment thereof specific for PD-1 or PD-L1.

[0035] Another aspect of the present invention provides a method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of an isolated monoclonal antibody or antigen-binding fragment thereof of the present invention and an antagonist of an immune checkpoint.

[0036] In certain embodiments, the immune checkpoint is the PD-1 / PD-L1 immune checkpoint.

[0037] In certain embodiments, the immune checkpoint antagonist is an antibody or antigen-binding fragment thereof specific for PD-1 or PD-L1.

[0038] In certain embodiments, the antibody is an anti-PD-1 antibody, such as cemiplimab, nivolumab, or pembrolizumab.

[0039] In certain embodiments, the antibody is an anti-PD-L1 antibody, such as avelumab, durvalumab, atezolizumab, KN035, or CK-301.

[0040] In certain embodiments, the immune checkpoint antagonist is a (non-antibody) peptide inhibitor of PD-1 / PD-L1, such as AUNP12; a small molecule inhibitor of PD-L1, such as CA-170, or a macrocyclic peptide, such as BMS-986189.

[0041] In certain embodiments, the cancer is a hematological cancer (such as AML and DLBCL) or a solid tumor (such as breast cancer, head and neck cancer, lung cancer, melanoma (including uveal melanoma), colon cancer, renal cancer, ovarian cancer, liver cancer, and prostate cancer).

[0042] In certain embodiments, the method further comprises administering to the patient a chemotherapeutic agent, an anti-angiogenic agent, a growth inhibitory agent, an immuno-oncology therapeutic agent, and / or an anti-neoplastic composition.

[0043] Another aspect of the invention provides polynucleotides encoding the heavy or light chains of the invention, or antigen-binding portions thereof.

[0044] In certain embodiments, the polynucleotide is codon-optimized for expression in human cells.

[0045] Another aspect of the present invention provides a vector comprising a polynucleotide of the present invention.

[0046] In certain embodiments, the vector is an expression vector (eg, a mammalian expression vector, a yeast expression vector, an insect expression vector, or a bacterial expression vector).

[0047] Another aspect of the present invention provides a method for promoting, enhancing, restoring, or rescuing effector T cell proliferation and / or enhancing effector T cell activity in a patient diagnosed with cancer, at risk of developing cancer or cancer recurrence, or for identifying and treating a patient with cancer, comprising administering to the patient an effective amount of an isolated monoclonal antibody or antigen-binding fragment thereof of the present invention upon identifying the patient as having a level of galectin-9 in a sample from the patient that is higher than the baseline level of galectin-9 in a healthy or control individual.

[0048] In certain embodiments, the method further comprises identifying the patient as having a level of galectin-9 in the sample that is higher than the reference level by comparing the level of galectin-9 in the sample with the reference level.

[0049] In certain embodiments, the method further comprises administering to the patient an antagonist of an immune checkpoint.

[0050] In certain embodiments, the immune checkpoint is the PD-1 / PD-L1 immune checkpoint.

[0051] In certain embodiments, the immune checkpoint antagonist is an antibody or antigen-binding fragment thereof specific for PD-1 or PD-L1.

[0052] In certain embodiments, the antibody is an anti-PD-1 antibody, such as cemiplimab, nivolumab, or pembrolizumab.

[0053] In certain embodiments, the antibody is an anti-PD-L1 antibody, such as avelumab, durvalumab, atezolizumab, KN035, or CK-301.

[0054] In certain embodiments, the immune checkpoint antagonist is a (non-antibody) peptide inhibitor of PD-1 / PD-L1, such as AUNP12; a small molecule inhibitor of PD-L1, such as CA-170, or a macrocyclic peptide, such as BMS-986189.

[0055] In certain embodiments, the cancer is a hematological cancer (such as AML and DLBCL) or a solid tumor (such as breast cancer, head and neck cancer, lung cancer, melanoma (including uveal melanoma), colon cancer, renal cancer, ovarian cancer, liver cancer, and prostate cancer).

[0056] In certain embodiments, the patient is an AML patient with Fab M0, M1, M4, or M5, or the patient is not an AML patient with Fab M2 or M3.

[0057] In certain embodiments, the sample is a blood sample, a plasma sample, or a serum sample.

[0058] Another aspect of the present invention is a method for rescuing or promoting effector T cell proliferation and / or enhancing effector T cell activity in patients diagnosed with AML, who are at risk of developing or relapsing from AML, or for identifying and treating patients with AML, comprising determining the level of galectin-9-encoding mRNA in bone marrow (BM)-derived mononuclear cell (MNC) samples from the patient relative to the level of BM-derived MNC or CD34 mRNA in healthy or control individuals. + Upon identifying the patient as having a statistically significantly higher or lower than baseline level in the cells, a method is provided comprising administering to the patient an effective amount of an isolated monoclonal antibody or antigen-binding fragment thereof of the invention.

[0059] In certain embodiments, the level of galectin-9-encoding mRNA in a BM-derived MNC sample from a patient is significantly higher than the baseline level if the patient is an AML patient with Fab M0, M1, M2, M4, or M5.

[0060] In certain embodiments, the level of galectin-9 encoding mRNA in a BM-derived MNC sample from a patient is significantly lower than the baseline level when the patient is an AML patient with Fab M3.

[0061] Another aspect of the present invention provides an antibody or antigen-binding portion thereof directed against or specific for galectin-9 for use in the treatment of cancer, wherein the antibody or antigen-binding portion thereof rescues effector T cell proliferation and / or enhances effector T cell activity.

[0062] In a specific embodiment, the effector T cells are Th1 cells.

[0063] Another aspect of the present invention provides a method of rescuing or promoting effector T cell proliferation and / or enhancing effector T cell activity, comprising contacting an effector T cell with an isolated monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0064] In a specific embodiment, the effector T cells are Th1 cells.

[0065] Another aspect of the present invention provides methods and related compositions for inducing or promoting immune memory that results in anti-tumor (anti-cancer) activity. In certain embodiments, the methods comprise administering to a subject an amount of a composition (such as a pharmaceutical composition comprising an antibody of the present invention) effective to induce, stimulate, or promote immune memory that effectively reduces or inhibits the onset, progression, or recurrence of tumors or cancer in the subject.

[0066] It is to be understood that any one embodiment of the invention described herein, including those set forth solely in the examples or claims, can be combined with any one or more additional embodiments of the invention, unless expressly stated otherwise or otherwise inappropriate. [Brief explanation of the drawings]

[0067] [Figure 1] FIG. 1 shows a sequence alignment of various anti-human galectin-9 humanized antibodies. [Figure 2] FIG. 2 shows a sequence alignment of various anti-human galectin-9 humanized antibodies. [Figure 3] Figure 3 shows the binding affinity (measured as EC50 in nM) of various anti-human galectin-9 humanized antibodies to recombinant human Gal9. Isotype-matched antibodies against different antigens are used as negative controls. In some cases, the original human-mouse chimeric antibody is also included for comparison. [Figure 4] Figure 4 shows the binding affinity (measured as EC50 in nM) of various anti-human galectin-9 humanized antibodies to recombinant mouse Gal9. Isotype-matched antibodies against different antigens are used as negative controls. In some cases, the original human-mouse chimeric antibody is also included for comparison. [Figure 5] FIG. 5 shows that various anti-human galectin-9 humanized antibodies of the present invention can block binding to TIM3 and CDC44. [Figure 6] FIG. 6 shows the ability of various anti-human galectin-9 humanized antibodies of the present invention to neutralize Gal9-induced Th1 cell apoptosis. [Figure 7] FIG. 7 shows the ability of various anti-human galectin-9 humanized antibodies of the invention to neutralize Gal9-induced Treg proliferation. [Figure 8] Figure 8 shows the ability of various anti-human galectin-9 humanized antibodies of the present invention to promote long-term survival when used in combination with an anti-PD1 antibody. The data demonstrate that combination therapy with an anti-PD1 antibody resulted in significantly better / synergistic therapeutic efficacy as measured by inhibition of tumor volume growth. [Figure 9] Figure 9 shows the ability of various humanized anti-human galectin-9 antibodies of the present invention to promote long-term survival when used in combination with an anti-PD1 antibody. The data demonstrate that combination therapy with an anti-PD1 antibody resulted in significantly better / synergistic therapeutic efficacy as measured by survival over time. [Figure 10] Figure 10 shows the levels of galectin-9 in serum or plasma from AML patients and healthy individuals. The data demonstrate that galectin-9 levels in plasma from AML patients at diagnosis or in the relapsed / refractory (R / R) stage were significantly higher than those in plasma from healthy patients and AML patients in complete remission after chemotherapy treatment. [Figure 11] Figure 11 shows the levels of galectin-9 in plasma or serum from AML patients stratified according to the French-American-British (Fab) classification. The data show that galectin-9 protein levels in plasma from AML patients with Fab M2 or Fab M3 at diagnosis were significantly lower than those observed in plasma from AML patients with Fab M0, M1, M4, or M5. Galectin-9 protein levels in plasma from AML patients with Fab M3 at diagnosis were within the normal physiological range. [Figure 12] Figure 12 shows the levels of galectin-9-encoding mRNA (LGALS9) in bone marrow (BM)-derived mononuclear cells (MNCs) from AML patients stratified according to the French-American-British (Fab) classification or from healthy individuals. The data show that galectin-9-encoding mRNA levels in BM-derived MNCs (all considered Fabs) from AML patients at the time of diagnosis were higher than the mRNA levels observed in BM-derived MNCs or CD34+ cells from healthy individuals. Galectin-9-encoding mRNA levels in BM-derived MNCs from AML patients with Fab M3 at the time of diagnosis were significantly lower than those observed in BM-derived MNCs from AML patients with Fab M0, M1, M4, or M5, or in BM-derived MNCs or CD34+ cells from healthy individuals. [Figure 13]Figure 13 shows the anti-tumor activity of antibodies of the present invention as monotherapy. This experiment demonstrates that anti-Gal9 monoclonal antibodies of the present invention are effective in inhibiting tumor growth in vivo in a xenograph mouse model. Specifically, approximately 500,000 cancer cells were inoculated into experimental mice, and tumor masses were allowed to grow to a predetermined size. Mice were then randomized and intraperitoneally (ip) injected with one of two antibodies: (1) an IgG isotype control at a dose of 10 mg / kg, or (2) the anti-Gal9 antibody HFB9-2 at a dose of 10 mg / kg. The first dose of antibody for various groups was administered on day 1, and subsequent doses were administered every three days for a total of eight doses for all groups with anti-HFB9-2 and control antibodies. Data are presented as mean ± sem (N = 10 mice / group). It is clear that the subject anti-Gal9 antibodies demonstrated an inhibitory effect on tumor growth in vivo. [Figure 14] Figure 14 further demonstrates the anti-tumor activity of antibodies of the invention as monotherapy with respect to survival: all mice in the control group died, and 40% (4 of 10) of the mice in the HFB9-2 treatment group were tumor-free and survived at the end of week 6. [Figure 15] Figure 15 shows the immunological memory of the anti-tumor activity of the antibody of the present invention. After the first tumor inoculation / challenge, naive animals developed tumors, and two of four animals reached the human endpoint tumor volume of 3000 mm within 31 days. Four animals with complete tumor regression that had previously been treated and cured with the antibody of the present invention, HFB9-2, completely rejected a second Wehi-164 tumor challenge inoculated 63 days after the first tumor challenge. These data suggest long-term immunological memory induced by HFB9-2 treatment. DETAILED DESCRIPTION OF THE INVENTION

[0068] Detailed Description of the Invention 1. Overview Monoclonal antibodies targeting immune checkpoints have demonstrated clinical success in a wide range of tumor types, but durable responses are only observed in a fraction of patients due to primary or secondary resistance to treatment.

[0069] Applicants believe that galectin-9 (Gal-9) is a key factor present in the tumor microenvironment that renders tumors resistant to current immunotherapies. Among other evidence, high Gal-9 expression has been reported in various cancer types, including hematological malignancies such as AML and ALL, and several solid tumors.

[0070] The invention described herein provides antibodies targeting Gal-9 that overcome resistance and improve clinical responses in at least a subset of cancer patients. The monoclonal antibodies of the invention specifically bind to human Gal9 with subnanomolar affinity, recognize recombinant Gal9 and Gal9 produced by human tumor cells, and are cross-reactive with mouse and monkey Gal9 orthologs. Furthermore, the monoclonal antibodies of the invention dose-dependently block the interaction of Gal9 with its receptors, TIM3 and CD44. These two receptors have been shown to mediate Gal9 immunosuppressive signals in effector T cells and regulatory T cells. Treatment of human PBMCs from healthy donors with the antibodies of the invention prevents Gal9-induced Th1 cell apoptosis and suppresses regulatory T cell proliferation.

[0071] Certain humanized versions of the antibodies of the present invention exhibit more favorable characteristics with respect to stability and pharmacokinetic (PK) profiles, making them uniquely suitable for further development as therapeutic antibodies. Specifically, such humanized antibodies exhibited stability for at least 14 days at 40°C, for several hours at low pH, and after several freeze-thaw cycles. Meanwhile, high plasma exposure following administration of a single 10 mg / kg dose to C57BL / 6 mice was observed for the humanized antibodies.

[0072] The antibodies of the present invention can be used to treat some cancers, such as AML. Gal9 has been reported to play a dual role in AML, both as a self-renewal factor for leukemia stem cells and as a suppressor of anti-cancer immunity. Therefore, antagonizing Gal9 function by using the Gal9 neutralizing antibodies of the present invention represents an attractive therapeutic approach for treating AML.

[0073] Taken together, the data presented herein demonstrate that neutralization of Gal9 by the antibodies of the present invention blocks an important immunosuppressive mechanism known to limit the efficacy of current immunotherapies.

[0074] Detailed aspects of the invention are further described individually in various sections below, however, it should be understood that any one embodiment of the invention, including embodiments described only in the examples or drawings and embodiments described only in one section below, can be combined with any other embodiment of the invention.

[0075] 2.Definition The term "antibody" in its broadest sense encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies). The term "antibody" can also broadly refer to a molecule comprising heavy chain complementarity-determining regions (CDRs) 1, 2, and 3, and light chain CDRs 1, 2, and 3, and capable of binding to an antigen. The term "antibody" also includes, but is not limited to, chimeric antibodies, humanized antibodies, human antibodies, and antibodies of various species, including mice, humans, cynomolgus monkeys, and others.

[0076] However, in a narrower sense, "antibody" refers to various monoclonal antibodies, including chimeric, humanized, and human monoclonal antibodies, particularly the humanized monoclonal antibodies of the present invention.

[0077] In some embodiments, the antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR). In some embodiments, the antibody comprises at least one heavy chain (HC) comprising a heavy chain variable region and at least a portion of a heavy chain constant region, and at least one light chain (LC) comprising a light chain variable region and at least a portion of a light chain constant region. In some embodiments, the antibody comprises two heavy chains (each heavy chain comprising a heavy chain variable region and at least a portion of a heavy chain constant region) and two light chains (each light chain comprising a light chain variable region and at least a portion of a light chain constant region).

[0078] As used herein, a single-chain Fv (scFv) or any other antibody that comprises, for example, a single polypeptide chain that comprises all six CDRs (three heavy chain CDRs and three light chain CDRs) is considered to have a heavy chain and a light chain. In some such embodiments, the heavy chain is the region of the antibody that comprises the three heavy chain CDRs, and the light chain is the region of the antibody that comprises the three light chain CDRs.

[0079] As used herein, the term "heavy chain variable region (HCVR)" refers to a region comprising at least heavy chain CDR1 (CDR-H1), framework 2 (HFR2), CDR2 (CDR-H2), FR3 (HFR3), and CDR3 (CDR-H3). In some embodiments, the heavy chain variable region also comprises at least a portion (e.g., the entirety) of FR1 (HFR1) located N-terminal to CDR-H1 and / or at least a portion (e.g., the entirety) of FR4 (HFR4) located C-terminal to CDR-H3.

[0080] As used herein, the term "heavy chain constant region" refers to a region comprising at least three heavy chain constant domains, CH1, CH2, and CH3. Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha. Non-limiting exemplary heavy chain constant regions include epsilon and mu. Each heavy constant region corresponds to an antibody isotype. For example, an antibody comprising a gamma constant region is an IgG antibody, a delta constant region is an IgD antibody, an alpha constant region is an IgA antibody, an epsilon constant region is an IgE antibody, and a mu constant region is an IgM antibody.

[0081] A particular isotype can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 antibodies (containing a γ1 constant region), IgG2 antibodies (containing a γ2 constant region), IgG3 antibodies (containing a γ3 constant region), and IgG4 antibodies (containing a γ4 constant region). IgA antibodies include, but are not limited to, IgAl antibodies (containing an α1 constant region) and IgA2 antibodies (containing an α2 constant region). IgM antibodies include, but are not limited to, IgM1 (containing a μ1 constant region) and IgM2 (containing a μ2 constant region).

[0082] As used herein, the term "heavy chain" refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain includes at least a portion of a heavy chain constant region. As used herein, the term "full-length heavy chain" refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence, and with or without a C-terminal lysine.

[0083] As used herein, the term "light chain variable region (LCVR)" refers to a region comprising light chain CDR1 (CDR-L1), framework (FR) 2 (LFR2), CDR2 (CDR-L2), FR3 (LFR3), and CDR3 (CDR-L3). In some embodiments, the light chain variable region also comprises at least a portion (e.g., the entirety) of FR1 (LFR1) and / or at least a portion (e.g., the entirety) of FR4 (LFR4).

[0084] As used herein, the term "light chain constant region" refers to a light chain constant domain C L Non-limiting exemplary light chain constant regions include lambda and kappa.

[0085] As used herein, the term "light chain" refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. As used herein, the term "full-length light chain" refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0086] The term "antibody fragment" or "antigen-binding portion" (of an antibody) includes, but is not limited to, fragments capable of binding to antigen, such as Fv, single-chain Fv (scFv), Fab, Fab', and (Fab')2. In certain embodiments, antibody fragments include Fab, Fab', F(ab')2, F d , single chain Fv or scFv, disulfide bond F v , V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mab2, (scFv)2, or scFv-Fc.

[0087] The term "Fab" refers to an antibody fragment with a molecular weight of approximately 50,000 daltons that has antigen-binding activity. Fab contains approximately the N-terminal half of the heavy chain and the entire light chain, linked by disulfide bridges. Fab can be obtained by treating immunoglobulins with the protease papain.

[0088] The term "F(ab')2" refers to a fragment of approximately 100,000 daltons and antigen-binding activity. This fragment is slightly larger than two Fab fragments linked via disulfide bridges in the hinge region. These fragments can be obtained by treating immunoglobulins with the protease pepsin. Fab fragments can be obtained from F(ab')2 fragments by cleavage of the disulfide bridges in the hinge region.

[0089] A single Fv chain "scFv" refers to a VH:VL polypeptide synthesized using genes encoding the VL and VH domains and a sequence encoding a peptide intended to bind to these domains. The scFv of the present invention comprises CDRs maintained in the appropriate conformation, for example, using genetic engineering techniques.

[0090] An "scFv" dimer refers to two scFv molecules linked together by a peptide bond. The Fv chains are often the result of expression of a fusion gene containing VH and VL encoding genes linked by a peptide-encoding linker sequence. Human scFv fragments can contain CDR regions maintained in the proper conformation, preferably by the use of genetic engineering techniques.

[0091] A "dsFv" fragment is a VH-VL heterodimer stabilized by disulfide bridges; a "dsFv" fragment can be bivalent (dsFv2). Bivalent Sc(Fv)2 or multivalent antibody fragments can form spontaneously by association of monovalent scFvs or can be generated by linking scFv fragments via peptide bond sequences.

[0092] The Fc fragment is the carrier of the antibody's biological properties, particularly its ability to be recognized by immune effectors or to activate complement. The Fc fragment consists of the constant fragment of the heavy chain beyond the hinge region.

[0093] The term "diabody" refers to a small antibody fragment with two antigen-fixing sites. The fragment comprises a variable heavy domain, VH, connected to a variable light domain, VL, in the same VH-VL polypeptide chain. Using a linker sequence that is too short to allow matching of the two domains on the same chain forces matching with the two complementary domains on another chain, thus creating two antigen-fixing sites.

[0094] An "antibody that binds to the same epitope" as a reference antibody can be determined by an antibody competition assay. An antibody that binds to the same epitope refers to an antibody that inhibits the binding of the reference antibody to the antigen by 50% or more in a competition assay, and conversely, a reference antibody that inhibits the binding of the antibody to the antigen by 50% or more in a competition assay. The term "compete," when used in the context of antibodies competing for the same epitope, means that competition between antibodies is determined by an assay in which the antibody being tested prevents or inhibits the specific binding of the reference antibody to a common antigen.

[0095] Many types of competitive binding assays can be used, including, for example, solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competition assays (see, e.g., Stahl et al., 1983, Methods in Enzymology, 9:242-253); solid-phase direct biotin-avidin EIAs (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619); solid-phase direct label assays; solid-phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); 125Solid-phase direct label RIA using labels (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct label RIA (Moldenhauer et al., 1990, Scand. J. Immunol.).

[0096] Typically, such assays involve the use of purified antigen bound to a solid surface or cells bearing either an unlabeled test antigen-binding protein or a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antibody. The test antibody is usually present in excess. Antibodies identified by competitive assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to an adjacent epitope sufficiently close to the epitope bound by the reference antibody to create steric hindrance. In some embodiments, a competing antibody, when present in excess, will inhibit specific binding of the reference antibody to a common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some cases, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more.

[0097] The term "antigen" refers to a molecule or portion of a molecule capable of being bound by a selective binding agent, such as an antibody or an immunologically functional fragment thereof, and which can additionally be used in a mammal to generate antibodies capable of binding to the antigen. An antigen can have one or more epitopes capable of interacting with an antibody.

[0098] The term "epitope" refers to the portion of an antigen molecule that is bound by a selective binding agent, such as an antibody or fragment thereof. The term includes any determinant capable of specific binding to an antibody. Epitopes can be contiguous or discontinuous (e.g., in a polypeptide, amino acid residues that are not contiguous with each other within the polypeptide sequence but that, in the context of the molecule, are bound by the antigen-binding protein). In some embodiments, an epitope may be a mimetic in that it comprises a three-dimensional structure similar to the epitope used to generate the antibody, but does not include, or only includes, a subset of, the amino acid residues found in the epitope used to generate the antibody. Epitope determinants may include chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural and / or charge characteristics.

[0099] In some embodiments, an "epitope" is defined by the method used to determine the epitope. For example, in some embodiments, an antibody binds to the same epitope as a reference antibody if it binds to the same region of the antigen, as determined by hydrogen-deuterium exchange (HDX).

[0100] For example, the epitope sequences of the parent antibodies HFB9-1 and HFB9-2 are disclosed as SEQ ID NO: 9 in U.S. Patent Application Publication No. 2017 / 0283499A1 (incorporated herein by reference). This sequence corresponds to the P4 peptide, covering the end of the binding peptide and the beginning of the C-terminal portion of galectin-9. It is present in three isoforms of galectin-9 (e.g., amino acids 166-178 of the S isoform, amino acids 178-190 of the M isoform, and amino acids 210-222 of the L isoform). The humanized antibodies of the present invention can bind to at least one, and preferably all, isoforms of galectin-9.

[0101] In certain embodiments, an antibody binds to the same epitope as a reference antibody if it binds to the same region of the antigen as determined by x-ray crystallography.

[0102] As used herein, a "chimeric antibody" refers to an antibody that comprises at least one variable region derived from a first species (e.g., mouse, rat, cynomolgus monkey, etc.) and at least one constant region derived from a second species (e.g., human, cynomolgus monkey, chicken, etc.). In some embodiments, a chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, all of the variable regions of a chimeric antibody are derived from the first species, and all of the constant regions of the chimeric antibody are derived from the second species.

[0103] As used herein, "humanized antibody" refers to an antibody in which at least one amino acid in the framework region of a non-human variable region (e.g., mouse, rat, cynomolgus monkey, chicken, etc.) has been replaced with the corresponding amino acid from a human variable region. In some embodiments, a humanized antibody comprises at least one human constant region or fragment thereof. In some embodiments, the humanized antibody fragment is a Fab, scFv, (Fab')2, etc.

[0104] As used herein, "CDR-grafted antibody" refers to a humanized antibody in which one or more complementarity-determining regions (CDRs) of a first (non-human) species have been grafted onto framework regions (FRs) of a second (human) species.

[0105] As used herein, "human antibody" refers to antibodies produced in humans, antibodies produced in non-human animals that contain human immunoglobulin genes, e.g., XENOMOUSE®, and antibodies selected using in vitro methods, e.g., phage display, where the antibody repertoire is based on human immunoglobulin sequences.

[0106] "Host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. Host cells can be prokaryotic or eukaryotic. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells, as well as their derivatives, such as 293-6E and DG44 cells, respectively.

[0107] As used herein, the term "isolated" refers to a molecule that is separated from at least some of the components typically found in nature or that is separated from at least some of the components typically produced. For example, a polypeptide is referred to as "isolated" if it is separated from at least some of the components of the cell in which it is produced. If a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to "isolate" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" if it is not part of a larger polynucleotide in which it is typically found in nature (e.g., in the case of a DNA polynucleotide, genomic DNA or mitochondrial DNA, etc.), or, for example, in the case of an RNA polynucleotide, if it is separated from at least some of the components of the cell in which it is produced. Thus, a DNA polynucleotide contained within a vector within a host cell may be referred to as "isolated" so long as the polynucleotide is not found within the vector in nature.

[0108] The terms "subject" and "patient" are used interchangeably herein to refer to mammals, such as humans. In some embodiments, methods of treating other non-human mammals are also provided, including, but not limited to, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, lab mammals, livestock mammals, sport mammals, and pet mammals. In some instances, "subject" or "patient" refers to a (human) subject or patient in need of treatment for a disease or disorder.

[0109] As used herein, the term "sample" or "patient sample" refers to a substance obtained from or derived from a subject of interest that contains cellular and / or other molecular entities to be characterized and / or identified, e.g., based on physical, biochemical, chemical, and / or physiological properties. For example, the phrase "disease sample" and variations thereof refers to any sample obtained from a subject of interest that is suspected or known to contain the cellular and / or molecular entities to be characterized.

[0110] "Tissue or cell sample" refers to a collection of similar cells obtained from a subject's or patient's tissue. The source of the tissue or cell sample can be fresh, frozen, and / or preserved organ or tissue sample or solid tissue from a biopsy or aspirate; blood or any blood component; bodily fluids such as sputum, cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any point in a subject's pregnancy or development. Additionally, the tissue sample can be primary cells or cell lines, or cultured cells or cell lines. If desired, the tissue or cell sample is obtained from a diseased tissue / organ. The tissue sample may contain compounds not naturally associated with the tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0111] As used herein, a "reference sample," "reference cell," or "reference tissue" refers to a sample, cell, or tissue obtained from a source known or believed to be free of the disease or condition that the methods or compositions of the present invention are to be used to identify. In one embodiment, the reference sample, reference cell, or reference tissue is obtained from a healthy part of the body of the same subject or patient in whom a disease or condition is to be identified using the compositions or methods of the present invention. In one embodiment, the reference sample, reference cell, or reference tissue is obtained from a healthy part of the body of at least one individual who is not the subject or patient in whom a disease or condition is to be identified using the compositions or methods of the present invention. In some embodiments, the reference sample, reference cell, or reference tissue is previously obtained from a patient before the onset of the disease or condition or at an early stage of the disease or condition.

[0112] A "disorder" or "disease" is any condition that would benefit from treatment with one or more Gal-9 antagonists of the present invention. This includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question. Non-limiting examples of disorders that may be treated herein include cancer.

[0113] "Diseases associated with the suppressive activity of regulatory T lymphocytes" refers to any disease (not autoimmune) in which the suppressive activity of regulatory T lymphocytes plays a role, particularly by promoting the onset or persistence of the disease. In particular, it has been demonstrated that the suppressive activity of regulatory T lymphocytes promotes tumor development. Thus, the present invention is more specifically directed to cancers in which the suppressive activity of T lymphocytes plays a role.

[0114] The term "cancer" is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth. Cancers can be benign (also referred to as benign tumors), pre-malignant, or malignant. Cancer cells can be solid cancer cells (i.e., that form solid tumors) or leukemic cancer cells. The term "cancer growth" is used herein to refer to proliferation or growth by one or more cells that comprise a cancer, resulting in a corresponding increase in the size or extent of the cancer.

[0115] Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific, non-limiting examples of such cancers include squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, brain cancer, endometrial cancer, testicular cancer, bile duct cancer, gallbladder cancer, gastric cancer, melanoma, and various types of head and neck cancer.

[0116] The French-American-British (Fab) classification of acute myeloid leukemia (ALM) divides ALM into different stages of the disease. The Fab subtypes are shown in Table 1. [Table 1]

[0117] In certain embodiments, cancer as used herein includes hematological cancers (such as AML and DLBCL) or solid tumors (such as breast cancer, head and neck cancer, lung cancer, melanoma (including uveal melanoma), colon cancer, renal cancer, ovarian cancer, liver cancer, and prostate cancer).

[0118] A "chemotherapeutic agent" is a compound that may be useful in the treatment of cancer. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, metoledopa, and uredopa; ethyleneimines and methylameramines (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine); acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 2). syn8); dolastatins; duocarmycins (including synthetic analogs, KW-2189, and CB1-TM1); eleutherobin; pancratistatin; sarcodictine; spongistatin; nitrogen mustards, e.g., chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, e.g., enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gumol and calicheamicin omegaol (e.g., Agnew, Chem See Intl. Ed. Engl, 33:183-186 (1994); dynemycins (including dynemycin A); bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detrevicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin , and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, e.g., mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folic acid analogs, e.g., denopterin, methotrexate, protease inhibitors, teropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as floric acid; Aceglatone; Aldophosphamide glycosides; Aminolevulinic acid; Eniluracil; Amsacrine; Bestravcil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diaziquone; Elfomitin; Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidynin; Maytansinoids, such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerin; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; Podophyllic acid;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® Cremophor Free, an albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (5- including treatment regimens of irinotecan with FU and leucovorin); the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin (including oxaliplatin treatment regimens (FOLFOX)); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (TARCEVA®)) and VEGF-A that reduce cell proliferation, and pharmaceutically acceptable salts, acids or derivatives of any of the above;

[0119] Further non-limiting exemplary chemotherapeutic agents include antihormonal agents that act to regulate or inhibit hormone action on cancer, such as antiestrogens and selective estrogen receptor modulators (SERMs), such as tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® toremifene; aromatase inhibitors, which inhibit the enzyme aromatase (which regulate estrogen production in the adrenal glands); For example, 4(5)-imidazole, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestany, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those used to treat aberrant cells (abherent antisense oligonucleotides that inhibit expression of genes in signaling pathways involved in cell proliferation, e.g., PKC-alpha, Ralf, and H-Ras; ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME® ribozyme) and HER2 expression inhibitors; vaccines, e.g., gene therapy vaccines, e.g., ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitors; ABARELIX® rmRH; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0120] "Anti-angiogenic agent" or "angiogenesis inhibitor" refers to a low molecular weight substance, polynucleotide (including, for example, inhibitory RNA (RNAi or siRNA)), polypeptide, isolated protein, recombinant protein, antibody, or conjugate or fusion protein thereof, that directly or indirectly inhibits angiogenesis, vasculogenesis, or undesired vascular permeability. Anti-angiogenic agents should be understood to include agents that bind to angiogenic factors or their receptors and block their angiogenic activity. For example, anti-angiogenic agents include antibodies or other antagonists against angiogenesis agents, such as antibodies against VEGF-A (e.g., bevacizumab (AVASTIN®)) or against a VEGF-A receptor (e.g., KDR receptor or Flt-1 receptor), anti-PDGFR inhibitors such as GLEEVEC® (imatinib mesylate), small molecules that block VEGF receptor signaling (e.g., PTK787 / ZK2284, SU6668, SUTENT® / SU1 1248 (sunitinib malate), AMG706, or those described in, for example, WO 2004 / 113304). Anti-angiogenic agents also include natural angiogenesis inhibitors, such as angiostatin, endostatin, and the like. See, e.g., Klagsbrun and D'Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22:3172-3179 (e.g., Table 3, listing anti-angiogenic therapies in malignant melanoma); Ferrara & Alitalo (1999) Nature Medicine 5(12):1359-1364; Tonini et al. (2003) Oncogene 22:6549-6556 (e.g., Table 2, listing known anti-angiogenic factors); and Sato (2003) Int. J. Clin. Oncol. 8:200-206 (e.g., Table 1, listing anti-angiogenic agents used in clinical trials).

[0121] As used herein, a "growth inhibitory agent" refers to a compound or composition that inhibits the growth of cells (such as cells expressing VEGF) either in vitro or in vivo. Thus, a growth inhibitory agent can significantly reduce the proportion of cells in S phase (such as cells expressing VEGF). Examples of growth inhibitory agents include, but are not limited to, agents that block cell cycle progression (at a location other than S phase), such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Additionally, agents that arrest G1, such as DNA alkylating agents, such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C, extend to S-phase arrest. Further information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1 (titled "Cell cycle regulation, oncogenes, and antitineoplastic drugs") (Murakami et al. (WB Saunders, Philadelphia, 1995), e.g., p. 13). The taxanes (paclitaxel and docetaxel) are both anticancer drugs derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer) is derived from the European yew and is a semisynthetic analog of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, resulting in the inhibition of mitosis in cells.

[0122] The term "anti-neoplastic composition" refers to a composition useful for treating cancer, comprising at least one active therapeutic agent. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenic agents, cancer immunotherapeutics (also called immuno-oncology agents), apoptotic agents, anti-tubulin agents, and other agents for treating cancer, such as anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (TARCEVA®)), platelet-derived growth factor inhibitors (e.g., GLEEVEC® (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, CTLA4 inhibitors (e.g., anti-CTLA antibody I). These include pilimumab (YERVOY®), PD-1 inhibitors (e.g., anti-PD1 antibodies, BMS-936558), PDL1 inhibitors (e.g., anti-PDL1 antibodies, MPDL3280A), PDL2 inhibitors (e.g., anti-PDL2 antibodies), VISTA inhibitors (e.g., anti-VISTA antibodies), cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets: ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA, PD-1, PDL1, PDL2, CTLA4, VISTA, or VEGF receptors, TRAIL / Apo2, and other bioactive agents and organic chemical agents, among others. Combinations thereof are also encompassed by the present invention.

[0123] "Treatment" refers to therapeutic treatment, for example, where a subject slows (alleviates) the targeted pathological condition or disorder, and for example, where a subject inhibits the recurrence of symptoms or disorders. "Treatment" encompasses any administration or use of a therapeutic agent for a disease (also referred to herein as a "disorder" or "symptom") in a mammal, including a human, and includes inhibiting a disease or its progression, inhibiting or slowing a disease or its progression, arresting its onset, partially or completely alleviating a disease, partially or completely alleviating one or more symptoms of a disease, or restoring or repairing a lost, deficient, or defective function; or stimulating an inefficient process. The term "treatment" also includes reducing the severity of any phenotypic characteristic and / or reducing the incidence, degree, or likelihood of the characteristic. Those in need of treatment include those already with the disorder and those at risk of recurrence of the disorder, or those in whom recurrence of the disorder is to be prevented or slowed.

[0124] The term "effective amount" or "therapeutically effective amount" refers to the amount of drug effective to treat a disease or disorder in a subject. In some embodiments, an effective amount refers to an amount effective at the necessary dosage and for the necessary period to achieve the desired therapeutic or preventive result. The therapeutically effective amount of the Gal9 antagonist of the present invention may vary according to factors such as the individual's disease state, age, sex, and weight, and the ability of the antagonist to induce the desired response in the individual. A therapeutically effective amount includes an amount in which any toxic or adverse effects of the Gal9 antagonist are outweighed by the therapeutically beneficial effects.

[0125] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, although not necessarily, the prophylactically effective amount will be less than the therapeutically effective amount, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease.

[0126] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier conventional in the art used in therapeutic agents that together comprise a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations used and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation used. For example, if the therapeutic agent is to be administered orally, the carrier may be a gel capsule. If the therapeutic agent is to be administered subcutaneously, the carrier ideally is not irritating to the skin and does not cause injection site reactions.

[0127] An "article of manufacture" is any product (e.g., package or container) or kit that includes at least one reagent, e.g., a pharmaceutical agent for treating a disease or disorder, or a probe for specifically detecting a biomarker described herein. In some embodiments, the article of manufacture or kit is promoted, distributed, or sold as a unit for performing a method described herein.

[0128] 3. Methods for treating cancer The invention described herein provides Gal9 antagonists (such as anti-Gal9 antibodies) for use in methods of treating humans and other non-human mammals.

[0129] In pathological situations, Tregs can cause inappropriate immune suppression, which can promote, for example, tumor growth. Tregs have been implicated in promoting the development of numerous cancer types by reducing anti-tumor immune responses, particularly by inappropriately inhibiting the activity of effector T lymphocytes.

[0130] During activation, galectin-9 is directly expressed by Tregs, whereas it is expressed very weakly or not at all by effector T lymphocytes. Therefore, by targeting galectin-9, for example, using a Gal-9-specific antibody, it is possible to specifically inhibit the suppressive activity of regulatory T lymphocytes without the risk of causing depletion of effector T lymphocytes. Therefore, antibodies according to the present invention directed against galectin-9 and inhibiting the suppressive activity of regulatory T lymphocytes can be used to treat diseases or conditions associated with the suppressive activity of regulatory T lymphocytes, in particular cancer.

[0131] In some embodiments, methods of treating or preventing cancer are provided, comprising administering an effective amount of a Gal9 antagonist to a subject in need of cancer treatment.

[0132] In some embodiments, a method of treating cancer is provided, comprising administering a Gal9 antagonist to a subject with cancer.

[0133] In some embodiments, there is provided a use of a Gal9 antagonist for treating cancer.

[0134] Cancers that can be treated by the method / use of the present invention include cancers in which regulatory T lymphocytes exert their suppressive activity, for example, cancers in which a relatively large amount of regulatory T lymphocytes exist in tumor tissue or in the circulatory system.The proliferation of regulatory T lymphocytes (which can be measured by the frequency of Treg) generally correlates with the increase in Treg activation.The frequency of regulatory T lymphocytes can be evaluated by any method known in the art, for example, by flow cytometry (FACS) analysis of intratumoral or circulating lymphocytes, or by immunohistological staining of tumor tissue.

[0135] Non-limiting exemplary cancers that can be treated with Gal9 antagonists are described herein and include carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific, non-limiting examples of such cancers include melanoma, cervical cancer, squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, brain cancer, endometrial cancer, testicular cancer, bile duct cancer, gallbladder cancer, stomach cancer, melanoma, and various types of head and neck cancer.

[0136] In certain embodiments, the methods / uses of the present invention may be used to treat cancers in which high levels of regulatory T lymphocytes are known and / or the cancer / tumor is clearly associated with a poor prognosis including chronic myeloid leukemia (CML), colon cancer, melanoma, uterine cancer, breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, primary lymphoma of the central nervous system, multiple myeloma, prostate cancer, Hodgkin's lymphoma, or hepatocellular carcinoma.

[0137] In certain embodiments, the methods / uses of the present invention can be used to treat cancers that produce large amounts of exosomes carrying galectin-9, which acts as an immunosuppressant. Non-limiting examples of such cancers include virus-induced cancers, such as nasopharyngeal carcinoma associated with EBV (Epstein-Barr virus), or hepatocellular carcinoma (CHC) associated with HCV (hepatitis C virus) or HBV (hepatitis B virus).

[0138] In some embodiments, the cancer is a hematological cancer (such as AML and DLBCL) or a solid tumor (such as breast cancer, head and neck cancer, lung cancer, melanoma (including uveal melanoma), colon cancer, renal cancer, ovarian cancer, liver cancer, and prostate cancer).

[0139] In a particular embodiment, the method / use of the present invention can be used to treat recurrence of fibrosis due to hepatitis C, since it has also been demonstrated that an increase in the frequency of regulatory T lymphocytes is a predictor of the recurrence of such fibrosis.

[0140] In some embodiments, the Gal9 antagonist is an anti-Gal9 antibody, or simply a "Gal9 antibody."

[0141] In some embodiments, the Gal9 antagonist for treating cancer can be a non-antibody protein, such as a soluble version of the Gal9 protein or a portion thereof (e.g., ECD) that inhibits the interaction of Gal9 with its ligand, and optionally further comprises a fusion partner to form a fusion molecule. Various exemplary Gal9 antagonists are described in more detail in the following sections.

[0142] In some embodiments, the Gal9 antagonists of the present invention can be used alone or in combination with any other suitable compound known to be capable of treating a disease or indication.

[0143] Thus, according to a particular embodiment of the present invention, an antibody directed against galectin-9 as defined above and inhibiting the suppressive activity of regulatory T lymphocytes is used in combination with a second therapeutic agent, e.g., an anti-cancer agent, for treating a disease associated with the suppressive activity of regulatory T lymphocytes.

[0144] That is, when the use is the treatment of cancer, the antibody can be used in combination with known treatments for cancer, such as surgery, radiation therapy, chemotherapy, or a combination thereof. For example, the antibody can be used in combination with adoptive immunotherapy, which consists of one or more injections of effector lymphocytes against tumor antigens, particularly EBV antigens. According to some embodiments, other anti-cancer agents used in combination with the antibodies directed against galectin-9 according to the present invention for cancer treatment include anti-angiogenic agents. According to certain embodiments, the antibody can be co-administered with cytokines, for example, cytokines that stimulate anti-tumor immune responses.

[0145] In such combination therapy, the antibodies of the invention may be used before, after, or simultaneously with the second therapeutic agent. See further section below regarding combination therapy.

[0146] 4. Route of Administration and Carriers In various embodiments, the Gal9 antagonist (e.g., Gal9 Ab) can be administered subcutaneously or intravenously. For brevity, the term "Gal9 antagonist" herein refers narrowly to a Gal1 antibody of the present invention, e.g., a humanized Gal9 antibody of the present invention.

[0147] In some embodiments, the Gal9 antagonist may be administered in vivo by various routes, including, but not limited to, oral, intraarterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, inhalation, intradermal, topical, transdermal, and intrathecal, or by other methods, such as by implantation.

[0148] In some embodiments, the Gal9 antagonist is an anti-Gal9 antibody or antigen-binding fragment thereof and is administered intravenously (iv) or subcutaneously (sc).

[0149] The subject compositions may be formulated into solid, semi-solid, liquid, or gaseous form preparations, including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols.

[0150] In various embodiments, compositions containing Gal9 antagonists are provided in formulations with a wide variety of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th Edition (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd Edition, Pharmaceutical Press (2000)). A variety of pharmaceutically acceptable carriers, including vehicles, adjuvants, and diluents, are available. In addition, a variety of pharmaceutically acceptable auxiliary agents, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, and wetting agents, are also available. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0151] In various embodiments, compositions containing Gal9 antagonists can be formulated for injection, including subcutaneous administration, by dissolving, suspending, or emulsifying them in aqueous or non-aqueous solvents such as vegetable or other oils, synthetic aliphatic acid glycerides, esters of higher fatty acids, or propylene glycol (along with conventional additives, e.g., solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives, if desired).

[0152] In various embodiments, the compositions can be formulated for inhalation using pressurized acceptable propellants, such as, for example, dichlorodifluoromethane, propane, and nitrogen.

[0153] In various embodiments, the composition can also be formulated into sustained-release microcapsules, for example, with biodegradable or non-biodegradable polymers. Non-limiting exemplary biodegradable formulations include polylactic-glycolic acid (PLGA) polymers. Non-limiting exemplary non-biodegradable formulations include polyglycerol fatty acid esters. Specific methods for producing such formulations are described, for example, in EP 1 125 584 A1.

[0154] Also provided are pharmaceutical dosage packs comprising one or more containers, each containing one or more doses of a Gal9 antagonist. In some embodiments, unit dosages are provided containing a predetermined amount of a composition comprising a Gal9 antagonist, with or without one or more additional agents. In some embodiments, such unit dosages are provided in a single-use pre-filled syringe for injection. In various embodiments, the composition contained within the unit dosage may comprise a buffer, such as saline or sucrose; a phosphate buffer; and / or be formulated within a stable and effective pH range. In addition, in some embodiments, the composition may be provided as a lyophilized powder that can be reconstituted by adding an appropriate liquid, such as sterile water. In some embodiments, the composition comprises one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. In some embodiments, the compositions of the present invention comprise heparin and / or proteoglycan.

[0155] The pharmaceutical composition is administered in an amount effective for treating or preventing a particular indication. The therapeutically effective amount typically depends on the weight of the subject to be treated, the physical condition or health of the subject, the extent of the condition to be treated, or the age of the subject to be treated.

[0156] In some embodiments, the Gal9 antagonist may be administered in an amount ranging from about 50 μg / kg to about 50 mg / kg of body weight per dose. In some embodiments, the Gal9 antagonist may be administered in an amount ranging from about 100 μg / kg to about 50 mg / kg of body weight per dose. In some embodiments, the Gal9 antagonist may be administered in an amount ranging from about 100 μg / kg to about 20 mg / kg of body weight per dose. In some embodiments, the Gal9 antagonist may be administered in an amount ranging from about 0.5 mg / kg to about 20 mg / kg of body weight per dose.

[0157] In some embodiments, the Gal9 antagonist may be administered in an amount ranging from about 10 mg to about 1,000 mg per dose. In some embodiments, the Gal9 antagonist may be administered in an amount ranging from about 20 mg to about 500 mg per dose. In some embodiments, the Gal9 antagonist may be administered in an amount ranging from about 20 mg to about 300 mg per dose. In some embodiments, the Gal9 antagonist may be administered in an amount ranging from about 20 mg to about 200 mg per dose.

[0158] The Gal9 antagonist composition can be administered to a subject when needed. In some embodiments, an effective amount of the Gal9 antagonist is administered to a subject one or more times. In various embodiments, an effective amount of the Gal9 antagonist is administered to a subject once a month, less than once a month, for example, once every two months, every three months, or every six months. In other embodiments, an effective amount of the Gal9 antagonist is administered more than once a month, for example, every two weeks, every week, twice a week, three times a week, daily, or multiple times a day. An effective amount of the Gal9 antagonist is administered to a subject at least once. In some embodiments, an effective amount of the Gal9 antagonist can be administered multiple times, including for a period of at least one month, at least six months, or at least one year. In some embodiments, the Gal9 antagonist is administered to a subject when needed to alleviate one or more symptoms of a condition.

[0159] 5. Combination therapy The Gal9 antagonist of the present invention (including any antibody and its functional fragment) can be administered to a subject in need of the Gal9 antagonist of the present invention in combination with other biologically active substances or other treatment procedures for treating diseases. For example, the Gal9 antagonist can be administered alone or together with other treatment modalities. The Gal9 antagonist can be administered before, substantially simultaneously with, or after other treatment modalities such as radiation therapy.

[0160] For the treatment of cancer, the Gal9 antagonist may be administered in combination with one or more anti-cancer agents, such as immune checkpoint inhibitors, chemotherapeutic agents, growth inhibitors, anti-angiogenic agents, or anti-neoplastic compositions.

[0161] In certain embodiments, the Gal9 antagonist specifically binds to Gal9 ("Gal9-binding antagonist"). For example, a Gal9 antagonist antibody or its antigen-binding fragment is administered together with a second antagonist, such as an immune checkpoint inhibitor (e.g., an inhibitor of the PD-1 or PD-L1 pathway), to a subject with a disease in which stimulating the immune system would be beneficial, such as cancer or an infectious disease. The two antagonists can be administered simultaneously or sequentially, for example, as described below for the combination of a Gal9 antagonist with an immuno-oncology therapeutic agent. One or more additional therapeutic agents, such as a checkpoint modulator, can be added to the treatment with a Gal9-binding antagonist to treat cancer or an infectious disease.

[0162] In certain embodiments, the Gal9 antagonist is administered to a subject, for example, a subject with cancer, simultaneously with or sequentially with another treatment. For example, the Gal9 antagonist can be administered with one or more of radiation therapy, surgery, or chemotherapy, for example, targeted chemotherapy, or immunotherapy.

[0163] Immunotherapy, for example, cancer immunotherapy, includes cancer vaccines and immunotumor therapeutic agents.Gal9 antagonists can be, for example, proteins, antibodies, antibody fragments, or small molecules that bind to Gal9.Gal9 antagonists can be antibodies or antigen-binding fragments thereof that specifically bind to Gal9.

[0164] In certain embodiments, the method of treating a subject with cancer comprises administering to the subject with cancer one or more immuno-oncology therapeutic agents, such as a Gal9 antagonist, e.g., a Gal9 antibody, and an immune checkpoint inhibitor.

[0165] Immunotherapy, such as treatment with immuno-oncology therapeutic agents, is effective in enhancing, stimulating, and / or upregulating immune responses in subjects. In one embodiment, administration of a Gal9 antagonist with an immuno-oncology therapeutic agent (such as a PD-1 inhibitor) has a synergistic effect in treating cancer, for example, in inhibiting tumor growth.

[0166] In one embodiment, the Gal9 antagonist is administered sequentially before the administration of the immuno-oncology therapeutic agent. In one embodiment, the Gal9 antagonist is administered simultaneously with the immuno-oncology therapeutic agent (such as a PD-1 inhibitor). In a further embodiment, the Gal9 antagonist is administered sequentially after the administration of the immuno-oncology therapeutic agent (such as a PD-1 inhibitor). The administration of the two agents may begin, for example, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days, 7 days, or one or more weeks apart, and the administration of the second agent may begin, for example, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days, 7 days, or one or more weeks after the administration of the first agent.

[0167] In certain embodiments, the Gal9 antagonist and the immuno-oncology therapeutic agent (e.g., a PD-1 inhibitor) are administered simultaneously, e.g., infused simultaneously into a patient over, for example, 30 or 60 minutes. The Gal9 antagonist can be co-formulated with the immuno-oncology therapeutic agent (such as a PD-1 inhibitor).

[0168] Examples of immuno-oncology therapeutic agents include small molecule drugs, antibodies or fragments thereof, or other biomolecules or small molecules. Examples of biological immuno-oncology therapeutic agents include, but are not limited to, antibodies, antibody fragments, vaccines, and cytokines. In one embodiment, the antibody is a monoclonal antibody. In a specific embodiment, the monoclonal antibody is a humanized or human antibody.

[0169] In one embodiment, the immuno-oncology therapeutic agent is (i) an agonist of a stimulatory (including costimulatory) molecule (e.g., a receptor or ligand) on an immune cell, e.g., a T cell, or (ii) an antagonist of an inhibitory (including co-inhibitory) molecule (e.g., a receptor or ligand), both of which result in the amplification of antigen-specific T cell responses. In a specific embodiment, the immuno-oncology therapeutic agent is (i) an agonist of a stimulatory (including co-stimulatory) molecule (e.g., a receptor or ligand) on a cell involved in innate immunity, e.g., a NK cell, or (ii) an antagonist of an inhibitory (including co-inhibitory) molecule (e.g., a receptor or ligand), and the immuno-oncology therapeutic agent enhances innate immunity. Such immuno-oncology therapeutic agents are often referred to as immune checkpoint modulators, e.g., immune checkpoint inhibitors or immune checkpoint stimulators.

[0170] In certain embodiments, the immuno-oncology therapeutic agent may be an agent that targets (or specifically binds to) a member of the B7 family of membrane-bound ligands, including B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5, and B7-H6, or a costimulatory or co-inhibitory receptor that specifically binds to a member of the B7 family. The immuno-oncology therapeutic agent may be an agent that targets a member of the TNF family of membrane-bound ligands, or a costimulatory or co-inhibitory receptor that specifically binds to a member of the TNF receptor family. Exemplary TNF and TNFR family members that can be targeted by immuno-oncology therapeutics include CD40 and CD40L, OX-40, OX-40L, GITR, GITRL, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RA These include NK, RANKL, TWEAKR / Fnl4, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTfiR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin alpha / TNP beta, TNFR2, TNFα, LTfiR, lymphotoxin alpha 1 beta 2, FAS, FASL, RELT, DR6, TROY, and NGFR. Immuno-oncology therapeutic agents that can be used in combination with Gal9 antagonist agents to treat cancer can include agents, such as antibodies, that target B7 family members, B7 receptor family members, TNF family members, or TNFR family members, such as those listed above.

[0171] In one embodiment, the Gal9 antagonist is selected from the group consisting of: (i) proteins that inhibit T cell activation, such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM3, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PDIH, LAIR1, TIM-1, TIM-4, and PSGL-1; and (ii) an antagonist of a protein (e.g., an immune checkpoint inhibitor), and one or more of an agonist of a protein that stimulates T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, CD40L, DR3, and CD28H.

[0172] In one embodiment, the immuno-oncology therapeutic agent is an agent that inhibits (i.e., is an antagonist of) cytokines that inhibit T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF, and other immunosuppressive cytokines) or is an agonist of cytokines that stimulate T cell activation and stimulate the immune response, such as IL-2, IL-7, IL-12, IL-15, IL-21, and IFNα (e.g., the cytokines themselves).

[0173] Other agents that can be combined with Gal9 antagonists to stimulate the immune system, for example, for the treatment of cancer and infectious diseases, include antagonists of inhibitory receptors on NK cells, or agonists that activate receptors on NK cells. For example, anti-Gal9 antagonists can be combined with antagonists of KIR.

[0174] Still other agents for combination therapy include agents that inhibit or deplete macrophages or monocytes, including CSF-IR antagonists such as CSF-IR antagonist antibodies, including, but not limited to, RG7155 (WO 11 / 70024, WO 11 / 107553, WO 11 / 131407, WO 13 / 87699, WO 13 / 119716, WO 13 / 132044) or FPA008 (WO 11 / 140249; WO 13 / 169264; WO 14 / 036357).

[0175] Immuno-oncology therapeutic agents also include agents that inhibit TGF-β signaling.

[0176] Additional agents that can be combined with Gal9 antagonists include agents that enhance tumor antigen presentation, such as dendritic cell vaccines, GM-CSF-secreting cellular vaccines, CpG oligonucleotides, and imiquimod, or therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines).

[0177] Still other therapies that can be combined with Gal9 antagonists include therapies that deplete or block Treg cells, such as agents that specifically bind to CD25.

[0178] Another therapy that can be combined with Gal9 antagonists is one that inhibits metabolic enzymes such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase.

[0179] Another class of agents that can be used includes agents that inhibit the formation of adenosine or that inhibit the adenosine A2A receptor.

[0180] Other therapies that can be combined with Gal9 antagonists to treat cancer include therapies that reverse / prevent T cell anergy or exhaustion, and therapies that trigger innate immune activation and / or inflammation at the tumor site.

[0181] Gal9 antagonists can be combined with multiple immuno-oncology therapeutics (such as immune checkpoint inhibitors), e.g., combinatorial approaches that target multiple components of the immune pathway, such as one or more of the following: therapies that enhance tumor antigen presentation (e.g., dendritic cell vaccines, GM-CSF-secreting cellular vaccines, CpG oligonucleotides, imiquimod); therapies that inhibit negative immune regulation, e.g., by inhibiting the CTLA-4 and / or PD1 / PD-L1 / PD-L2 pathways and / or by depleting or blocking Tregs or other immune suppressive cells; therapies that stimulate positive immune regulation, e.g., by agonists that stimulate the CD-137, OX-40, and / or GITR pathways and / or stimulate T cell effector function; therapies that increase the frequency of anti-tumor T cells overall (e.g., cytotoxic T cells, immune deficiency syndrome (COVID-19), immune deficiency syndrome (COVID-19), immune deficiency syndrome (COVID-19), immune deficiency syndrome (COVID-19), immune deficiency syndrome (COVID-19), immune deficiency syndrome (COVID-19), immune deficiency syndrome (COVID-19), immune deficiency syndrome (COVID-19), immune deficiency syndrome (COVID-19), immune deficiency syndrome (UNESCO ... therapies that physically augment T cells; therapies that deplete or inhibit Tregs, e.g., Tregs in tumors, using, for example, CD25 antagonists (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion; therapies that affect the function of suppressor myeloid cells in tumors; therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines); adoptive T cell transfer or NK cell transfer (CAR-T therapy) including genetically modified cells, e.g., cells modified with chimeric antigen receptors; therapies that inhibit metabolic enzymes such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase; therapies that reverse / prevent T cell anergy or exhaustion; therapies that trigger innate immune activation and / or inflammation at the tumor site; administration of immunostimulatory cytokines or blocking immunosuppressive cytokines.

[0182] For example, a Gal9 antagonist can be used in conjunction with one or more agonist agents that ligate positive costimulatory receptors; one or more antagonists (blocking agents) that attenuate signaling through inhibitory receptors, such as antagonists that overcome different immunosuppressive pathways within the tumor microenvironment (e.g., blocking PD-L1 / PD-1 / PD-L2 interactions); one or more agents that systemically increase the frequency of anti-tumor immune cells, such as T cells, and deplete or inhibit Tregs (e.g., by inhibiting CD25); one or more agents that inhibit metabolic enzymes, such as IDO; one or more agents that reverse / prevent T cell anergy or exhaustion; and one or more agents that trigger innate immune activation and / or inflammation at the tumor site.

[0183] In one embodiment, a subject with a disease that can benefit from immune system stimulation, such as cancer or an infectious disease, is treated by administering to the subject a Gal9 antagonist and an immuno-oncology therapeutic agent, where the immuno-oncology therapeutic agent is a CTLA-4 antagonist, such as an antagonistic CTLA-4 antibody. Examples of suitable CTLA-4 antibodies include YERVOY (ipilimumab) or tremelimumab.

[0184] In one embodiment, a subject with a disease that can benefit from immune system stimulation, such as cancer or an infectious disease, is treated by administering a Gal9 antagonist and an immuno-oncology therapeutic agent to the subject, where the immuno-oncology therapeutic agent is a PD-1 antagonist, such as an antagonistic PD-1 antibody. Examples of suitable PD-1 antibodies include OPDIVO (nivolumab), KEYTRUDA (pembrolizumab), or MEDI-0680 (AMP-514; WO 2012 / 145493). Another immuno-oncology therapeutic agent may include pidilizumab (CT-011). Another approach to targeting the PD-1 receptor is a recombinant protein called AMP-224, which is composed of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1.

[0185] In one embodiment, a subject with a disease that can benefit from immune system stimulation, such as cancer or an infectious disease, is treated by administering to the subject a Gal9 antagonist and an immuno-oncology therapeutic agent, where the immuno-oncology therapeutic agent is a PD-L1 antagonist, such as an antagonistic PD-L1 antibody. Examples of suitable PD-L1 antibodies include MPDL3280A (RG7446; WO 2010 / 077634), durvalumab (MEDI4736), BMS-936559 (WO 2007 / 005874), MSB0010718C (WO 2013 / 79174), or rHigM12B7.

[0186] In one embodiment, a subject with a disease that can benefit from immune system stimulation, such as cancer or an infectious disease, is treated by administering a Gal9 antagonist and an immuno-oncology therapeutic agent to the subject, where the immuno-oncology therapeutic agent is a LAG-3 antagonist, such as an antagonistic LAG-3 antibody. Examples of suitable LAG3 antibodies include BMS-986016 (WO 10 / 19570, WO 14 / 08218), or IMP-731 or IMP-321 (WO 8 / 132601, WO 9 / 44273).

[0187] In one embodiment, a subject with a disease that can benefit from immune system stimulation, such as cancer or an infectious disease, is treated by administering to the subject a Gal9 antagonist and an immuno-oncology therapeutic agent, where the immuno-oncology therapeutic agent is a CD137 (4-1BB) agonist, such as an agonistic CD137 antibody. Examples of suitable CD137 antibodies include urelumab or PF-05082566 (WO 12 / 32433).

[0188] In one embodiment, a subject with a disease that can benefit from immune system stimulation, such as cancer or an infectious disease, is treated by administering a Gal9 antagonist and an immuno-oncology therapeutic agent to the subject, where the immuno-oncology therapeutic agent is a GITR agonist, such as an agonistic GITR antibody. Examples of suitable GITR antibodies include TRX-518 (WO 06 / 105021, WO 09 / 009116), MK-4166 (WO 11 / 028683), or the GITR antibodies disclosed in WO 2015 / 031667.

[0189] In one embodiment, a subject with a disease that can benefit from immune system stimulation, such as cancer or an infectious disease, is treated by administering to the subject a Gal9 antagonist and an immuno-oncology therapeutic agent, where the immuno-oncology therapeutic agent is an OX40 agonist, such as an agonistic OX40 antibody. Examples of suitable OX40 antibodies include MEDI-6383, MEDI-6469, or MOXR0916 (RG7888; WO 06 / 029879).

[0190] In one embodiment, a subject with a disease that can benefit from immune system stimulation, such as cancer or an infectious disease, is treated by administering to the subject a Gal9 antagonist and an immuno-oncology therapeutic agent, where the immuno-oncology therapeutic agent is a CD40 agonist, such as an agonistic CD40 antibody. In a specific embodiment, the immuno-oncology therapeutic agent is a CD40 antagonist, such as an antagonistic CD40 antibody. Examples of suitable CD40 antibodies include lucatumumab (HCD122), dacetuzumab (SGN-40), CP-870,893, or Chi Lob 7 / 4.

[0191] In one embodiment, a subject with a disease that can benefit from immune system stimulation, such as cancer or an infectious disease, is treated by administering to the subject a Gal9 antagonist and an immuno-oncology therapeutic agent, where the immuno-oncology therapeutic agent is a CD27 agonist, such as an agonistic CD27 antibody. Examples of suitable CD27 antibodies include valilumab (CDX-1127).

[0192] In one embodiment, a subject with a disease that can benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering to the subject a Gal9 antagonist and an immuno-oncology therapeutic agent, the immuno-oncology therapeutic agent being MGA271 (against B7H3) (WO 11 / 109400).

[0193] In one embodiment, a subject with a disease that can benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering to the subject a Gal9 antagonist and an immuno-oncology therapeutic agent, wherein the immuno-oncology therapeutic agent is a KIR antagonist such as lirilumab.

[0194] In one embodiment, a subject with a disease that can benefit from immune system stimulation, such as cancer or infectious disease, is treated by administering a Gal9 antagonist and an immuno-oncology therapeutic agent to the subject, and the immuno-oncology therapeutic agent is an IDO antagonist.Examples of suitable IDO antagonists include INCB-024360 (WO 2006 / 122150, WO 07 / 75598, WO 08 / 36653, WO 08 / 36642), indoximod, NLG-919 (WO 9 / 73620, WO 9 / 1156652, WO 11 / 56652, WO 12 / 142237) or F001287.

[0195] In one embodiment, a subject with a disease that can benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering to the subject a Gal9 antagonist and an immuno-oncology therapeutic agent, where the immuno-oncology therapeutic agent is a Toll-like receptor agonist, such as a TLR2 / 4 agonist (e.g., Bacillus Calmette-Guerin); a TLR7 agonist (e.g., hirutonol or imiquimod); a TLR7 / 8 agonist (e.g., resiquimod); or a TLR9 agonist (e.g., CpG7909).

[0196] In one embodiment, a subject having a disease that can benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering to the subject a Gal9 antagonist and an immuno-oncology therapeutic agent, wherein the immuno-oncology therapeutic agent is a TGF-β inhibitor, e.g., GC1008, LY2157299, TEW7197, or IMC-TR1.

[0197] 6. Exemplary Gal9 Antagonists In some embodiments, the Gal9 antagonist is a Gal9 antibody. In some embodiments, the Gal9 antagonist for treating cancer can be a non-antibody protein, such as soluble Gal9 or a portion thereof (e.g., ECD), which inhibits the interaction of Gal9 with its ligand, and optionally further comprises a fusion partner, in the form of a fusion molecule. In other embodiments, the antagonist can be a small molecule or a small peptide.

[0198] Gal9 antibody In some embodiments, an antibody is provided that blocks the binding of Gal9 to its ligand. In some embodiments, an antibody is provided that inhibits Gal9-mediated signal transduction. In some such embodiments, the antibody is a Gal9 antibody. In some embodiments, the Gal9 antibody inhibits the binding of Gal9 to its ligand. In some embodiments, the Gal9 antibody inhibits Gal9-mediated signal transduction.

[0199] In some embodiments, the Gal9 antibodies of the invention have a dissociation constant (K d ) is ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 In certain embodiments, the Gal9 antibody has a dissociation constant (K M ) for Gal9, e.g., for human Gal9. d) is ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 M).

[0200] In some embodiments, a Gal9 antibody having any of the characteristics provided herein inhibits at least 25%, 50%, 75%, 80%, 90%, or 100% of Gal9 signaling.

[0201] In some embodiments, the antibody binds to Gal9 from multiple species, for example, in some embodiments, the antibody binds to human Gal9 and also binds to Gal9 from at least one non-human mammal selected from mouse, rat, dog, guinea pig, and cynomolgus monkey.

[0202] In some embodiments, multispecific antibodies are provided. In some embodiments, bispecific antibodies are provided. Non-limiting exemplary bispecific antibodies include antibodies comprising a first arm comprising a heavy chain / light chain combination that binds to a first antigen and a second arm comprising a heavy chain / light chain combination that binds to a second antigen. Further non-limiting exemplary multispecific antibodies are dual variable domain antibodies. In some embodiments, the bispecific antibody comprises a first arm that inhibits Gal9 binding and a second arm that stimulates T cells, for example, by binding to CD3. In some embodiments, the first arm binds to Gal9.

[0203] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof (including humanized monoclonal antibody or antigen-binding fragment thereof) of the present invention contains one or more point mutations in its amino acid sequence designed to improve the antibody's developability. For example, Raybould et al. (Five computational developability guidelines for therapeutic antibody profiling, PNAS 116(10):4025-4030, 2019) described the Therapeutic Antibody Profiler (TAP), a computational tool that builds downloadable homology models of variable domain sequences, tests them against five developability guidelines, and reports potential sequence liabilities and canonical forms. The authors further provide freely available TAP at opig.stats.ox.ac.uk / webapps / sabdab-sabpred / TAP.php.

[0204] Beyond achieving the desired affinity for the antigen, there are many barriers to developing therapeutic mAbs. These barriers include inherent immunogenicity, chemical and conformational instability, self-association, high viscosity, multispecificity, and poor expression. For example, high levels of hydrophobicity, particularly in the highly variable complementarity-determining regions (CDRs), have been repeatedly linked to aggregation, viscosity, and multispecificity. Net charge asymmetry in the heavy and light chain variable domains also correlates with self-association and viscosity at high concentrations. Patches of positive and negative charges in the CDRs are associated with high clearance rates and low expression levels. Product heterogeneity (e.g., due to oxidation, isomerization, or glycosylation) is often attributed to specific sequence motifs that are prone to post- or co-translational modifications. Computational tools are available to facilitate the identification of sequence barriers. Also, Warszawski et al. (Optimizing antibody affinity and stability by the automated design of the variable light-heavy chain interfaces. PLoS Comput Biol 15(8):e1007207. https: / / doi.org / 10.1371 / journal.pcbi.1007207) described a method for optimizing antibody affinity and stability by automated design of variable light-variable heavy chain interfaces. Further methods are available for identifying potential developability problems of candidate antibodies, and in preferred embodiments of the invention, such problems can be addressed by introducing one or more point mutations into the candidate antibody via conventional methods, resulting in an optimized therapeutic antibody of the invention.

[0205] The sequences of certain representative antibodies (including the light chain (LC) and heavy chain (HC) variable regions, CDR regions, and framework regions (FR)) are listed below. [ka]

[0206] For all antibody heavy chain sequences, framework region sequences HFR1 to HFR4 are defined by the VH-CDR sequences. For example, HFR1 is the HCVR sequence located on the N-terminal side of VH-CDR1. HFR2 is the HCVR sequence located between VH-CDR1 and VH-CDR2. HFR3 is the HCVR sequence located between VH-CDR2 and VH-CDR3. HFR4 is the HCVR sequence located at the most C-terminal side.

[0207] Similarly, for all antibody light chain sequences, the framework region sequences LFR1 to LFR4 are defined by the VL-CDR sequences. For example, LFR1 is the LCVR sequence located on the N-terminal side of VL-CDR1. LFR2 is the LCVR sequence located between VL-CDR1 and VL-CDR2. LFR3 is the LCVR sequence located between VL-CDR2 and VL-CDR3. LFR4 is the C-terminalmost sequence of the LCVR.

[0208] The HFR1 to HFR4 sequences of HFB9-1hz1-hG1AA are SEQ ID NOs: 1, 3, 5, and 7. The LFR1 to LFR4 sequences of HFB9-1hz1-hG1AA are SEQ ID NOs: 9, 11, 13, and 15. [ka]

[0209] The HFR1 to HFR4 sequences of HFB9-1hz2-hG1AA are SEQ ID NOs: 17, 19, 21, and 23. The LFR1 to LFR4 sequences of HFB9-1hz2-hG1AA are SEQ ID NOs: 25, 27, 29, and 31. [ka] [ka]

[0210] The HFR1 to HFR4 sequences of HFB9-1hz3-hG1AA are SEQ ID NOs: 33, 35, 37, and 39. The LFR1 to LFR4 sequences of HFB9-1hz3-hG1AA are SEQ ID NOs: 41, 43, 45, and 47. [ka]

[0211] The HFR1 to HFR4 sequences of HFB9-1hz4-hG1AA are SEQ ID NOs: 49, 51, 53, and 55. The LFR1 to LFR4 sequences of HFB9-1hz4-hG1AA are SEQ ID NOs: 57, 59, 61, and 63. [ka] [ka]

[0212] The HFR1 to HFR4 sequences of HFB9-2hz11-hG1AA are SEQ ID NOs: 65, 67, 69, and 71. The LFR1 to LFR4 sequences of HFB9-2hz11-hG1AA are SEQ ID NOs: 73, 75, 77, and 79. [ka]

[0213] The HFR1 to HFR4 sequences of HFB9-2hz12-hG1AA are SEQ ID NOs: 81, 83, 85, and 87. The LFR1 to LFR4 sequences of HFB9-2hz12-hG1AA are SEQ ID NOs: 89, 91, 93, and 95. [ka] [ka]

[0214] The HFR1 to HFR4 sequences of HFB9-2hz13-hG1AA are SEQ ID NOs: 97, 99, 101, and 103. The LFR1 to LFR4 sequences of HFB9-2hz13-hG1AA are SEQ ID NOs: 105, 107, 109, and 111. [ka]

[0215] The HFR1 to HFR4 sequences of HFB9-2hz14-hG1AA are SEQ ID NOs: 113, 115, 117, and 119. The LFR1 to LFR4 sequences of HFB9-2hz14-hG1AA are SEQ ID NOs: 121, 123, 125, and 127.

[0216] 7. Humanized antibodies In some embodiments, the Gal9 antibody is a humanized antibody. Humanized antibodies are useful as therapeutic molecules because they reduce or eliminate immune responses to antibody therapeutics and human immune responses to non-human antibodies (such as human anti-mouse antibody (HAMA) responses) that can result in reduced efficacy of the therapeutic.

[0217] Antibody can be humanized by any standard method.Non-limiting exemplary methods of humanization include, for example, the methods described in U.S. Patent No. 5,530,101; U.S. Patent No. 5,585,089; U.S. Patent No. 5,693,761; U.S. Patent No. 5,693,762; U.S. Patent No. 6,180,370; Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-27 (1988); Verhoeyen et al., Science 239:1534-36 (1988); and U.S. Patent Application Publication No. 2009 / 0136500.All are incorporated by reference.

[0218] A humanized antibody is an antibody in which at least one amino acid in a framework region of a non-human variable region is substituted with an amino acid from the corresponding position in a human framework region, hi some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least 12, at least 15, or at least 20 amino acids in the framework region of the non-human variable region are substituted with amino acids from one or more corresponding positions in one or more human framework regions.

[0219] In some embodiments, the corresponding human amino acids used for substitution are derived from framework regions of different human immunoglobulin genes. That is, in some such embodiments, one or more of the non-human amino acids may be substituted with corresponding amino acids from the human framework region of a first human antibody or encoded by a first human immunoglobulin gene, one or more of the non-human amino acids may be substituted with corresponding amino acids from the human framework region of a second human antibody or encoded by a second human immunoglobulin gene, one or more of the non-human amino acids may be substituted with corresponding amino acids from the human framework region of a third human antibody or encoded by a third human immunoglobulin gene, etc. Furthermore, in some embodiments, all of the corresponding human amino acids used for substitution within a single framework region, e.g., FR2, need not be derived from the same human framework. However, in some embodiments, all of the corresponding human amino acids used for substitution are derived from the same human antibody or encoded by the same human immunoglobulin gene.

[0220] In some aspects, antibodies are humanized by replacing one or more entire framework regions with corresponding human framework regions. In some embodiments, the human framework region with the highest level of homology to the non-human framework region to be replaced is selected. In some embodiments, such humanized antibodies are CDR-grafted antibodies.

[0221] In some aspects, after CDR grafting, one or more framework amino acids are returned to the corresponding amino acids in the mouse framework region. Such "back mutations" are made in some embodiments to retain one or more mouse framework amino acids that are likely to contribute to the structure of one or more CDRs, and / or that may be involved in antigen contact and / or that are likely to be involved in the overall structural integrity of the antibody. In some embodiments, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 1, or 0 back mutations are made to the framework region of the antibody after CDR grafting.

[0222] In some embodiments, a humanized antibody also comprises a human heavy chain constant region and / or a human light chain constant region.

[0223] 8. Human antibodies In some embodiments, the Gal9 antibody is a human antibody. Human antibodies can be produced by any suitable method. Non-limiting exemplary methods include producing human antibodies in transgenic mice containing human immunoglobulin loci. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551-55 (1993); Jakobovits et al., Nature 362:255-8 (1993); Onberg et al., Nature 368:856-9 (1994); and U.S. Patent Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807; 6,300,129; 6,255,458; 5,877,397; 5,874,299; and 5,545,806.

[0224] Non-limiting exemplary methods also include producing human antibodies using phage display libraries (see, e.g., Hoogenboom et al., J. Mol. Biol. 227:381-8 (1992); Marks et al., J. Mol. Biol. 222:581-97 (1991); and WO 99 / 10494).

[0225] Human antibody constant region In some embodiments, the humanized, chimeric, or human antibodies described herein comprise one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype selected from K and λ. In some embodiments, the antibodies described herein comprise a human IgG constant region, such as human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody or Fc fusion partner comprises, for example, a C237S mutation in the IgG1 constant region. In some embodiments, the antibodies described herein comprise a human IgG2 heavy chain constant region. In some such embodiments, the IgG2 constant region comprises a P331S mutation as described in U.S. Patent No. 6,900,292. In some embodiments, the antibodies described herein comprise a human IgG4 heavy chain constant region. In some such embodiments, the antibodies described herein comprise a S241P mutation in the human IgG4 constant region. See, for example, Angal et al., Mol. Immunol. 30(1):105-108 (1993). In some embodiments, the antibodies described herein comprise a human IgG4 constant region and a human kappa light chain.

[0226] The choice of heavy chain constant region can determine whether an antibody will have effector functions in vivo. Such effector functions, in some embodiments, can include antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), which can result in the killing of cells to which the antibody binds. Typically, antibodies containing human IgG1 or IgG3 heavy chains have effector functions.

[0227] In some embodiments, effector function is undesired. For example, in some embodiments, effector function may be undesired in the treatment of inflammatory conditions and / or autoimmune disorders. In some such embodiments, a human IgG4 or IgG2 heavy chain constant region is selected or engineered. In some embodiments, the IgG4 constant region comprises the S241P mutation.

[0228] Any of the antibodies described herein can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include antigens and / or epitopes to which the antibody binds, as well as ligands that bind to the antibody constant region. For example, Protein A, Protein G, Protein A / G, or antibody affinity columns can be used to purify antibodies by binding to the constant region.

[0229] In some embodiments, hydrophobic interaction chromatography (HIC), such as a butyl or phenyl column, is also used to purify some polypeptides. Many methods of purifying polypeptides are known in the art.

[0230] Additionally, in some embodiments, the antibodies described herein are produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498:229-44 (2009); Spirin, Trends Biotechnol. 22:538-45 (2004); Endo et al., Biotechnol. Adv. 21:695-713 (2003).

[0231] 9. Nucleic acid molecules encoding Gal9 antagonists The present invention also provides nucleic acid molecules comprising polynucleotides encoding one or more chains of an antibody described herein, such as a Gal9 antibody. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding the heavy chain or the light chain of an antibody described herein. In some embodiments, the nucleic acid molecule comprises both a polynucleotide encoding the heavy chain and a polynucleotide encoding the light chain of an antibody described herein. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide encoding the heavy chain and a second nucleic acid molecule comprises a second polynucleotide encoding the light chain.

[0232] In some such embodiments, the heavy and light chains are expressed as two separate polypeptides from one nucleic acid molecule or from two separate nucleic acid molecules. In some embodiments, for example, when the antibody is an scFv, a single polynucleotide encodes a single polypeptide comprising both the heavy and light chains linked together.

[0233] In some embodiments, a polynucleotide encoding a heavy or light chain of an antibody described herein comprises a nucleotide sequence encoding a leader sequence, which, when translated, is located at the N-terminus of the heavy or light chain. As noted above, the leader sequence may be the native heavy or light chain leader sequence, or may be another heterologous leader sequence.

[0234] The nucleic acid molecule can be constructed using recombinant DNA techniques conventional in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell, such as a mammalian cell.

[0235] 10. Vector Provided herein are vectors comprising polynucleotides encoding the heavy and / or light chains of the antibodies described herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, the vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy and light chains are expressed from the vector as two separate polypeptides. In some embodiments, the heavy and light chains are expressed as part of a single polypeptide, for example, when the antibody is an scFv.

[0236] In some embodiments, a first vector comprises a polynucleotide encoding a heavy chain, and a second vector comprises a polynucleotide encoding a light chain. In some embodiments, the first vector and the second vector are transfected into host cells in similar amounts (such as similar molar amounts or similar masses). In some embodiments, a molar or mass ratio of 5:1 to 1:5 between the first vector and the second vector is transfected into host cells. In some embodiments, a mass ratio of 1:1 to 1:5 is used for the vector encoding the heavy chain and the vector encoding the light chain. In some embodiments, a mass ratio of 1:2 is used for the vector encoding the heavy chain and the vector encoding the light chain.

[0237] In some embodiments, the vector is selected to be optimized for the expression of polypeptide in CHO or CHO-derived cells or in NSO cells.Such exemplary vector is described, for example, in Running Deer et al., Biotechnol.Prog.20:880-889(2004).In some embodiments, the vector is selected for the in vivo expression of Gal9 antagonist in animals, including humans.In some such embodiments, the expression of polypeptide(s) is under the control of promoter(s) that function in tissue-specific manner.For example, liver-specific promoter is described, for example, in WO2006 / 076288.

[0238] 11.Host cells In various embodiments, the heavy and / or light chains of the antibodies described herein can be expressed in prokaryotic cells, such as bacterial cells; or eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression can be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells (including COS7 cells); 293 cells (including 293-6E cells); CHO cells (including CHO-S and DG44 cells); PER.C6® cells (Crucell); and NSO cells. In some embodiments, the heavy and / or light chains of the antibodies described herein can be expressed in yeast. See, e.g., U.S. Patent Application Publication No. 2006 / 0270045A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy and / or light chains of the Gal9 antibody. For example, in some embodiments, CHO cells produce polypeptides that have higher levels of sialylation than the same polypeptides produced in 293 cells.

[0239] Introduction of one or more nucleic acids into desired host cells can be achieved by any method, including, but not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc., non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press (2001). Nucleic acids may be transiently or stably transfected into desired host cells according to any suitable method.

[0240] In some embodiments, one or more polypeptides may be produced in vivo in an animal that has been engineered or transfected with one or more nucleic acid molecules encoding the polypeptides, according to any suitable method. [Example]

[0241] Example 1 Humanized anti-Gal9 antibodies have high binding affinity to human and mouse galectin-9 This example demonstrates that various anti-human Gal9 humanized antibodies of the present invention have high binding affinity to both human Gal9 and mouse Gal9.

[0242] Antibody binding affinity was measured using the Octet system, commercially available from Fortebio (Creative Biolabs). According to Creative Biolabs' description, the Octet platform is based on biolayer interferometry (BLI) technology, utilizing an entire system including instruments, biosensors, reagents, and assay kits to support the evaluation of biomolecular interactions in 96-well or 384-well microplates. The Octet system uses a dip-and-read assay mode, bypassing the need for microfluidics and enabling real-time, label-free analysis of affinity and kinetics. There are three biosensor-based assay orientations that can be used to examine antibody interactions: tandem blocking, premix blocking, and classic sandwich. Compared to Biacore, the Octet dip-and-read assay allows for a longer analyte binding step and facilitates analyte rebinding to the ligand-coated sensor. Meanwhile, faster association times mean less sample is consumed, saving valuable protein.

[0243] Also, as explained by Creative Biolabs, the principle of BLI technology is based on the optical interference pattern of white light reflected from two surfaces: a layer of immobilized proteins and an internal reference layer. Binding of a ligand immobilized on the biosensor tip surface to an analyte in solution results in an increase in optical thickness at the biosensor tip, which in turn results in a shift in the interference pattern measured in nanometers. The wavelength shift (ΔΛ) is a direct measure of the change in optical thickness of the biolayer. Measuring this shift over a period of time and plotting its magnitude as a function of time yields a classic association / dissociation curve. This interaction is measured in real time, providing the ability to monitor binding specificity, association and dissociation rates, and concentration with great precision and accuracy.

[0244] Using this system, antibody affinity to recombinant human and mouse Gal9 was measured for selected humanized antibodies of the present invention. The results are summarized in the table below. The data show that the humanized antibodies tested have high affinity, in the mid- to low-nM range, for both human and mouse Gal9 proteins / antigens. [Table 2]

[0245] Sequence alignment of selected humanized antibodies of the present invention was performed using standard sequence alignment software. The results are shown in Figures 1 and 2. In particular, in Figure 1, the V H Area and V L The regions were aligned to identify the original V region of the human-mouse chimeric antibody HFB9-1. H Area and V L The amino acid residue changes compared to the heavy and light chain variable regions are shown. Humanization primarily altered the amino acid sequences within the framework regions of the heavy and light chain variable regions (HCVR and LCVR). However, there were also extensive changes within the heavy chain CDR2 sequence (see Figure 1).

[0246] Similarly, in Figure 2, the V of the six humanized antibodies H Area and V L The regions were aligned to identify the original V region of the human-mouse chimeric antibody HFB9-2. H Area and V L The amino acid residue changes compared to the heavy and light chain variable regions are shown. Humanization primarily altered the amino acid sequences within the framework regions of the heavy and light chain variable regions (HCVR and LCVR). However, there were also extensive changes within the heavy chain CDR2 sequence, and one residue change within the heavy chain CDR1 sequence for one humanized antibody (see Figure 2).

[0247] Example 2 Anti-Gal9 antibodies exhibit subnanomolar (nM) affinity for Gal9 This experiment demonstrates that the humanized antibodies of the present invention exhibit very high (subnanomolar) affinity for recombinant human Gal9 and cross-react with recombinant mouse and monkey Gal9 (data not shown). The EC50 values for each humanized antibody tested were determined at increasing concentrations of each antibody, and the results are summarized in the tables of Figure 3 for binding to recombinant human Gal9 and Figure 4 for binding to recombinant mouse Gal9.

[0248] It is clear that all three humanized variants of HFB9-1, except for the 1hz4 antibody, exhibited sub-nM affinity for both human and mouse Gal-9 (see Figures 3 and 4). On the other hand, five of the six humanized HFB9-2 antibodies (excluding 2hz12) exhibited sub-nM affinity for human Gal9, but only four of the five (excluding 2h14) maintained sub-nM affinity for mouse Gal9.

[0249] Strong cross-reactivity to the monkey ortholog Gal9 was also observed (data not shown).

[0250] The sequences of these representative antibodies (including the light chain (LC) and heavy chain (HC) variable regions, CDR regions, and framework regions (FR)) are listed below. [ka]

[0251] For all antibody heavy chain sequences, framework region sequences HFR1 to HFR4 are defined by the VH-CDR sequences. For example, HFR1 is the HCVR sequence located on the N-terminal side of VH-CDR1. HFR2 is the HCVR sequence located between VH-CDR1 and VH-CDR2. HFR3 is the HCVR sequence located between VH-CDR2 and VH-CDR3. HFR4 is the HCVR sequence located at the most C-terminal side.

[0252] Similarly, for all antibody light chain sequences, the framework region sequences LFR1 to LFR4 are defined by the VL-CDR sequences. For example, LFR1 is the LCVR sequence located on the N-terminal side of VL-CDR1. LFR2 is the LCVR sequence located between VL-CDR1 and VL-CDR2. LFR3 is the LCVR sequence located between VL-CDR2 and VL-CDR3. LFR4 is the C-terminalmost sequence of the LCVR.

[0253] The HFR1 to HFR4 sequences of HFB9-1hz1-hG1AA are SEQ ID NOs: 1, 3, 5, and 7. The LFR1 to LFR4 sequences of HFB9-1hz1-hG1AA are SEQ ID NOs: 9, 11, 13, and 15. [ka]

[0254] The HFR1 to HFR4 sequences of HFB9-1hz2-hG1AA are SEQ ID NOs: 17, 19, 21, and 23. The LFR1 to LFR4 sequences of HFB9-1hz2-hG1AA are SEQ ID NOs: 25, 27, 29, and 31. [ka]

[0255] The HFR1 to HFR4 sequences of HFB9-1hz3-hG1AA are SEQ ID NOs: 33, 35, 37, and 39. The LFR1 to LFR4 sequences of HFB9-1hz3-hG1AA are SEQ ID NOs: 41, 43, 45, and 47. [ka]

[0256] The HFR1 to HFR4 sequences of HFB9-1hz4-hG1AA are SEQ ID NOs: 49, 51, 53, and 55. The LFR1 to LFR4 sequences of HFB9-1hz4-hG1AA are SEQ ID NOs: 57, 59, 61, and 63. [ka]

[0257] The HFR1 to HFR4 sequences of HFB9-2hz11-hG1AA are SEQ ID NOs: 65, 67, 69, and 71. The LFR1 to LFR4 sequences of HFB9-2hz11-hG1AA are SEQ ID NOs: 73, 75, 77, and 79. [ka]

[0258] The HFR1 to HFR4 sequences of HFB9-2hz12-hG1AA are SEQ ID NOs: 81, 83, 85, and 87. The LFR1 to LFR4 sequences of HFB9-2hz12-hG1AA are SEQ ID NOs: 89, 91, 93, and 95. [ka]

[0259] The HFR1 to HFR4 sequences of HFB9-2hz13-hG1AA are SEQ ID NOs: 97, 99, 101, and 103. The LFR1 to LFR4 sequences of HFB9-2hz13-hG1AA are SEQ ID NOs: 105, 107, 109, and 111. [ka]

[0260] The HFR1 to HFR4 sequences of HFB9-2hz14-hG1AA are SEQ ID NOs: 113, 115, 117, and 119. The LFR1 to LFR4 sequences of HFB9-2hz14-hG1AA are SEQ ID NOs: 121, 123, 125, and 127.

[0261] Example 3 Binding by anti-Gal9 antibodies blocks Gal9 binding to receptors TIM3 and CD44 This experiment demonstrates that the anti-Gal9 antibodies of the present invention block Gal9 binding to its receptors TIM3 and CD44, and are therefore capable of antagonizing downstream signaling from Gal9.

[0262] The data in Figure 5 clearly demonstrate that the humanized antibodies of the present invention block Gal9 binding to both the TIM3 and CD44 receptors in a dose-dependent manner.

[0263] Example 4 Anti-Gal9 antibody neutralizes Gal9-induced Th1 apoptosis YANG et al. (INFLAMMATION 40(3):1062-1071, 2017) reported that elevated galectin-9 suppresses Th1 effector function and inhibits activated CD4+ in osteoarthritis. + reported that Gal9 induces apoptosis of T cells. This experiment demonstrates that the anti-Gal9 antibody of the present invention neutralizes Gal9-induced Th1 apoptosis.

[0264] Specifically, human PBMCs were isolated from healthy donors and incubated with increasing antibody concentrations in the presence of Gal9 at an amount that induces T cell apoptosis in the absence of the antibody. The percentage of apoptotic CD4+ T cells was determined across a range of antibody concentrations to determine the EC50 value of the antibody. The results are summarized in Figure 6. The data clearly demonstrate that treatment of human PBMCs from healthy donors with the antibody of the invention prevents Gal9-induced Th1 cell apoptosis in a dose-dependent manner.

[0265] Example 5 Anti-Gal9 antibody suppresses Gal9-induced Treg proliferation As mentioned above, galectin-9 is directly expressed by Tregs, and their activation is associated with increased expression of Gal9. This experiment demonstrates that inhibition of galectin-9 by the anti-Gal9 antibody of the present invention suppresses Treg proliferation.

[0266] Specifically, human PBMCs were isolated from healthy donors and incubated with increasing concentrations of antibodies of the present invention in the presence of Gal9 at an amount that stimulates Treg proliferation in the absence of the antibodies of the present invention. + Ki67 HIGH The percentage of T cells was determined across a range of antibody concentrations to determine the EC50 values of the antibodies. The results are summarized in Figure 7. The data clearly show that treatment of human PBMCs from healthy donors with the antibodies of the invention dose-dependently suppressed Gal-9-induced Treg proliferation, in that higher antibody concentrations were associated with a lower percentage of proliferating Tregs, based on the expression of the Foxp3 and Ki67 marker genes.

[0267] Example 6 Combined treatment with anti-Gal9 and anti-PD-1 antibodies demonstrated synergistic effects in inhibiting tumor growth in vivo and prolonging survival This experiment demonstrates that the anti-Gal9 monoclonal antibody of the present invention and the anti-PD-1 antibody have a synergistic effect in inhibiting tumor growth in vivo in a xenograph mouse model.

[0268] Specifically, approximately 500,000 cancer cells were inoculated into experimental mice, and tumor masses were allowed to grow to a predetermined size. Mice were then randomized and injected intraperitoneally (ip) with one of four antibodies or antibody combinations: (1) an IgG isotype control at a dose of 10 mg / kg, (2) the anti-Gal9 antibody HFB9-2 (clone RMP1-14) at a dose of 10 mg / kg, (3) an anti-mPD-1 antibody at a dose of 10 mg / kg, or (4) a combination of an anti-mPD-1 antibody at 10 mg / kg and an anti-HFB9-2 antibody at 10 mg / kg.

[0269] The first dose of antibody for the various groups was administered on day 1, with subsequent doses administered every three days for a total of four doses for all groups with anti-mPD-1 antibodies and seven doses for all groups with anti-HFB9-2 and control antibodies. Data are shown as mean ± sem (n = 8 / group) (Figure 8).

[0270] The subject anti-Gal9 and anti-mPD-1 antibodies demonstrated that the combination treatment reduced tumor growth by 500 mm during the 7-week study period. 3 The synergistic effect of the anti-HFB9-2 and anti-mPD-1 groups on tumor growth in vivo was evident in that the two groups essentially completely suppressed tumor growth in vivo, whereas tumor growth in the control and anti-HFB9-2 groups exceeded this level as early as 2 weeks, and in the anti-mPD-1 group as early as 4 weeks.

[0271] Furthermore, with regard to survival (Figure 9), all mice in the control group died toward the end of week 3, all mice in the anti-HFB9-2 antibody group died toward the end of week 5, and only 25% (2 of 8) of the mice in the anti-mPD-1 group were tumor-free at the end of week 7. However, at the same time, in the combination therapy group, 5 of 8 mice were tumor-free and 1 mouse had a tumor of approximately 100 mm. 3 had a 75% survival rate, including having tumors of

[0272] This surprising finding strongly suggests that simultaneous inhibition of Gal-9 function and the PD-1 / PD-L1 immune checkpoints can synergistically inhibit tumor growth in vivo and prolong survival.

[0273] Example 7 Anti-Gal9 antibodies are stable To confirm that the subject humanized anti-Gal9 antibodies are stable during storage and therefore suitable for further development as therapeutics, various developability assays were performed on selected humanized antibodies.

[0274] In the first experiment, 1 to 2.75 mg / mL of the subject humanized antibodies, HFB9-1hz1-hG1AA, HFB9-1hz2-hG1AA, HFB9-1hz3-hG1AA, HFB9-2hz11-hG1AA, and HFB9-2hz13-hG1AA, were stored in PBS (pH 7.4) at 25 or 40° C., and the stability of the various antibodies was determined on days 0, 3, 7, and 14. Results (not shown) demonstrated that all antibodies tested were stable under the conditions tested.

[0275] In a second experiment, the same antibodies were tested for stability under low pH conditions (100 mM AcH, pH 3.5, 25° C.) for 0, 3, and 6 hours. Results (not shown) again demonstrated that all antibodies tested were stable under the conditions tested.

[0276] In a third experiment, the same antibodies were subjected to one, two, or three freeze-thaw cycles, and the results (not shown) again demonstrated that all antibodies tested were stable under the conditions tested.

[0277] Example 8 Galectin-9 Levels in Plasma and Serum from AML Patients To determine the levels of Gal-9 in patient plasma and serum, peripheral blood samples from AML patients were obtained from the Clinical Hematology Department of the Gustave Roussy Institute (Villejuif, France) according to a protocol approved by the Institutional Review Board. Informed consent was obtained from all patients in accordance with the Declaration of Helsinki. Patients were stratified according to the French-American-British (Fab) classification criteria. Plasma or serum from peripheral blood from AML patients was prepared according to standard procedures. For healthy donors, plasma or serum samples were obtained from commercial sources.

[0278] Galectin-9 protein levels in plasma or serum were assessed by ELISA using R&D SYSTEMS®'s "Quantikine® ELISA Human Galectin-9," and statistical analysis (unpaired two-tailed t-test) was performed using PRISM® 5 for Windows software.

[0279] As shown in Figure 10, galectin-9 protein levels in the plasma of AML patients at the time of disease diagnosis or in the relapsed / refractory stage (R / R) were significantly higher than those observed in plasma from healthy individuals. Galectin-9 protein levels in the plasma of AML patients in complete remission after chemotherapy were close to the normal physiological range.

[0280] As shown in Figure 11, galectin-9 protein levels in the plasma of AML patients with Fab M2 or Fab M3 at diagnosis were significantly lower than those observed in the plasma from AML patients with Fab M0, M1, M4, or M5. Galectin-9 protein levels in the plasma of AML patients with Fab M3 at diagnosis were within the normal physiological range.

[0281] In addition, LGALS9 mRNA expression levels were investigated in AML patients and healthy individuals. LGALS9 mRNA expression levels were extracted from the publicly available "AML_Ohsu_Nature 2018" dataset deposited by Tyner et al. (see PMID 30333627 for a complete description of the study). Data are presented in Figure 12 as normalized log 2 RPKM. The dotted lines represent the median LGALS9 levels in BM-derived MNCs from healthy individuals or AML patients, respectively.

[0282] As shown in Figure 12, the levels of galectin-9-encoding mRNA in BM-derived MNCs (all considered Fabs) from AML patients at diagnosis were higher than the mRNA levels observed in BM-derived MNCs or CD34+ cells from healthy individuals. In addition, the levels of galectin-9-encoding mRNA in BM-derived MNCs from AML patients with Fab M3 at diagnosis were higher than the levels observed in BM-derived MNCs from AML patients with Fab M0, M1, M4, or M5, or in BM-derived MNCs or CD34+ cells from healthy individuals. + This was significantly lower than that observed in cells. The present invention provides, for example, the following items. (Item 1) 1. An isolated monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof is specific for galectin-9, the monoclonal antibody comprising: (1a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 2, the HCVR CDR2 sequence of SEQ ID NO: 4, and the HCVR CDR3 sequence of SEQ ID NO: 6; and (1b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 10, the LCVR CDR2 sequence of SEQ ID NO: 12, and the LCVR CDR3 sequence of SEQ ID NO: 14; or (2a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 18, the HCVR CDR2 sequence of SEQ ID NO: 20, and the HCVR CDR3 sequence of SEQ ID NO: 22; and (2b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 26, the LCVR CDR2 sequence of SEQ ID NO: 28, and the LCVR CDR3 sequence of SEQ ID NO: 30; or (3a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 34, the HCVR CDR2 sequence of SEQ ID NO: 36, and the HCVR CDR3 sequence of SEQ ID NO: 38; and (3b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 42, the LCVR CDR2 sequence of SEQ ID NO: 44, and the LCVR CDR3 sequence of SEQ ID NO: 46; or (4a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 50, the HCVR CDR2 sequence of SEQ ID NO: 52, and the HCVR CDR3 sequence of SEQ ID NO: 54; and (4b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 58, the LCVR CDR2 sequence of SEQ ID NO: 60, and the LCVR CDR3 sequence of SEQ ID NO: 62; or (5a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 66, the HCVR CDR2 sequence of SEQ ID NO: 68, and the HCVR CDR3 sequence of SEQ ID NO: 70; and (5b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 74, the LCVR CDR2 sequence of SEQ ID NO: 76, and the LCVR CDR3 sequence of SEQ ID NO: 78; or (6a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 82, the HCVR CDR2 sequence of SEQ ID NO: 84, and the HCVR CDR3 sequence of SEQ ID NO: 86; and (6b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 90, the LCVR CDR2 sequence of SEQ ID NO: 92, and the LCVR CDR3 sequence of SEQ ID NO: 94; or (7a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 98, the HCVR CDR2 sequence of SEQ ID NO: 100, and the HCVR CDR3 sequence of SEQ ID NO: 102; and (7b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 106, the LCVR CDR2 sequence of SEQ ID NO: 108, and the LCVR CDR3 sequence of SEQ ID NO: 110; or (8a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 114, the HCVR CDR2 sequence of SEQ ID NO: 116, and the HCVR CDR3 sequence of SEQ ID NO: 118; and (8b) A light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 122, the LCVR CDR2 sequence of SEQ ID NO: 124, and the LCVR CDR3 sequence of SEQ ID NO: 128. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising: (Item 2) (1c) the antibody or antigen-binding fragment thereof of (1a) and (1b) further comprises the HFR3 sequence of SEQ ID NO: 5, and optionally further comprises the HFR1 sequence of SEQ ID NO: 1; or (2c) the antibody or antigen-binding fragment thereof of (2a) and (2b) further comprises the HFR3 sequence of SEQ ID NO: 21, and optionally further comprises the HFR1 sequence of SEQ ID NO: 17; or (3c) the antibody or antigen-binding fragment thereof of (3a) and (3b) further comprises the HFR3 sequence of SEQ ID NO: 37, and optionally further comprises the HFR1 sequence of SEQ ID NO: 33; or (4c) the antibody or antigen-binding fragment thereof of (4a) and (4b) further comprises the HFR3 sequence of SEQ ID NO: 53, and optionally further comprises the HFR1 sequence of SEQ ID NO: 49; or (5c) the antibody or antigen-binding fragment thereof of (5a) and (5b) further comprises the HFR3 sequence of SEQ ID NO: 69, and optionally further comprises the HFR1 sequence of SEQ ID NO: 65; or (6c) the antibody or antigen-binding fragment thereof of (6a) and (6b) further comprises the HFR3 sequence of SEQ ID NO: 85, and optionally further comprises the HFR1 sequence of SEQ ID NO: 81; or (7c) The antibody or antigen-binding fragment thereof of (7a) and (7b) further comprises an HFR3 sequence of SEQ ID NO: 101, and optionally further comprises an HFR1 sequence of SEQ ID NO: 97; or (8c) The antibody or antigen-binding fragment thereof of (8a) and (8b) further comprises the HFR3 sequence of SEQ ID NO: 117, and optionally further comprises the HFR1 sequence of SEQ ID NO: 113. Item 1. The isolated monoclonal antibody or antigen-binding fragment thereof according to item 1. (Item 3) (1A) the HCVR sequence is SEQ ID NO: 8; and / or (1B) the LCVR sequence is SEQ ID NO: 16, or (2A) the HCVR sequence is SEQ ID NO: 24; and / or (2B) the LCVR sequence is SEQ ID NO: 32, or (3A) the HCVR sequence is SEQ ID NO: 40; and / or (3B) the LCVR sequence is SEQ ID NO: 48, or (4A) the HCVR sequence is SEQ ID NO: 56; and / or (4B) the LCVR sequence is SEQ ID NO: 64, or (5A) the HCVR sequence is SEQ ID NO: 72; and / or (5B) the LCVR sequence is SEQ ID NO: 80, or (6A) the HCVR sequence is SEQ ID NO: 88; and / or (6B) the LCVR sequence is SEQ ID NO: 96, or (7A) the HCVR sequence is SEQ ID NO: 104; and / or (7B) the LCVR sequence is SEQ ID NO: 112, or (8A) the HCVR sequence is SEQ ID NO: 120; and / or (8B) The LCVR sequence is SEQ ID NO: 128. 3. The isolated monoclonal antibody or antigen-binding fragment thereof according to item 1 or 2. (Item 4) is a humanized antibody, and (1) the HCVR sequence of SEQ ID NO: 8 and the LCVR sequence of SEQ ID NO: 16; or (2) the HCVR sequence of SEQ ID NO: 72 and the LCVR sequence of SEQ ID NO: 80 4. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 3, comprising: (Item 5) The antigen-binding fragment thereof is Fab, Fab', F(ab') 2 、F d , single chain Fv or scFv, disulfide bond F v , V-NAR domain, IgNar, intrabody, IgGΔCH 2 , minibody, F(ab') 3 , tetrabodies, triabodies, diabodies, single domain antibodies, DVD-Ig, Fcab, mAb 2 , (scFv) 2 5. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 4, wherein the monoclonal antibody or antigen-binding fragment thereof is a Fc-Fc or scFv-Fc. (Item 6) 6. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 5, wherein the monoclonal antibody or antigen-binding fragment thereof cross-reacts with mouse Gal9. (Item 7) 7. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 6, wherein the monoclonal antibody or antigen-binding fragment thereof binds to human Gal9 with an EC50 of about 0.1 to 0.2 nM and / or binds to mouse Gal9 with an EC50 of about 0.5 to 1.0 nM. (Item 8) the monoclonal antibody or antigen-binding fragment thereof has a K of less than about 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 2 nM, or 1 nM for human Gal9. d 8. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 7, wherein the antibody or antigen-binding fragment binds to the antibody. (Item 9) 9. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of items 1 to 8, which binds to Gal9 and inhibits binding of Gal9 to a Gal9 receptor (e.g., TIM3 or CD44). (Item 10) T cells (CD4 + 10. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 9, which neutralizes Gal-9-induced Th1 apoptosis of T cells or the like. (Item 11) 11. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 10, which inhibits Gal9-induced Treg proliferation. (Item 12) 12. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of items 1 to 11, which synergizes with an immune checkpoint antagonist to inhibit tumor growth in vivo and / or prolong survival in mice bearing xenograft tumors. (Item 13) 13. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of items 1 to 12, wherein the antagonist of the immune checkpoint is an antibody or antigen-binding fragment thereof specific for PD-1 or PD-L1. (Item 14) 14. A method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 13 and an antagonist of an immune checkpoint. (Item 15) 15. The method of claim 14, wherein the immune checkpoint is the PD-1 / PD-L1 immune checkpoint, and optionally the antagonist of the immune checkpoint is an antibody or antigen-binding fragment thereof specific for PD-1 or PD-L1; a peptide inhibitor of PD-1 / PD-L1; a small molecule inhibitor of PD-L1; a macrocyclic peptide; or any combination thereof. (Item 16) 16. The method according to any one of items 14 to 15, wherein the cancer is a blood cancer (such as AML and DLBCL) or a solid tumor (such as breast cancer, head and neck cancer, lung cancer, melanoma (including uveal melanoma), colon cancer, renal cancer, ovarian cancer, liver cancer, and prostate cancer). (Item 17) 17. The method of any one of items 14 to 16, further comprising administering to said patient a chemotherapeutic agent, an anti-angiogenic agent, a growth inhibitory agent, an immuno-oncology therapeutic agent, and / or an anti-neoplastic composition. (Item 18) 14. A polynucleotide encoding the heavy or light chain or antigen-binding portion thereof as defined in any one of items 1 to 13. (Item 19) 19. The polynucleotide of item 18, which is codon-optimized for expression in human cells. (Item 20) 20. A vector, such as an expression vector (e.g., a mammalian expression vector, a yeast expression vector, an insect expression vector, or a bacterial expression vector), comprising the polynucleotide of item 18 or 19. (Item 21) 14. A method for rescuing or promoting effector T cell proliferation and / or enhancing effector T cell activity in a patient diagnosed with cancer, at risk of developing cancer or cancer recurrence, or for identifying and treating a patient with cancer, comprising administering to the patient an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 13, upon identifying the patient as having a level of galectin-9 in a sample from the patient that is higher than a reference level of galectin-9 in a healthy or control individual. (Item 22) 22. The method of claim 21, further comprising identifying the patient as having a level of galectin-9 in the sample that is higher than the reference level by comparing the level of galectin-9 in the sample to the reference level. (Item 23) 23. The method of item 21 or 22, further comprising administering to the patient an antagonist of an immune checkpoint, optionally wherein the immune checkpoint is the PD-1 / PD-L1 immune checkpoint. (Item 24) 24. The method of claim 23, wherein the antagonist of the immune checkpoint is an antibody or antigen-binding fragment thereof specific for PD-1 or PD-L1; a (non-antibody) peptide inhibitor of PD-1 / PD-L1; a small molecule inhibitor of PD-L1; a macrocyclic peptide; or any combination thereof. (Item 25) 25. The method according to any one of items 21 to 24, wherein the cancer is a blood cancer (such as AML and DLBCL) or a solid tumor (such as breast cancer, head and neck cancer, lung cancer, melanoma (including uveal melanoma), colon cancer, renal cancer, ovarian cancer, liver cancer, and prostate cancer). (Item 26) 26. The method of item 25, wherein the patient is an AML patient with Fab M0, M1, M4, or M5, or the patient is not an AML patient with Fab M2 or M3. (Item 27) 27. The method according to any one of items 21 to 26, wherein the sample is a blood sample, a plasma sample, or a serum sample. (Item 28) 1. A method for rescuing or promoting effector T cell proliferation and / or enhancing effector T cell activity in patients diagnosed with AML, who are at risk of developing or relapsing from AML, or for identifying and treating patients with AML, comprising determining whether levels of galectin-9-encoding mRNA in bone marrow (BM)-derived mononuclear cell (MNC) samples from said patients are higher than levels of galectin-9-encoding mRNA in BM-derived MNC or CD34 in healthy or control individuals. + 14. A method comprising administering to a patient an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 13, upon identifying the patient as having a statistically significantly higher or lower than a reference level in a cell. (Item 29) 29. The method of claim 28, wherein (1) the level of galectin-9-encoding mRNA in the BM-derived MNC sample from the patient is significantly higher than the reference level when the patient is an AML patient with Fab M0, M1, M2, M4, or M5, or (2) the level of galectin-9-encoding mRNA in the BM-derived MNC sample from the patient is significantly lower than the reference level when the patient is an AML patient with Fab M3. (Item 30) An antibody or antigen-binding portion thereof directed against or specific for galectin-9 for use in the treatment of cancer, wherein the antibody or antigen-binding portion thereof rescues effector T cell proliferation and / or enhances effector T cell activity, and optionally the effector T cell is a Th1 cell. (Item 31) 14. A method for rescuing or promoting effector T cell proliferation and / or enhancing effector T cell activity, comprising contacting the effector T cells with the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 13, wherein optionally the effector T cells are Th1 cells. (Item 32) 14. A method for inducing or promoting immunological memory in a subject, comprising administering to the subject an effective amount of a composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of items 1 to 13, wherein the immunological memory is effective to inhibit or reduce tumor progression or recurrence, or cancer cell proliferation in the subject.

Claims

1. 1. An isolated monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof is specific for galectin-9, the monoclonal antibody comprising: (5a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 66, the HCVR CDR2 sequence of SEQ ID NO: 68, and the HCVR CDR3 sequence of SEQ ID NO: 70; and (5b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 74, the LCVR CDR2 sequence of SEQ ID NO: 76, and the LCVR CDR3 sequence of SEQ ID NO:

78. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising: (5c) The antibody or antigen-binding fragment thereof of (5a) and (5b) further comprises an HFR3 sequence of SEQ ID NO:

69. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1. (5A) the heavy chain variable region (HCVR) sequence is SEQ ID NO: 72; and / or (5B) The light chain variable region (LCVR) sequence is SEQ ID NO:

80.

3. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2.

4. The antigen-binding fragment thereof may be Fab, Fab', F(ab') ２ , single chain Fv or scFv, disulfide-linked F ｖ , intrabody, minibody, F(ab') ３ , tetrabodies, triabodies, diabodies, (scFv) ２ 4. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which is a scFv-Fc or scFv-Fc.

5. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the monoclonal antibody or antigen-binding fragment thereof cross-reacts with mouse Gal9.

6. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the monoclonal antibody or antigen-binding fragment thereof binds to human Gal9 with an EC50 of 0.1 to 0.2 nM and / or binds to mouse Gal9 with an EC50 of 0.5 to 1.0 nM.

7. The monoclonal antibody or antigen-binding fragment thereof has a K of less than 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 2 nM, or 1 nM for human Gal9. ｄ The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which binds to

8. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which binds to Gal9 and inhibits binding of Gal9 to a Gal9 receptor.

9. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which neutralizes Gal-9-induced Th1 apoptosis of T cells.

10. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, which inhibits Gal9-induced Treg proliferation.

11. 11. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, which synergizes with an antagonist of an immune checkpoint to inhibit tumor growth in vivo and / or prolong survival in mice bearing xenograft tumors, wherein the antagonist of the immune checkpoint is an antibody or antigen-binding fragment thereof specific for PD-1.

12. 12. A combination comprising the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 and an antagonist of an immune checkpoint, for treating cancer in a patient in need thereof, wherein said antagonist of said immune checkpoint is an antibody or antigen-binding fragment thereof specific for PD-1.

13. The combination of claim 12, wherein the cancer is a blood cancer or a solid tumor.

14. 12. A composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 11 for use in a method of promoting effector T cell proliferation and / or enhancing effector T cell activity in a patient diagnosed with cancer, at risk of developing cancer or cancer recurrence, or for use in a method of identifying and treating a patient with cancer, the method comprising administering the composition to the patient upon identifying the patient as having a level of galectin-9 in a sample from the patient that is higher than a reference level of galectin-9 in a healthy individual.

15. 15. The composition of claim 14, wherein the method further comprises identifying the patient as having a level of galectin-9 in the sample that is higher than the reference level by comparing the level of galectin-9 in the sample with the reference level.

16. 16. The composition of claim 14 or 15, wherein the method further comprises administering to the patient an antagonist of an immune checkpoint, wherein the antagonist of the immune checkpoint is an antibody or antigen-binding fragment thereof specific for PD-1.

17. The composition of any one of claims 14 to 16, wherein the cancer is a blood cancer or a solid tumor.

18. 18. The composition of claim 17, wherein the patient is an FAB M0, M1, M4, or M5 AML patient, or the patient is not an FAB M2 or M3 AML patient.

19. The composition of any one of claims 14 to 18, wherein the sample is a blood sample, a plasma sample, or a serum sample.

20. 12. A composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 11 for use in a method of promoting effector T cell proliferation and / or enhancing effector T cell activity in a patient diagnosed with AML, who is at risk of developing or relapsing from AML, or for use in a method of identifying and treating patients with AML, wherein the level of galectin-9-encoding mRNA in a bone marrow (BM)-derived mononuclear cell (MNC) sample from said patient is higher than that of BM-derived MNC or CD34 in a healthy individual. ＋ Upon identifying the patient as having a statistically significantly higher or lower level in the cells than the reference level, administering the composition to the patient.

21. The composition of claim 20, wherein the method comprises: (1) identifying the patient as an AML patient with FAB M0, M1, M2, M4, or M5 if the level of galectin-9-encoding mRNA in the BM-derived MNC sample from the patient is significantly higher than the reference level; or (2) identifying the patient as an AML patient with FAB M3 if the level of galectin-9-encoding mRNA in the BM-derived MNC sample from the patient is significantly lower than the reference level.

22. 1. A composition comprising an antibody or antigen-binding portion thereof directed against or specific for galectin-9 for use in the treatment of cancer, wherein the antibody or antigen-binding portion thereof promotes effector T cell proliferation and / or enhances effector T cell activity; The antibody comprises: (5a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 66, the HCVR CDR2 sequence of SEQ ID NO: 68, and the HCVR CDR3 sequence of SEQ ID NO: 70; and (5b) A composition comprising a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 74, the LCVR CDR2 sequence of SEQ ID NO: 76, and the LCVR CDR3 sequence of SEQ ID NO:

78.

23. 12. A composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 11 for use in a method of promoting effector T cell proliferation and / or enhancing effector T cell activity, the method comprising contacting the effector T cell with the isolated monoclonal antibody or antigen-binding fragment thereof.

24. 12. A composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 11 for inducing or promoting immunological memory in a subject, wherein the immunological memory is effective to inhibit or reduce tumor progression or recurrence, or cancer cell proliferation in the subject.

25. 12. A composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 11 for treating cancer in a patient in need thereof, wherein the composition is administered in combination with an antagonist of an immune checkpoint, wherein the antagonist of the immune checkpoint is an antibody or antigen-binding fragment thereof specific for PD-1.

26. The composition of claim 25, wherein the cancer is a blood cancer or a solid tumor.

27. 1. A composition comprising an isolated monoclonal antibody or antigen-binding fragment thereof for treating cancer in a patient in need thereof, wherein the monoclonal antibody or antigen-binding fragment thereof is specific for Galectin-9, and wherein the monoclonal antibody comprises: (5a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 66, the HCVR CDR2 sequence of SEQ ID NO: 68, and the HCVR CDR3 sequence of SEQ ID NO: 70; and (5b) a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 74, the LCVR CDR2 sequence of SEQ ID NO: 76, and the LCVR CDR3 sequence of SEQ ID NO:

78. A composition comprising:

28. (5c) The antibody or antigen-binding fragment thereof of (5a) and (5b) further comprises an HFR3 sequence of SEQ ID NO:

69.

28. The composition of claim 27.

29. (5A) the heavy chain variable region (HCVR) sequence is SEQ ID NO: 72; and / or (5B) The light chain variable region (LCVR) sequence is SEQ ID NO:

80.

29. The composition of claim 27 or 28.

30. A pharmaceutical composition comprising the composition of any one of claims 27 to 29 and a pharmaceutically acceptable carrier.

31. 1. A combination comprising an isolated monoclonal antibody or antigen-binding fragment thereof and an antagonist of an immune checkpoint for treating cancer in a patient in need thereof, wherein the antagonist of the immune checkpoint is an antibody or antigen-binding fragment thereof specific for PD-1, and the monoclonal antibody or antigen-binding fragment thereof is specific for Galectin-9, wherein the monoclonal antibody is: (5a) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 66, the HCVR CDR2 sequence of SEQ ID NO: 68, and the HCVR CDR3 sequence of SEQ ID NO: 70; and (5b) LCVR CDR1 sequence of SEQ ID NO: 74, LCVR CDR2 sequence of SEQ ID NO: 76 and a light chain variable region (LCVR) comprising the LCVR CDR3 sequence of SEQ ID NO:

78. A combination comprising:

32. (5c) The antibody or antigen-binding fragment thereof of (5a) and (5b) further comprises an HFR3 sequence of SEQ ID NO:

69.

32. The combination of claim 31.

33. (5A) the heavy chain variable region (HCVR) sequence is SEQ ID NO: 72; and / or (5B) The light chain variable region (LCVR) sequence is SEQ ID NO:

80.

33. A combination according to claim 31 or 32.

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