Anti-galectin-9 antibody and its uses

Abstract

The present invention provides a humanized monoclonal antibody against Gal9, and methods of using the antibody to treat cancer, including combination therapies with antagonists of the PD-1 / PD-L1 immune checkpoint.

Description

[0001] Citation of Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 957,910, filed on January 7, 2020, the entire contents of which, including all sequences and drawings, are incorporated herein by reference. Background of the Invention

[0004] 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 members in humans. Galectin-9 is a soluble 34-39 kDa protein without a leader peptide but is still secreted by a non-classical mechanism. It preferentially interacts with β-galactoside residues of glycoproteins and glycolipids. In humans, galectin-9 exists in three isoforms (long, medium, and short).

[0005] Galectin-9 is one of the most studied ligands of HAVCR2 (TIM-3) and is expressed on various hematological malignancies such as CLL, MDS, Hodgkin and non-Hodgkin lymphomas, AML, and solid tumors such as lung cancer, breast cancer, and hepatocellular carcinoma.

[0006] The HAVCR2 / galectin-9 interaction has been found to attenuate T cell expansion and effector function in the tumor microenvironment and chronic infections. In addition, galectin-9 promotes tumorigenesis through tumor cell transformation, cell cycle regulation, angiogenesis, and cell adhesion.

[0007] Galectin-9 is also directly expressed by regulatory T lymphocytes (or Tregs), and its expression increases during Treg activation. At the same time, galectin-9 is very weakly expressed by effector T lymphocytes (such as CD8 + CTL), and this expression disappears during effector T lymphocyte activation. The inhibition of galectin-9 by anti-Gal9 antibodies has been found to inhibit the inhibitory activity of Tregs.

[0008] Wu et al. (Immunity 41(2):270-282, 2014) reported that Gal-9 is crucial in regulating immune responses. Gal-9 is highly expressed by induced regulatory T cells (iTregs), and is essential for the generation and function of iTreg cells, but not for natural regulatory T (nTreg) cells. Gal-9 expression in iTREG cells is driven by the transcription factor Smad3, forming a feed-forward loop that further promotes Foxp3 expression. Gal-9 increases the stability and function of iTREG cells by directly binding to its receptor CD44, which forms a complex with transforming growth factor-β (TGF-β) receptor I (TGF-βRI) and activates Smad3. Further findings show that GAL-9 signaling regulates iTreg cell induction mainly by acting through the CNS1 region of the Foxp locus. Exogenous GAL-9, in addition to being an effector molecule of Treg cells, also acts in concert with TGF-β to enhance iTreg cell differentiation and maintenance.

[0009] Various types of T lymphocytes generally develop into, for example, "effector" cells or effector T lymphocytes, which will perform specialized immune functions to protect the host organism. Thus, CD4 + T lymphocytes or helper T lymphocytes secrete major cytokines, particularly assisting the humoral function of B lymphocytes (production of specific antibodies) and the cytotoxic activity of CD8 + T lymphocytes.

[0010] Another population of CD4 + T lymphocytes consists of natural regulatory T lymphocytes or "regulatory T lymphocytes (Tregs)". They constitutively overexpress the CD25 molecule (therefore, also known as "CD4 + CD25 + "), and the Foxp3 transcription factor. This small proportion of CD4 + CD25 + T lymphocytes have the property of acting as negative regulators of immune response agents that will recognize various autoantigens through their TCR. Regulatory T lymphocytes also play a major role in the physiology of the immune system, particularly in protecting the organism from the emergence of autoimmune diseases. In other words, Tregs are a subset of natural regulatory T lymphocytes (or "nTregs"), characterized by the expression consisting of CD25, CTLA-4, and GITR, and the specific expression of the transcription factor FoxP3.

[0011] Tregs exert immunosuppressive activity on effector T lymphocytes. Once this activity is activated, it promotes tumor growth in pathological conditions such as tumors. Therefore, the inhibitory activity of Tregs can be understood as reducing the activity of anti-tumor immune responses by inhibiting the function of effector T lymphocytes.

[0012] Galectin-9 is mainly associated with tumor immunosuppression caused by the interaction of different immune receptors. For example, Gal9 inhibits Th1 responses and induces peripheral tolerance, as demonstrated by reduced apoptosis of Th1 cells upon Gal9 blockade, increased susceptibility to CIA (collagen-induced arthritis) in Gal9 knockout mice, and prolonged graft survival and AID inhibition upon Gal9 administration. Gal9 also regulates peripheral NK cell function to promote maternal-fetal tolerance, promotes the expansion of MDSCs, and synergizes with TGF-β to promote Treg expansion.

[0013] The circulating levels of Gal9 are significantly higher in certain cancer patients compared to healthy controls.

[0014] Summary of the Invention

[0015] The invention described herein relates to humanized antibodies against galectin-9 and their use for the treatment of diseases associated with the inhibitory activity of regulatory T lymphocytes (Tregs).

[0016] In particular, the invention described herein provides anti-Gal9 neutralizing antibodies that release immunosuppression in the TME (tumor microenvironment), eliciting anti-tumor activity and clinical responses in cancer patients.

[0017] The anti-Gal9 neutralizing antibodies of the present invention are derivative antibodies based on two anti-Gal9 neutralizing antibodies (designated Ab1 and Ab2, respectively) disclosed in US 2017-0283499 A1 (filed on June 5, 2015, incorporated herein by reference), both of which bind recombinant human Gal9 with sub-nM EC 50 values and both of which block Gal9-induced apoptosis of human CD4 + T cells or the expansion of Tregs in peripheral blood from healthy donors. However, these two antibodies differ in that Ab2 blocks the interaction of recombinant human Gal9 with two immune receptors (R1 and R2), while Ab1 does not.

[0018] The invention described herein provides a variety of humanized monoclonal antibodies based on Ab1 and Ab2. These humanized monoclonal antibodies bind to recombinant human and / or murine Gal9, block Gal9-induced Th1 cell apoptosis, and block Gal9-induced Treg expansion. More importantly, the humanized monoclonal antibodies of the invention act in synergy with antibodies targeting the PD-1 / PD-L1 immune checkpoint, thereby providing a therapeutic advantage in overcoming resistance encountered in immunotherapy (e.g., resistance in the ineffectiveness of using PD-1 and PD-L1 antagonists).

[0019] The antibodies of the invention have broad utility in the treatment of 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, kidney cancer, ovarian cancer, liver cancer, and prostate cancer.

[0020] Accordingly, 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 wherein 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) 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.;

[0021] In certain embodiments, in an isolated monoclonal antibody or antigen-binding fragment thereof: (1c) the antibody or antigen-binding fragment 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 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 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 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 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 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 of (7a) and (7b) further comprises the HFR3 sequence of SEQ ID NO: 101 and optionally further comprises the HFR1 sequence of SEQ ID NO: 97; or (8c) the antibody or antigen-binding fragment 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.

[0022] In certain embodiments, in an isolated monoclonal antibody or an 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: 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.

[0023] In certain embodiments, the isolated monoclonal antibody or an 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.

[0024] In certain embodiments, the antigen-binding fragment is Fab, Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNAR, intrabodies, IgGΔCH2, minibodies, F(ab')3, tetrabody, tribody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2 or scFv-Fc.

[0025] In some embodiments, the monoclonal antibody or an antigen-binding fragment thereof of the invention has an engineered Fc region that eliminates immune effector functions. For example, the engineered Fc region of the subject antibody can have the "LALA" double mutation (Leu234Ala together with Leu235Ala) and thus has attenuated effector functions. Such an antibody can have the name G1AA due to the LALA double mutation on IgG1.

[0026] Other recombinant human IgG antibodies that partially or completely do not bind to Fcγ receptors (FcγR) and complement protein C1q and thus have abrogated immune effector functions are known in the art and are used in various therapeutic applications to reduce FcγR activation and Fc-mediated toxicity. Some such Fc-engineered antibodies / fragments have partially achieved this goal, while other antibodies / fragments have completely abrogated FcγR activation and Fc-mediated toxicity. In certain embodiments, the antibodies / fragments of the present invention have an engineered hIgG Fc domain comprising the hIgG1-P329G LALA or hIgG4-P329G SPLE (human IgG4 S228P / L235E variant of IgG4) mutation, with completely abrogated FcγR and C1q interactions and with unaffected FcRn interactions and Fc stability. The P329G Fc mutation disrupts the formation of the proline sandwich motif with FcγR. Since this motif is present at the interface of all IgG Fc / FcγR complexes, its disruption can be applied to all human and most other mammalian IgG subclasses to generate effector-silent IgG molecules. Thus, in certain embodiments, the subject antibodies / fragments have any one of the IgG subclasses with such effector-silent Fc mutations.

[0027] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof cross-reacts with murine Gal9.

[0028] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof binds to human Gal9 with an EC of about 0.1 - 0.2 nM, and / or 50 binds to murine Gal9 with an EC of about 0.5 - 1.0 nM. 50

[0029] 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.

[0030] In certain embodiments, the monoclonal antibody or antigen-binding fragment of the present invention comprises one or more point mutations in its amino acid sequence, which are designed to improve the developability of the antibody. For example, in certain embodiments, the one or more point mutations render the antibody more stable during its expression in host cells, its purification and / or formulation process during preparation, and / or its administration to a subject patient. In certain embodiments, the one or more point mutations render the antibody less likely to aggregate during the preparation and / or formulation process.

[0031] In certain embodiments, the present invention provides therapeutic antibodies with minimized or reduced developability issues, for example, by replacing one or more amino acids in their sequences (e.g., in one or more of their CDRs) to remove or reduce hydrophobicity and / or optimize charge.

[0032] In certain embodiments, an isolated monoclonal antibody or an antigen-binding fragment thereof binds to GAL9 and inhibits the binding of GAL9 to a GAL9 receptor (e.g., TIM3 or CD44).

[0033] In certain embodiments, an isolated monoclonal antibody or an antigen-binding fragment thereof neutralizes Gal-9-induced Th1 apoptosis of T cells (such as CD4 + T cells).

[0034] In certain embodiments, an isolated monoclonal antibody or an antigen-binding fragment thereof inhibits Gal9-induced Treg expansion.

[0035] In certain embodiments, an isolated monoclonal antibody or an antigen-binding fragment thereof synergistically inhibits in vivo tumor growth and / or prolongs survival in mice with xenograft tumors in combination with an antagonist of an immune checkpoint.

[0036] In certain embodiments, the antagonist of the immune checkpoint is an antibody or an antigen-binding fragment thereof specific for PD-1 or PD-L1.

[0037] Another aspect of the present invention provides a method of treating cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of an isolated monoclonal antibody or an antigen-binding fragment thereof of the present invention and an antagonist of an immune checkpoint.

[0038] In certain embodiments, the immune checkpoint is the PD-1 / PD-L1 immune checkpoint.

[0039] In certain embodiments, the antagonist of the immune checkpoint is an antibody or an antigen-binding fragment thereof specific for PD-1 or PD-L1.

[0040] In certain embodiments, the antibody is an anti-PD-1 antibody, such as cemiplimab, nivolumab, or pembrolizumab.

[0041] In certain embodiments, the antibody is an anti-PD-L1 antibody, such as avelumab, durvalumab, atezolizumab, KN035, or CK-301.

[0042] In certain embodiments, the antagonist of the immune checkpoint 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.

[0043] In certain embodiments, 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, kidney cancer, ovarian cancer, liver cancer, and prostate cancer).

[0044] In certain embodiments, the method further comprises administering to the patient a chemotherapeutic agent, an anti-angiogenic agent, a growth inhibitory agent, an immune-oncology agent, and / or an anti-tumor composition.

[0045] Another aspect of the invention provides a polynucleotide encoding a heavy chain or a light chain of the invention or an antigen-binding portion thereof.

[0046] In certain embodiments, the polynucleotide is codon-optimized for expression in human cells.

[0047] Another aspect of the invention provides a vector comprising the polynucleotide of the invention.

[0048] In certain embodiments, the vector is an expression vector (e.g., a mammalian expression vector, a yeast expression vector, an insect expression vector, or a bacterial expression vector).

[0049] Another aspect of the 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 having a risk of cancer recurrence, or a method for identifying and treating a patient with cancer, the method comprising: when identifying a patient as having a level of galectin-9 in a sample from the patient higher than a reference level of galectin-9 in a healthy or control individual, administering to the patient an effective amount of the isolated monoclonal antibody of the invention or an antigen-binding fragment thereof.

[0050] In certain embodiments, the method further comprises identifying a patient as having a level of galectin-9 in the sample higher than the reference level by comparing the level of galectin-9 in the sample with the reference level.

[0051] In certain embodiments, the method further comprises administering to the patient an antagonist of the immune checkpoint.

[0052] In certain embodiments, the immune checkpoint is the PD-1 / PD-L1 immune checkpoint.

[0053] In certain embodiments, the antagonist of the immune checkpoint is an antibody specific for PD-1 or PD-L1 or an antigen-binding fragment thereof.

[0054] In certain embodiments, the antibody is an anti-PD-1 antibody, such as cemiplimab, nivolumab, or pembrolizumab.

[0055] In certain embodiments, the antibody is an anti-PD-L1 antibody, such as avelumab, durvalumab, atezolizumab, KN035, or CK-301.

[0056] In certain embodiments, the antagonist of the immune checkpoint 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.

[0057] In certain embodiments, the cancer is a hematologic 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, kidney cancer, ovarian cancer, liver cancer, and prostate cancer).

[0058] In certain embodiments, the patient is a Fab M0, M1, M4, or M5 AML patient, or a patient who is not a Fab M2 or M3 AML patient.

[0059] In certain embodiments, the sample is a blood sample, a plasma sample, or a serum sample.

[0060] Another aspect of the present invention provides a method for rescuing or promoting effector T cell proliferation and / or enhancing effector T cell activity in a patient diagnosed with AML, in the process of developing AML, or at risk of AML recurrence, or a method for identifying and treating a patient with AML, the method comprising: when it is identified that the level of galectin-9-encoding mRNA in a sample of monocytes (MNCs) derived from bone marrow (BM) of the patient is statistically significantly higher or lower than the reference level in BM-derived MNCs or CD34 + cells of healthy or control individuals, administering to the patient an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0061] In certain embodiments, when the patient is a Fab M0, M1, M2, M4, or M5 AML patient, the level of galectin-9-encoding mRNA in a sample of BM-derived MNCs from the patient is significantly higher than the reference level.

[0062] In certain embodiments, when the patient is a Fab M3 AML patient, the level of galectin-9-encoding mRNA in the BM-derived MNC sample from the patient is significantly lower than the reference level.

[0063] Another aspect of the invention provides an antibody or an antigen-binding portion thereof that targets galectin-9 or is specific for galectin-9 for treating cancer, wherein the antibody or its antigen-binding portion rescues effector T cell proliferation and / or enhances effector T cell activity.

[0064] In certain embodiments, the effector T cell is a Th1 cell.

[0065] Another aspect of the invention provides a method of rescuing or promoting effector T cell proliferation and / or enhancing effector T cell activity, which comprises contacting the effector T cell with the isolated monoclonal antibody or an antigen-binding fragment thereof of the invention.

[0066] In certain embodiments, the effector T cell is a Th1 cell.

[0067] Another aspect of the invention provides methods and related compositions for inducing or promoting the generation of immune memory with anti-tumor (anti-cancer) activity. In certain embodiments, the method comprises administering to a subject a composition (e.g., a pharmaceutical composition comprising an antibody of the invention) in an amount effective to induce, stimulate, or promote immune memory, the immune memory being effective to reduce or inhibit the initiation, progression, or recurrence of a tumor or cancer in the subject.

[0068] It should be understood that any one embodiment of the invention described herein, including those described only in the examples or claims, may be combined with any one or more other embodiments of the invention, unless explicitly disclaimed or otherwise inapplicable. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Showing sequence alignments of various anti-human galectin-9 humanized antibodies.

[0071] Figure 2 Showing sequence alignments of various anti-human galectin-9 humanized antibodies.

[0072] Figure 3 Showing the binding affinity (measured as EC 50 (in nM)) of various anti-human galectin-9 humanized antibodies for recombinant human Gal-9. Isotype-matched antibodies against different antigens were used as negative controls. In one case, the original human-mouse chimeric antibody was also included for comparison.

[0073] Figure 4Show the binding affinity of various anti-human galectin-9 humanized antibodies to recombinant murine Gal-9 (measured as EC in nM). Isotype-matched antibodies against different antigens were used as negative controls. In one case, the original human-mouse chimeric antibody was also included for comparison. 50 )

[0074] Figure 5 Show that various anti-human galectin-9 humanized antibodies of the present invention can block the binding to TIM3 and CDC44.

[0075] Figure 6 Show the ability of various anti-human galectin-9 humanized antibodies of the present invention to neutralize Gal-9-induced apoptosis of Th1 cells.

[0076] Figure 7 Show the ability of various anti-human galectin-9 humanized antibodies of the present invention to neutralize Gal-9-induced Treg expansion.

[0077] Figure 8 Show the ability of various anti-human galectin-9 humanized antibodies of the present invention to promote long-term survival when used in combination with anti-PD1 antibody. Data confirm that combination therapy with anti-PD1 antibody results in significantly better / synergistic therapeutic efficacy, as measured by inhibition of tumor volume increase.

[0078] Figure 9 Show the ability of various anti-human galectin-9 humanized antibodies of the present invention to promote long-term survival when used in combination with anti-PD1 antibody. Data confirm that combination therapy with anti-PD1 antibody results in significantly better / synergistic therapeutic efficacy, as measured by survival over time.

[0079] Figure 10 Show the levels of galectin-9 in serum or plasma from AML patients and healthy individuals. Data confirm that the levels of galectin-9 in plasma from AML patients at the diagnostic or relapse / refractory (R / R) stage are significantly higher than those in plasma from healthy patients and AML patients in complete remission after chemotherapy treatment.

[0080] Figure 11 Show the levels of galectin-9 in plasma or serum from AML patients graded according to the French-American-British (FAB) classification. Data show that the levels of galectin-9 protein in plasma from Fab M2 or Fab M3 AML patients at diagnosis are significantly lower than those observed in plasma from Fab M0, M1, M4, or M5 AML patients. The levels of galectin-9 protein in plasma from Fab M3 AML patients at diagnosis are within the normal physiological range.

[0081] Figure 12 Show the levels of prolactin-9 encoding mRNA (LGALS9) in monocytes (MNCs) of bone marrow (BM) origin from AML patients classified according to the French-American-British (FAB) classification or from healthy individuals. The data confirm that, at diagnosis, the levels of galectin-9 encoding mRNA in BM-derived MNCs from AML patients (considering all FAB) are higher than those in BM-derived MNCs from healthy individuals or CD34 + as observed in those. At diagnosis, the levels of galectin-9 encoding mRNA in BM-derived MNCs from Fab M3 AML patients are significantly lower than those observed in BM-derived MNCs from Fab M0, M1, M4 or M5 AML patients or BM-derived MNCs from healthy individuals or CD34 + cells in BM-derived MNCs.

[0082] Figure 13 Show the anti-tumor activity of the antibody of the present invention as a single therapy. This experiment confirms that the anti-Gal9 monoclonal antibody of the present invention is effective in inhibiting tumor growth in vivo in a xenograft mouse model. In particular, approximately 500,000 cancer cells were inoculated into experimental mice, and the tumor mass was allowed to grow to a predetermined size. Then, the mice were randomized and injected intraperitoneally (i.p.) with one of two antibodies: (1) an IgG isotype control at a dose of 10 mg / kg, (2) the anti-Gal9 antibody HFB9-2 at a dose of 10 mg / kg. The first dose of antibody for each group was administered on day 1, and subsequent doses were administered every 3 days, for a total of eight doses for any group with the anti-HFB9-2 antibody and the control antibody. The data are presented as mean ± s.e.m. (n = 10 mice per group). Clearly, the subject anti-Gal9 antibody shows an inhibitory effect on tumor growth in vivo.

[0083] Figure 14 Further show the anti-tumor activity of the antibody of the present invention as a single therapy in terms of survival: all mice in the control group died, and 40% of the mice (4 out of 10) in the HFB9-2 treatment group survived tumor-free at the end of week 6.

[0084] Figure 15 Show the immune memory of the anti-tumor activity of the antibody of the present invention. After the first tumor inoculation / attack, untreated animals developed tumors, and 2 out of 4 animals reached 3000 mm within 31 days 3The humane endpoint of tumor volume. Four animals with complete tumor regression previously treated and cured with HFB9-2 (the antibody of the present invention) completely rejected a second Wehi-164 tumor challenge inoculated 63 days after the first tumor challenge. These data indicate long-term immune memory induced by treatment with HFB9-2. DETAILED DESCRIPTION OF THE INVENTION

[0086] 1. General Overview

[0087] Although monoclonal antibodies targeting immune checkpoints have demonstrated clinical success in a range of tumor types, sustained responses are observed only in a subset of patients due to primary or secondary resistance to treatment.

[0088] The applicant believes that galectin-9 (Gal-9) is a key factor present in the tumor microenvironment that renders tumors resistant to current immunotherapies. Along with other evidence, high Gal-9 expression has been reported in different types of cancer, including hematological malignancies such as AML and ALL, as well as a variety of solid tumors.

[0089] 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 present invention specifically bind human Gal-9 with sub-nanomolar affinity, recognize recombinant Gal-9 and Gal-9 produced by human tumor cells, and cross-react with mouse and monkey Gal-9 orthologs. They also block the interaction of Gal-9 with its receptors TIM3 and CD44 in a dose-dependent manner. Both of these receptors have been described to mediate effector and regulatory T cell Gal-9 immunosuppressive signals. Treatment of human PBMCs from healthy donors with the antibodies of the present invention prevents Gal-9-induced Th1 cell apoptosis and inhibits the expansion of regulatory T cells.

[0090] Certain humanized forms of the antibodies of the present invention exhibit further favorable characteristics in terms of stability and pharmacokinetic (PK) profiles and are thus uniquely suitable for further development as therapeutic antibodies. In particular, such humanized antibodies exhibit stability at 40 °C for at least 14 days, stability at low pH for several hours, and stability after several freeze-thaw cycles. At the same time, high plasma exposure of the humanized antibody was observed after single-dose administration of 10 mg / kg to C57BL / 6 mice.

[0091] The antibodies of the present invention can be used to treat many cancers, such as AML. It has been reported that Gal-9 plays a dual role in AML, acting both as a self-renewal factor for leukemia stem cells and as an inhibitor of anti-cancer immunity. Thus, antagonizing Gal-9 function by using the Gal-9-neutralizing antibodies of the present invention represents an attractive therapeutic approach for treating AML.

[0092] In summary, the data provided herein demonstrate that neutralizing Gal-9 with the antibodies of the present invention blocks key immunosuppressive mechanisms that are known to limit the efficacy of current immunotherapies.

[0093] The detailed aspects of the present invention are further and separately described in the various sections below. However, it should be understood that any one embodiment of the present invention, including embodiments described only in the examples or the figures, and embodiments described only under one section below, can be combined with any other embodiment of the present invention.

[0094] 2. Definitions

[0095] The term "antibody" encompasses, in the broadest sense, various 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 complementarity determining regions (CDRs) 1, 2, and 3 of the heavy chain and CDRs 1, 2, and 3 of the light chain, wherein the molecule is capable of binding an antigen. The term "antibody" also includes but is not limited to chimeric antibodies, humanized antibodies, human antibodies, and antibodies of various species such as mouse, human, cynomolgus monkey, etc.

[0096] However, in a narrower sense, "antibody" refers to various monoclonal antibodies, including chimeric monoclonal antibodies, humanized monoclonal antibodies, and human monoclonal antibodies, particularly the humanized monoclonal antibodies of the present invention.

[0097] 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 and two light chains, wherein each heavy chain comprises a heavy chain variable region and at least a portion of a heavy chain constant region, and wherein each light chain comprises a light chain variable region and at least a portion of a light chain constant region.

[0098] As used herein, a single-chain Fv (scFv) or any other antibody comprising, for example, a single polypeptide chain containing 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 containing the three heavy chain CDRs, and the light chain is the region of the antibody containing the three light chain CDRs.

[0099] As used herein, the term "heavy chain variable region (HCVR)" refers to a region that includes 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 further includes at least a portion (e.g., the entire portion) of FR1 (HFR1) at the N-terminus of CDR-H1 and / or at least a portion (e.g., the entire portion) of FR4 (HFR4) at the C-terminus of CDR-H3.

[0100] As used herein, the term "heavy chain constant region" refers to a region that includes at least three heavy chain constant domains CH1, CH2, and CH3. Non-limiting exemplary heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions further include ε and μ. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody that includes a γ constant region is an IgG antibody, an antibody that includes a δ constant region is an IgD antibody, an antibody that includes an α constant region is an IgA antibody, an antibody that includes an ε constant region is an IgE antibody, and an antibody that includes a μ constant region is an IgM antibody.

[0101] Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (including a γ1 constant region), IgG2 (including a γ2 constant region), IgG3 (including a γ3 constant region), and IgG4 (including a γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (including an α1 constant region) and IgA2 (including an α2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 (including a μ1 constant region) and IgM2 (including a μ2 constant region).

[0102] As used herein, the term "heavy chain" refers to a polypeptide that includes at least a heavy chain variable region, with or without a leader sequence. In some embodiments, the 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 that includes 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.

[0103] As used herein, the term "light chain variable region (LCVR)" refers to a region that includes 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 further includes at least a portion (e.g., the entire portion) of FR1 (LFR1) and / or at least a portion (e.g., the entire portion) of FR4 (LFR4).

[0104] As used herein, the term "light chain constant region" refers to a region that includes light chain constant region C Lregion. Non-limiting exemplary light chain constant regions include λ and κ.

[0105] As used herein, the term "light chain" refers to a polypeptide comprising at least one light chain variable region, with or without a leader sequence. In some embodiments, the 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.

[0106] The term "antibody fragment" or "antigen-binding portion" (of an antibody) includes, but is not limited to, fragments capable of binding an antigen, such as Fv, single-chain Fv (scFv), Fab', and (Fab')2. In certain embodiments, the antibody fragment includes Fab, Fab', F(ab')2, F d , single-chain Fv or scFv, disulfide-linked F v , V-NAR domain, IgNar, intracellular antibody, IgGΔCH2, microantibody, F(ab')3, tetrabody, tribody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2 or scFv-Fc.

[0107] The term "Fab" refers to an antibody fragment having a molecular weight of approximately 50,000 daltons and having the activity of binding an antigen. It comprises approximately half of the heavy chain N-terminal side and the entire light chain linked by a disulfide bridge. Fab can be particularly obtained by treating an immunoglobulin with the protease papain.

[0108] The term "F(ab')2" refers to a fragment of approximately 100,000 daltons having the activity of binding an antigen. This fragment is slightly larger than two Fab fragments linked by a disulfide bond in the hinge region. These fragments are obtained by treating an immunoglobulin with the protease pepsin. Fab fragments can be obtained from F(ab')2 fragments by cleaving the disulfide bridges in the hinge region.

[0109] A single Fv chain "scFv" corresponds to a VH:VL polypeptide synthesized using genes encoding the VL and VH domains and a sequence encoding a peptide designed to link these domains. According to the present invention, scFv includes, for example, CDRs maintained in a proper conformation using genetic recombination techniques.

[0110] A dimer of "scFv" corresponds to two scFv molecules linked together by a peptide bond. This Fv chain is typically the result of the expression of a fusion gene comprising genes encoding VH and VL linked by a linker sequence encoding a peptide. Human scFv fragments can include CDR regions preferably maintained in a proper conformation by using genetic recombination techniques.

[0111] The "dsFv" fragment is a VH-VL heterodimer stabilized by a disulfide bridge; it can be bivalent (dsFV2). Bivalent Sc(Fv)2 fragments or multivalent antibodies can form spontaneously by the association of monovalent scFvs, or be generated by linking scFv fragments with peptide-binding sequences.

[0112] The Fc fragment is the support for the biological properties of the antibody, particularly its ability to be recognized by immune effectors or activate complement. It consists of the constant heavy-chain fragments outside the hinge region.

[0113] The term "diabody" refers to small antibody fragments with two antigen-binding sites. These fragments contain a variable heavy-chain domain VH linked to a variable light-chain domain VL in the same VH-VL polypeptide chain. Using a linker sequence that is too short to allow the two domains of the same chain to pair, pairing with the two complementary domains of the other chain necessarily occurs, thus generating two antigen-binding sites.

[0114] An "antibody that binds the same epitope" as a reference antibody can be determined by an antibody competition assay. It refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. When used in the context of antibodies competing for the same epitope, the term "compete" refers to determining the competition between antibodies by an assay in which the tested antibody prevents or inhibits the specific binding of the reference antibody to a common antigen.

[0115] Many types of competitive binding assays can be used, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619); solid-phase direct label assay; solid-phase direct label sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); using I 125Labeled solid-phase direct-label RIA (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-labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol.).

[0116] Typically, such assays involve using a purified antigen bound to a solid surface or a cell carrying any of these, an unlabeled test antigen-binding protein, and a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cell in the presence of the test antibody. Typically, the test antibody is present in excess. Antibodies identified by competitive assays (competitor antibodies) include antibodies that bind the same epitope as the reference antibody and antibodies that bind an adjacent epitope that is close enough to the epitope bound by the reference antibody to cause steric hindrance. In some embodiments, when the competitor antibody is present in excess, it will inhibit the specific binding of the reference antibody to the common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some cases, the binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more.

[0117] The term "antigen" refers to a molecule or a portion of a molecule that can be bound by a selective binding agent (e.g., an antibody or an immunologically functional fragment thereof) and that can additionally be used in a mammal to generate an antibody capable of binding the antigen. An antigen can have one or more epitopes capable of interacting with an antibody.

[0118] The term "epitope" is the portion of an antigen molecule that is bound by a selective binding agent (e.g., an antibody or a fragment thereof). The term includes any determinant capable of specifically binding an antibody. Epitopes can be continuous or discontinuous (e.g., in a polypeptide, amino acid residues that are not contiguous in the polypeptide sequence but are bound by an antigen-binding protein in the context of the molecule). In some embodiments, epitopes can be mimetics in that they contain a three-dimensional structure similar to the epitope used to generate the antibody but do not contain or contain only some of the amino acid residues found in the epitope used to generate the antibody. Epitope determinants can include the chemically reactive surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural features and / or specific charge features.

[0119] In some embodiments, an "epitope" is defined by the method used to determine it. For example, in some embodiments, if an antibody binds the same region of an antigen as determined by hydrogen-deuterium exchange (HDX), the antibody binds the same epitope as the reference antibody.

[0120] For example, the epitope sequences of the parental antibodies HFB9-1 and HFB9-2 are disclosed as SEQ ID NO: 9 in US 2017-0283499A1 (incorporated herein by reference). This sequence corresponds to the P4 peptide and covers the terminus of the binding peptide and the start of the C-terminal portion of galectin-9. It exists in three isotypes of galectin-9 (e.g., amino acids 166 to 178 of the S isotype, amino acids 178 to 190 of the M isotype, amino acids 210 to 222 of the L isotype). The humanized antibodies of the present invention can bind to at least one isotype of galectin-9, preferably all isotypes.

[0121] In certain embodiments, as determined by X-ray crystallography, if antibodies bind to the same region of the antigen, they bind to the same epitope as the reference antibody.

[0122] As used herein, a "chimeric antibody" refers to an antibody that contains at least one variable region from a first species (such as mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (such as human, cynomolgus monkey, chicken, etc.). In some embodiments, the chimeric antibody contains at least one murine variable region and at least one human constant region. In some embodiments, all variable regions of the chimeric antibody are from the first species, and all constant regions of the chimeric antibody are from the second species.

[0123] As used herein, a "humanized antibody" refers to an antibody in which at least one amino acid in the framework region of a non-human variable region (such as mouse, rat, cynomolgus monkey, chicken, etc.) has been replaced with the corresponding amino acid from a human variable region. In some embodiments, the humanized antibody contains at least one human constant region or a fragment thereof. In some embodiments, the humanized antibody fragment is Fab, scFv, (Fab')2, etc.

[0124] As used herein, a "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 the framework region (FR) of a second (human) species.

[0125] As used herein, a "human antibody" refers to an antibody produced in a human, an antibody produced in a non-human animal containing human immunoglobulin genes, e.g., and an antibody selected using in vitro methods such as phage display, where the antibody library is based on human immunoglobulin sequences.

[0126] "Host cell" means a cell that can be or has been the recipient of a vector or isolated polynucleotide. A host cell can be a prokaryotic cell or a eukaryotic cell. 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 NS0 cells, cells (Crucell) and 293 and CHO cells, and derivatives thereof such as 293-6E and DG44 cells, respectively.

[0127] As used herein, the term "isolated" refers to a molecule that has been separated from at least some of the components with which it is normally found in nature, or has been separated from at least some of the components by which it is normally produced. For example, a polypeptide is said to be "isolated" when it has been separated from at least some of the components of the cell that produced it. When a polypeptide is secreted by a cell after expression, physical separation of the supernatant containing the polypeptide from the cell that produced it is considered to "isolate" the polypeptide. Similarly, a polynucleotide is said to be "isolated" when it is not part of a larger polynucleotide that is normally found in nature (e.g., genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide), or when it has been separated from at least some of the components of the cell that produced it, e.g., in the case of an RNA polynucleotide. Thus, a DNA polynucleotide contained in a vector within a host cell can be said to be "isolated" provided that the polynucleotide is not found in that vector in nature.

[0128] The terms "subject" and "patient" are used interchangeably herein and refer to a mammal such as a human. In some embodiments, methods of treating other non-human mammals are also provided, including but not limited to rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets. In some instances, a "subject" or "patient" refers to a (human) subject or patient in need of treatment for a disease or disorder.

[0129] As used herein, the term "sample" or "patient sample" refers to a material obtained from or derived from a target subject that contains cells and / or other molecular entities to be characterized and / or identified, e.g., based on physical, biochemical, chemical, and / or physiological characteristics. For example, the phrase "disease sample" and variations thereof refer to any sample obtained from a target subject that is expected or known to contain cells and / or molecular entities to be characterized.

[0130] "An "organ or cell sample" means a collection of cells obtained from the tissue of a subject or patient. The source of the organ or cell sample can be solid tissue from a fresh, frozen, and / or preserved organ or tissue sample or biopsy or aspirate; blood or any blood component; body fluid, such as sputum, cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time of the subject's pregnancy or development. The tissue sample can also be primary or cultured cells or cell lines. Optionally, the organ or cell sample is obtained from diseased tissue / organs. The tissue sample can contain compounds that are not naturally mixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0131] 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 not to be afflicted with the disease or disorder being identified using the methods or compositions of the present invention. In one embodiment, the reference sample, reference cell, or reference tissue is obtained from a healthy portion of the body of the same subject or patient being identified for the disease or disorder 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 portion of the body of at least one individual who is not the subject or patient being identified for the disease or disorder using the compositions or methods of the present invention. In some embodiments, the reference sample, reference cell, or reference tissue was previously obtained from the patient before the development of the disease or disorder or in the early stages of the disease or disorder.

[0132] A "disorder" or "disease" is any disorder 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 those pathological conditions that render a mammal susceptible to the disorder being discussed. Non-limiting examples of disorders to be treated herein include cancer.

[0133] A "disease associated with the inhibitory activity of regulatory T lymphocytes" refers to any disease (non-autoimmune) in which the inhibitory activity of regulatory T lymphocytes plays a role, particularly by promoting the development or persistence of the disease. In particular, it has been shown that the inhibitory activity of regulatory T lymphocytes promotes the development of tumors. Thus, the present invention is more particularly directed to cancers in which the inhibitory activity of T lymphocytes plays a role.

[0134] The term "cancer" is used herein to refer to a group of cells that exhibit an abnormally high level of proliferation and growth. Cancer can be benign (also known as a benign tumor), pre-malignant, or malignant. Cancer cells can be solid cancer cells (i.e., forming solid tumors) or leukemia cancer cells. The term "cancer growth" is used herein to refer to the proliferation or growth of one or more cells containing cancer, which results in a corresponding increase in the size or extent of the cancer.

[0135] 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 carcinoma, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, renal cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, brain cancer, endometrial cancer, testicular cancer, cholangiocarcinoma, gallbladder cancer, stomach cancer, melanoma, and various types of head and neck cancer.

[0136] The French-American-British (FAB) classification of acute myeloid leukemia (ALM) classifies ALM into different stages of the disease. The Fab subtypes are shown in Table 1.

[0137] Table 1: FAB Classification

[0138] FAB subtype Description M0 Undifferentiated acute myeloblastic leukemia M1 Acute myeloblastic leukemia with the lowest degree of maturity M2 Acute myeloblastic leukemia with a degree of maturity M3 Acute promyelocytic leukemia (APL) M4 Acute myelomonocytic leukemia M5 Acute monocytic leukemia M6 Acute erythroblastic leukemia M7 Acute megakaryoblastic leukemia

[0139] In certain embodiments, cancers as used herein include hematologic 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).

[0140] "Chemotherapeutic agent" refers to a chemical compound that can be used to treat cancer. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and Cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and ureteropa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; polyacetylenes (especially bryostatin and bryostatinone); camptothecin (including the synthetic analogue topotecan); bryostatin; calicheamicin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ω1I (see, e.g., Agnew, Chem lntl.Ed.Engl, 33:183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclarubicin, actinomycin, anthramycin, azaserine, bleomycin, actinomycin C, carubicin, carminomycin, carzinophilin, chromomycin, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Doxorubicin (including morpholino doxorubicin, cyano morpholino-doxorubicin, 2-pyrrolino doxorubicin, and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, mazethramycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rhodomycin, streptothricin, streptozocin, tubercidin, bestatin, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as dimethyl folate, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thioguanine, tioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxuridine, doxifluridine, enocitabine, floxuridine; androgenic hormones, such as calusterone, drostanolone propionate, thiotriol, methyltestosterone, testosterone lactone; antiadrenal, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as leucovorin; glucuronolactone; aldophosphamide glycoside; aminolevulinic acid; enuracil; aclarubicin; amsacrine; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; eflornithine; elisidepsin; epothilones; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansine alkaloids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2″-trichloroethylamine; trichothecenes (especially T-2 toxin, verrucarin A, baccharin A, and enniatin); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; carzelesin; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois) and docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; Gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; nogalamycin; teniposide; edatrexate; daunomycin; aminopterin; capecitabine; ibandronate; irinotecan (Camptosar, CPT-11) (including treatment regimens of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including oxaliplatin treatment regimens (FOLFOX); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib ) and VEGF-A, and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0141] Other non-limiting exemplary chemotherapeutic agents include antihormonal agents for modulating or inhibiting the hormonal effect on cancer, such as antiestrogens and selective estrogen receptor modulators (SERM), including for example tamoxifen (including tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, raloxifene hydrochloride (keoxifene), LY117018, onapristone and toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal gland, such as 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, formestane, fadrozole, vorozole, letrozole and anastrozole; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and zalcitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit gene expression in signal transduction pathways involved in abnormal cell proliferation, such as PKC-α, Ralf and H-Ras; ribozymes, such as VEGF expression inhibitors (e.g., ribozymes) and HER2 expression inhibitors; vaccines, such as gene therapy vaccines, e.g., vaccines, vaccines and vaccines, rIL-2; topoisomerase 1 inhibitors; rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0142] "Anti-angiogenic agent" or "angiogenesis inhibitor" refers to a small molecular weight substance, polynucleotide (including, for example, inhibitory RNA (RNAi or siRNA)), polypeptide, isolated protein, recombinant protein, antibody, or a conjugate or fusion protein thereof that directly or indirectly inhibits angiogenesis, vasculogenesis, or unwanted vascular permeability. It should be understood that anti-angiogenic agents include those substances that bind to angiogenic factors or their receptors and block their angiogenic activity. For example, anti-angiogenic agents are antibodies or other antagonists of angiogenic agents, such as antibodies against VEGF-A (e.g., bevacizumab ) or antibodies against VEGF-A receptors (e.g., KDR receptor or Flt-1 receptor), anti-PDGFR inhibitors such as (imatinib mesylate), small molecules that block VEGF receptor signaling (e.g., PTK787 / ZK2284, SU6668, (sunitinib malate), AMG706, or those small molecules described, for example, in International Patent Application WO 2004 / 113304). Anti-angiogenic agents also include natural angiogenesis inhibitors, such as angiostatin, endostatin, etc. See, for example, Klagsbrun and D'Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22:3172-3179 (e.g., anti-angiogenic therapy for malignant melanoma listed in Table 3); Ferrara and Alitalo (1999) Nature Medicine 5(12):1359-1364; Tonini et al. (2003) Oncogene 22:6549-6556 (e.g., known anti-angiogenic factors listed in Table 2); and Sato (2003) Int. J. Clin. Oncol. 8:200-206 (e.g., anti-angiogenic agents used in clinical trials listed in Table 1).

[0143] As used herein, "growth inhibitory agent" refers to a compound or composition that inhibits the growth of cells (e.g., cells expressing VEGF) in vitro or in vivo. Thus, a growth inhibitory agent can be an inhibitor that significantly reduces the percentage of cells (e.g., cells expressing VEGF) in the S phase. Examples of growth inhibitory agents include, but are not limited to, substances that block cell cycle progression (at positions other than in the S phase), such as substances that induce G1 arrest and M phase arrest. Typical M phase blockers include vinca alkaloids (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those substances that arrest G1 also result in S phase arrest, such as DNA alkylating agents like tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. More information can be found, for example, in "The Molecular Basis of Cancer" edited by Mendelsohn and Israel, Chapter 1, page 13, in the article titled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (W.B. Saunders, Philadelphia, 1995). Taxanes (paclitaxel and docetaxel) are anti-cancer drugs derived from the yew tree. Docetaxel, which is derived from Taxus baccata Rhone-Poulenc Rorer) is a semi-synthetic analogue of paclitaxel Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of tubulin dimers into microtubules and stabilize the microtubules by preventing disassembly, resulting in the inhibition of mitosis in cells.

[0144] The term "antineoplastic composition" refers to a composition that contains at least one active therapeutic agent and is useful for treating cancer. Examples of therapeutic agents include, but are not limited to, for example, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiotherapy, anti-angiogenic agents, cancer immunotherapeutic agents (also known as 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 platelet-derived growth factor inhibitors (e.g., (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, CTLA-4 inhibitors (e.g., the anti-CTLA antibody ipilimumab ), PD-1 inhibitors (e.g., anti-PD-1 antibody, BMS-936558), PD-L1 inhibitors (e.g., anti-PD-L1 antibody, MPDL3280A), PD-L2 inhibitors (e.g., anti-PD-L2 antibody), VISTA inhibitors (e.g., anti-VISTA antibody), cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets ErbB2, ErbB3, ErbB4, PDGFR-β, BlyS, APRIL, BCMA, PD-1, PD-L1, PD-L2, CTLA-4, VISTA, or VEGF receptor, TRAIL / Apo2, and other bioactive agents and organic chemical agents, etc. Combinations thereof are also included in the present invention.

[0145] "Treatment" refers to therapeutic treatment, the purpose of which is to prevent or slow down (alleviate) a targeted pathological condition or disorder, and for example, where the purpose is to inhibit the recurrence of the disorder or disease. "Treatment" encompasses any administration or application of a therapeutic agent for a disease (also referred to herein as a "disorder" or "condition") in a mammal (including a human), and includes inhibiting the disease or the progression of the disease, inhibiting or slowing down the disease or its progression, arresting its development, partially or completely relieving the disease, partially or completely relieving one or more symptoms of the disease, or restoring or repairing a lost, missing, 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 that characteristic. Those in need of treatment include those who already have the disorder and those at risk of recurrence of the disorder or in whom recurrence of the disorder is to be prevented or slowed down.

[0146] The term "effective amount" or "therapeutically effective amount" refers to the amount of a drug that is effective in treating a disease or disorder in a subject. In some embodiments, the effective amount refers to the amount that is effective in achieving the desired therapeutic or prophylactic outcome at the required dose and for the required period of time. The therapeutically effective amount of the GAL9 antagonist of the present invention can vary depending on factors such as, for example, the individual's disease state, age, gender, and body weight, and the ability of the antagonist to elicit the desired response in the individual. The therapeutically effective amount encompasses the amount in which the therapeutic beneficial effect exceeds any toxic or harmful effects of the GAL9 antagonist.

[0147] "Prophylactically effective amount" refers to the amount that is effective in achieving the desired prophylactic outcome at the required dose and for the required period of time. Generally but not necessarily, since the prophylactic dose is used in a subject before or at an early stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0148] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid or vehicle, which is conventional in the art and is used together with a therapeutic agent, and together they constitute a "pharmaceutical composition" for administration to a subject. The pharmaceutically acceptable carrier is non-toxic to the recipient at the administered doses and concentrations and is compatible with the other ingredients of the formulation. The pharmaceutically acceptable carrier is suitable for the formulation employed. For example, if the therapeutic agent is to be administered orally, the carrier can be a gelatin capsule. If the therapeutic agent is administered subcutaneously, the carrier is desirably non-irritating to the skin and does not cause injection site reactions.

[0149] "Manufacture" is any article (e.g., a package or container) or kit that contains at least one reagent (e.g., a drug for treating a disease or disorder, or a probe for specifically detecting a biomarker described herein). In some embodiments, the article or kit is promoted, distributed or sold as a unit for performing the methods described herein.

[0150] 3. Methods of treating cancer

[0151] The invention described herein provides Gal9 antagonists (e.g., anti-Gal9 antibodies) for use in methods of treating humans and other non-human mammals.

[0152] In pathological conditions, Tregs may cause inappropriate immunosuppression, which may, for example, promote tumor growth. Tregs are associated with reduced anti-tumor immune responses, particularly by inappropriately suppressing the activity of effector T lymphocytes, thereby promoting the development of many cancer types.

[0153] During activation, galectin-9 is directly expressed by Tregs, while it is expressed very weakly or not at all by effector T lymphocytes. Thus, by targeting galectin-9, for example, using a Gal-9-specific antibody, the inhibitory activity of regulatory T lymphocytes can be specifically inhibited without the risk of depletion of effector T lymphocytes. Thus, the antibodies according to the invention that target galectin-9 and inhibit the inhibitory activity of regulatory T lymphocytes can be used to treat diseases or disorders associated with the inhibitory activity of regulatory T lymphocytes, particularly for treating cancer.

[0154] In some embodiments, there is provided a method for treating or preventing cancer, which comprises administering to a subject in need of such treatment an effective amount of a Gal9 antagonist.

[0155] In some embodiments, there is provided a method of treating cancer, wherein the method comprises administering a Gal9 antagonist to a subject having cancer.

[0156] In some embodiments, there is provided the use of a Gal9 antagonist for treating cancer.

[0157] Cancers that can be treated by the methods / uses of the present invention include those cancers in which regulatory T lymphocytes exert their inhibitory activity, such as those cancers in which a relatively large amount of regulatory T lymphocytes are present in tumor tissue or circulation. The expansion of regulatory T lymphocytes (which can be measured by the frequency of Tregs) is generally associated with an increase in Treg activation. The frequency of regulatory T lymphocytes can be evaluated by any method known in the art, such as by flow cytometry (FACS) analysis of intratumoral lymphocytes or circulating lymphocytes, or by immunohistological staining of tumor tissue.

[0158] The present disclosure provides non-limiting exemplary cancers that can be treated with Gal9 antagonists, including 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 carcinoma, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, renal carcinoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, brain cancer, endometrial cancer, testicular cancer, cholangiocarcinoma, gallbladder cancer, gastric cancer, melanoma, and various types of head and neck cancers.

[0159] In certain embodiments, the methods / uses of the present invention can be used to treat cancers in which a known high level of regulatory T lymphocytes is present and / or whose cancers / tumors are significantly associated with poor prognosis, including: chronic myeloid leukemia (CML), colon cancer, melanoma, uterine cancer, breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, primary central nervous system lymphoma, multiple myeloma, prostate cancer, Hodgkin lymphoma, or hepatocellular carcinoma.

[0160] In certain embodiments, the methods / uses of the present invention can be used to treat cancers that produce a large amount of exosomes carrying galectin-9, and the exosomes exert an immunosuppressive effect. 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).

[0161] 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, kidney cancer, ovarian cancer, liver cancer, and prostate cancer).

[0162] In certain embodiments, the methods / uses of the present invention can be used to treat the recurrence of fibrosis caused by hepatitis C, as it has also been shown that increasing the frequency of regulatory T lymphocytes is a factor predictive of such fibrosis recurrence.

[0163] In some embodiments, the Gal9 antagonist is an anti-Gal9 antibody, or simply "GAL9 antibody" for short.

[0164] In some embodiments, the Gal9 antagonist for treating cancer can be a non-antibody protein, such as a soluble form of the Gal9 protein or a portion thereof (e.g., ECD) that inhibits the interaction between Gal9 and its ligand, optionally further comprising a fusion partner and in the form of a fusion molecule. Various exemplary Gal9 antagonists are described in more detail in the following sections.

[0165] In some embodiments, the Gal9 antagonist 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.

[0166] Thus, according to a particular embodiment of the present invention, an antibody that targets galectin-9 and inhibits the inhibitory activity of regulatory T lymphocytes, as previously defined, is used in combination with a second therapeutic agent (e.g., an anti-cancer agent) for treating a disease associated with the inhibitory activity of regulatory T lymphocytes.

[0167] That is, when the use is for treating cancer, the antibody can be used in combination with known therapies for cancer such as surgery, radiotherapy, 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 aspects, other anti-cancer agents used in combination with the antibody against galectin-9 according to the present invention for cancer treatment include anti-angiogenic agents. According to certain aspects, the antibody can be co-administered with cytokines, such as cytokines that stimulate an anti-tumor immune response.

[0168] In such combination therapies, the antibody of the present invention can be used before, after, or simultaneously with the second therapeutic agent. See other sections below regarding combination therapies.

[0169] 4. Routes of Administration and Carriers

[0170] In various embodiments, the Gal9 antagonist (e.g., Gal9 Ab) can be administered subcutaneously or intravenously. For simplicity, the "Gal9 antagonist" herein refers in a narrow sense to the Gal1 antibody of the present invention, such as the humanized Gal9 antibody of the present invention.

[0171] In some embodiments, the Gal9 antagonist can be administered in vivo by various routes, including but not limited to oral, intra-arterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, by inhalation, intradermal, topical, transdermal, and intrathecal, or otherwise, such as by implantation.

[0172] In some embodiments, the Gal9 antagonist is an anti-Gal9 antibody or an antigen-binding fragment thereof and is administered intravenously (i.v.) or subcutaneously (s.c.).

[0173] The subject compositions can be formulated into preparations in solid, semi-solid, liquid or gaseous forms; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols.

[0174] In various embodiments, compositions comprising a Gal9 antagonist are provided in formulations with a variety of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)). A variety of pharmaceutically acceptable carriers including vehicles, adjuvants, and diluents are available. In addition, various pharmaceutically acceptable auxiliary substances, such as pH regulators and buffers, tonicity regulators, stabilizers, wetting agents, etc. are also available. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0175] In various embodiments, compositions comprising a Gal9 antagonist can be formulated for injection, including subcutaneous administration, by dissolving, suspending, or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable oil or other oils, synthetic fatty acid glycerides, higher fatty acid esters, or propylene glycol; and, if desired, used in combination with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers, and preservatives.

[0176] In various embodiments, the composition can be formulated for inhalation, for example, using a pressurized acceptable propellant such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0177] In various embodiments, the composition can also be formulated as sustained-release microcapsules, for example, having a biodegradable or non-biodegradable polymer. Non-limiting exemplary biodegradable formulations include poly(lactic-co-glycolic acid) (PLGA) polymers. Non-limiting exemplary non-biodegradable formulations include polyglyceryl fatty acid esters. Some methods of preparing such formulations are described in, for example, EP 1125584 A1.

[0178] There is also provided a pharmaceutical dosage package comprising one or more containers, each container containing one or more doses of a Gal9 antagonist. In some embodiments, unit doses are provided, wherein the unit dose contains a predetermined amount of a composition comprising a Gal9 antagonist, with or without one or more additional agents. In some embodiments, such unit doses are provided in a disposable pre-filled syringe for injection. In various embodiments, the composition contained in the unit dose can comprise saline, sucrose, and the like; buffers such as phosphates, and the like; and / or be formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition can be provided as a lyophilized powder, which can be reconstituted upon addition of a suitable liquid (e.g., 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.

[0179] The pharmaceutical composition is administered in an amount effective for treating or preventing a particular indication. A therapeutically effective amount generally depends on the body weight of the subject being treated, his or her physical or health condition, the extent of the condition to be treated, or the age of the subject being treated.

[0180] In some embodiments, the Gal9 antagonist can be administered in an amount in the range of about 50 μg / kg body weight to about 50 mg / kg body weight per dose. In some embodiments, the Gal9 antagonist can be administered in an amount in the range of about 100 μg / kg body weight to about 50 mg / kg body weight per dose. In some embodiments, the Gal9 antagonist can be administered in an amount in the range of about 100 μg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the Gal9 antagonist can be administered in an amount in the range of about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose.

[0181] In some embodiments, the Gal9 antagonist can be administered in an amount in the range of about 10 mg to about 1,000 mg per dose. In some embodiments, the Gal9 antagonist can be administered in an amount in the range of about 20 mg to about 500 mg per dose. In some embodiments, the Gal9 antagonist can be administered in an amount in the range of about 20 mg to about 300 mg per dose. In some embodiments, the Gal9 antagonist can be administered in an amount in the range of about 20 mg to about 200 mg per dose.

[0182] The Gal9 antagonist composition can be administered to a subject as needed. In some embodiments, an effective dose of the Gal9 antagonist is administered to the subject one or more times. In various embodiments, an effective dose of the GAL9 antagonist is administered to the subject once a month, less than once a month (e.g., every two months, every three months, or every six months). In other embodiments, an effective dose of the Gal9 antagonist is administered more than once a month, such as every two weeks, weekly, twice a week, three times a week, once a day, or multiple times a day. An effective dose of the Gal9 antagonist is administered to the subject at least once. In some embodiments, an effective dose 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 the subject as needed to alleviate one or more symptoms of the disorder.

[0183] 5. Combination Therapy

[0184] The Gal9 antagonists of the present invention, including any antibodies and functional fragments thereof, can be administered in combination with other bioactive substances or other therapeutic procedures for treating diseases to a subject in need thereof. For example, the Gal9 antagonists can be administered alone or in combination with other treatment modalities. They can be provided before, substantially simultaneously with, or after other treatment modalities (e.g., radiotherapy).

[0185] For treating cancer, the Gal9 antagonist can be administered in combination with one or more anti-cancer agents, such as immune checkpoint inhibitors, chemotherapeutic agents, growth inhibitory agents, anti-angiogenic agents, or anti-tumor compositions.

[0186] In certain embodiments, a Gal9 antagonist that specifically binds Gal9 (“Gal9-binding antagonist”), e.g., a Gal9 antagonist antibody or an antigen-binding fragment thereof, is administered to a subject having a disease (e.g., cancer or an infectious disease) in which stimulation of the immune system would be beneficial, in combination with a second antagonist such as an immune checkpoint inhibitor (e.g., an inhibitor of the PD-1 or PD-L1 pathway). The two antagonists can be administered simultaneously or sequentially, e.g., as described below for combinations of GAL9 antagonists with immuno-oncology agents. One or more additional therapeutic agents (e.g., checkpoint modulators) can be added to the treatment of cancer or an infectious disease with a Gal9-binding antagonist.

[0187] In certain embodiments, a Gal9 antagonist is administered to a subject, e.g., a subject having cancer, simultaneously or sequentially with another therapy. For example, the Gal9 antagonist can be administered in combination with one or more of: radiotherapy, surgery, or chemotherapy, e.g., targeted chemotherapy or immunotherapy.

[0188] Immunotherapy, e.g., cancer immunotherapy, includes cancer vaccines and immuno-oncology agents. A Gal9 antagonist can be, e.g., a protein, antibody, antibody fragment, or small molecule that binds Gal9. A Gal9 antagonist can be an antibody or an antigen-binding fragment thereof that specifically binds Gal9.

[0189] In certain embodiments, a method of treating a subject having cancer comprises administering to the subject having cancer a Gal9 antagonist (e.g., a Gal9 antibody) and one or more immuno-oncology agents (such as an immune checkpoint inhibitor).

[0190] Immunotherapy (e.g., therapy with immuno-oncology agents) is effective for enhancing, stimulating, and / or upregulating the immune response in a subject. In one aspect, the administration of a Gal9 antagonist in combination with an immuno-oncology agent (such as a PD-1 inhibitor) has a synergistic effect in the treatment of cancer, e.g., in inhibiting tumor growth.

[0191] In one aspect, a Gal9 antagonist is administered sequentially before an immuno-oncology agent. In one aspect, the Gal9 antagonist is administered concurrently with an immuno-oncology agent (e.g., a PD-1 inhibitor). In yet another aspect, a Gal9 antagonist is administered sequentially after an immuno-oncology agent (such as a PD-1 inhibitor). Administration of the two agents can be initiated, for example, at intervals of 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, or administration of the second agent can be initiated, 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 administration of the first agent.

[0192] In certain aspects, the Gal9 antagonist and an immuno-oncology agent (e.g., a PD-1 inhibitor) are administered concurrently, such as by concurrent infusion, for example, over a period of 30 or 60 minutes to a patient. The Gal9 antagonist can be co-formulated with an immuno-oncology agent (such as a PD-1 inhibitor).

[0193] Immuno-oncology agents include, for example, small molecule drugs, antibodies or fragments thereof, or other biologics or small molecules. Examples of biologic immuno-oncology agents include, but are not limited to, antibodies, antibody fragments, vaccines, and cytokines. In one aspect, the antibody is a monoclonal antibody. In certain aspects, the monoclonal antibody is a humanized antibody or a human antibody.

[0194] In one aspect, the immuno-oncology agent is an agonist of (i) a stimulatory (including co-stimulatory) 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 amplification of an antigen-specific T cell response. In certain aspects, the immuno-oncology agent is an agonist of (i) a stimulatory (including co-stimulatory) molecule (e.g., a receptor or ligand) on a cell involved in innate immunity (e.g., an NK cell), or (ii) an antagonist of an inhibitory (including co-inhibitory) molecule (e.g., a receptor or ligand), and wherein the immuno-oncology agent enhances innate immunity. Such immuno-oncology agents are commonly referred to as immune checkpoint modulators, such as immune checkpoint inhibitors or immune checkpoint stimulators.

[0195] In certain embodiments, the immuno-oncology agent can be an agent that targets (or specifically binds to) a member of the B7 family of membrane-bound ligands, the members of which include 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 co-stimulatory or co-inhibitory receptor that specifically binds to a B7 family member. The immuno-oncology agent can be an agent that targets a member of the TNF family of membrane-bound ligands or a co-stimulatory or co-inhibitory receptor that specifically binds thereto (e.g., a member of the TNF receptor family). Exemplary TNF and TNFR family members that can be targeted by the immuno-oncology agent 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, RANK, RANKL, TWEAKR / Fnl4, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTfiR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin α / TNΡβ, TNFR2, TNFa, LTfiR, lymphotoxin a1β2, FAS, FASL, RELT, DR6, TROY, and NGFR. The immuno-oncology agent that can be combined with the Gal9 antagonist for treating cancer can be an agent that targets a B7 family member, a B7 receptor family member, a TNF family member, or a TNFR family member, such as an antibody, such as those described above.

[0196] In one aspect, the Gal9 antagonist is administered in combination with one or more of the following: (i) antagonists of 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 (e.g., immune checkpoint inhibitors), and (ii) agonists of proteins that stimulate T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, GITR, TRGIL, CD70, CD27, CD40L, DR3, and CD28H.

[0197] In one aspect, the immuno-oncology agent is an agent that inhibits (i.e., antagonizes) cytokines that inhibit T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF, and other immunosuppressive cytokines), or an agonist of cytokines that stimulate T cell activation and stimulate an immune response, such as IL-2, IL-7, IL-12, IL-15, IL-21, and IFNα (e.g., the cytokine itself).

[0198] Other agents that can be combined with a Gal9 antagonist for stimulating the immune system (e.g., for treating cancer and infectious diseases) include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, an anti-Gal9 antagonist can be combined with an antagonist of KIR.

[0199] Other agents for combination therapy also include agents that inhibit or deplete macrophages or monocytes, including but not limited to CSF-1R antagonists, such as CSF-1R antagonist antibodies, including RG7155 (WO11 / 70024, WO11 / 107553, WO11 / 131407, WO13 / 87699, WO13 / 119716, WO13 / 132044) or FPA008 (WO11 / 140249; WO13169264; WO14 / 036357).

[0200] The immuno-oncology agent also includes an agent that inhibits TGF-β signaling.

[0201] Additional agents that can be combined with a Gal9 antagonist include agents that enhance tumor antigen presentation, such as dendritic cell vaccines, GM-CSF secreting cell vaccines, CpG oligonucleotides, and imiquimod, or therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines).

[0202] Other therapies that can be combined with a Gal9 antagonist include therapies that deplete or block Treg cells, such as agents that specifically bind CD25.

[0203] Another therapy that can be combined with a Gal9 antagonist is a therapy that inhibits metabolic enzymes, such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase.

[0204] Another class of agents that can be used includes agents that inhibit adenosine formation or inhibit the adenosine A2A receptor.

[0205] Other therapies that can be combined with a Gal9 antagonist for treating 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.

[0206] Gal9 antagonists can be combined with more than one immuno - oncology reagent (e.g., immune checkpoint inhibitors), and can be combined, for example, with combination methods targeting multiple elements 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 cell vaccines, CpG oligonucleotides, imiquimod); therapies that inhibit negative immune regulation, such as by inhibiting the CTLA - 4 and / or PD1 / PD - L1 / PD - L2 pathways and / or depleting or blocking Tregs or other immunosuppressive cells; therapies that stimulate positive immune regulation, such as with agonists that stimulate the CD - 137, OX - 40, and / or GITR pathways and / or stimulate T - cell effector function; therapies that systemically increase the frequency of anti - tumor T cells; therapies that deplete or inhibit Tregs (e.g., Tregs in tumors), such as using antagonists of CD25 (e.g., daclizumab) or depletion by ex vivo anti - CD25 beads; therapies that affect the function of inhibitory myeloid cells in tumors; therapies that enhance the immunogenicity of tumor cells (e.g., anthracyclines); adoptive T - cell or NK - cell transfer, including genetically modified cells, such as cells modified by chimeric antigen receptors (CAR - T therapy); 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 of immunosuppressive cytokines.

[0207] For example, Gal9 antagonists can be used in combination with one or more agonists that ligate positive co - stimulatory receptors; one or more antagonists (blockers) that attenuate signaling through inhibitory receptors, such as antagonists that overcome different immunosuppressive pathways within the tumor microenvironment (e.g., blocking the PD - L1 / PD - 1 / PD - L2 interaction); one or more agents that systemically increase the frequency of anti - tumor immune cells (e.g., T cells), 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.

[0208] In one embodiment, a subject suffering from a disease (e.g., cancer or an infectious disease) that can benefit from stimulation of the immune system is treated by administering a Gal9 antagonist and an immuno - oncology reagent to the subject, wherein the immuno - oncology reagent is a CTLA - 4 antagonist, such as an antagonistic CTLA - 4 antibody. Suitable CTLA - 4 antibodies include, for example, YERVOY (ipilimumab) or tremelimumab.

[0209] In one embodiment, a subject suffering from a disease that would benefit from stimulation of the immune system (such as cancer or an infectious disease) is treated by administering a Gal9 antagonist and an immuno - oncology agent to the subject, wherein the immuno - oncology agent is a PD - 1 antagonist, such as an antagonistic PD - 1 antibody. Suitable PD - 1 antibodies include, for example, OPDIVO (nivolumab), KEYTRUDA (pembrolizumab), or MEDI - 0680 (AMP - 514; WO2012 / 145493). The immuno - oncology agent may also include pidilizumab (CT - 011). Another approach targeting the PD - 1 receptor is a recombinant protein consisting of the extracellular domain of PD - L2 (B7 - DC) fused to the Fc portion of IgG1, called AMP - 224.

[0210] In one embodiment, a subject suffering from a disease that would benefit from stimulation of the immune system (such as cancer or an infectious disease) is treated by administering a Gal9 antagonist and an immuno - oncology agent to the subject, wherein the immuno - oncology agent is a PD - L1 antagonist, such as an antagonistic PD - L1 antibody. Suitable PD - L1 antibodies include, for example, MPDL3280A (RG7446; WO2010 / 077634), durvalumab (MEDI4736), BMS - 936559 (WO2007 / 005874), MSB0010718C (WO2013 / 79174), or rHIgM12B7.

[0211] In one embodiment, a subject suffering from a disease that would benefit from stimulation of the immune system (such as cancer or an infectious disease) is treated by administering a Gal9 antagonist and an immuno - oncology agent to the subject, wherein the immuno - oncology agent is a LAG - 3 antagonist, such as an antagonistic LAG - 3 antibody. Suitable LAG3 antibodies include, for example, BMS - 986016 (WO10 / 19570, WO 14 / 08218) or IMP - 731 or IMP - 321 (WO08 / 132601, WO09 / 44273).).

[0212] In one embodiment, a subject suffering from a disease that would benefit from stimulation of the immune system (such as cancer or an infectious disease) is treated by administering a Gal9 antagonist and an immuno - oncology agent to the subject, wherein the immuno - oncology agent is a CD137 (4 - 1BB) agonist, such as an agonistic CD137 antibody. Suitable CD137 antibodies include, for example, urelumab or PF - 05082566 (WO12 / 32433).

[0213] In one embodiment, a subject having a disease that would benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering a Gal9 antagonist and an immuno-oncology agent to the subject, wherein the immuno-oncology agent is a GITR agonist, such as an agonistic GITR antibody. Suitable GITR antibodies include, for example, TRX-518 (WO06 / 105021, WO09 / 009116), MK-4166 (WO 11 / 028683), or the GITR antibodies disclosed in WO2015 / 031667.

[0214] In one embodiment, a subject having a disease that would benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering a Gal9 antagonist and an immuno-oncology agent to the subject, wherein the immuno-oncology agent is an OX40 agonist, such as an agonistic OX40 antibody. Suitable OX40 antibodies include, for example, MEDI-6383, MEDI-6469, or MOXR0916 (RG7888; WO06 / 029879).

[0215] In one embodiment, a subject having a disease that would benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering a Gal9 antagonist and an immuno-oncology agent to the subject, wherein the immuno-oncology agent is a CD40 agonist, such as an agonistic CD40 antibody. In certain embodiments, the immuno-oncology agent is a CD40 antagonist, such as an antagonistic CD40 antibody. Suitable CD40 antibodies include, for example, lucatumumab (HCD122), daratumumab (SGN-40), CP-870,893, or Chi Lob 7 / 4.

[0216] In one embodiment, a subject having a disease that would benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering a Gal9 antagonist and an immuno-oncology agent to the subject, wherein the immuno-oncology agent is a CD27 agonist, such as an agonistic CD27 antibody. Suitable CD27 antibodies include, for example, varlilumab (CDX-1127).

[0217] In one embodiment, a subject having a disease that would benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering a Gal9 antagonist and an immuno-oncology agent to the subject, wherein the immuno-oncology agent is MGA271 (against B7H3) (WO11 / 109400).

[0218] In one embodiment, a subject suffering from a disease that can benefit from stimulation of the immune system (such as cancer or an infectious disease) is treated by administering a Gal9 antagonist and an immuno-oncology agent to the subject, wherein the immuno-oncology agent is a KIR antagonist, such as lirilumab.

[0219] In one embodiment, a subject suffering from a disease that can benefit from stimulation of the immune system (such as cancer or an infectious disease) is treated by administering a Gal9 antagonist and an immuno-oncology agent to the subject, wherein the immuno-oncology agent is an IDO antagonist. Suitable IDO antagonists include, for example, INCB-024360 (WO2006 / 122150, WO07 / 75598, WO08 / 36653, WO08 / 36642), indoximod, NLG-919 (WO09 / 73620, WO09 / 1156652, WO11 / 56652, WO 12 / 142237) or F001287.

[0220] In one embodiment, a subject suffering from a disease that can benefit from stimulation of the immune system (such as cancer or an infectious disease) is treated by administering a Gal9 antagonist and an immuno-oncology agent to the subject, wherein the immuno-oncology agent is a Toll-like receptor agonist, such as a TLR2 / 4 agonist (such as Bacillus Calmette-Guerin); a TLR7 agonist (such as, Hiltonol or imiquimod); a TLR7 / 8 agonist (such as resiquimod); or a TLR9 agonist (such as CpG7909).

[0221] In one embodiment, a subject suffering from a disease that can benefit from stimulation of the immune system (such as cancer or an infectious disease) is treated by administering a Gal9 antagonist and an immuno-oncology agent to the subject, wherein the immuno-oncology agent is a TGF-β inhibitor, such as GC1008, LY2157299, TEW7197 or IMC-TR1.

[0222] 6. Exemplary Gal9 antagonists

[0223] 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 (such as, ECD) that inhibits the interaction between Gal9 and its ligand, optionally further comprising a fusion partner and in the form of a fusion molecule. In other embodiments, the antagonist can also be a small molecule or a small peptide.

[0224] Gal9 antibody

[0225] In some embodiments, antibodies that block the binding of Gal9 and its ligand are provided. In some embodiments, antibodies that inhibit Gal9-mediated signal transduction are provided. 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.

[0226] In some embodiments, for Gal9, such as human Gal9, the Gal9 antibody of the present invention has a dissociation constant (K d ) of ≤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., from 10 -8 M to 10 -13 M, e.g., from 10 -9 M to 10 -13 M). In certain embodiments, for Gal9, such as human Gal9, the Gal9 antibody has a dissociation constant (K d ) of ≤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., from 10 -8 M to 10 -13 M, e.g., from 10 -9 M to 10 -13 M).

[0227] In some embodiments, a Gal9 antibody having any of the features provided herein inhibits Gal9 signal transduction by at least 25%, 50%, 75%, 80%, 90%, or 100%.

[0228] In some embodiments, the antibody binds Gal9 from multiple species. For example, in some embodiments, the antibody binds human Gal9 and also binds Gal9 from at least one non-human mammal selected from the group consisting of mouse, rat, dog, guinea pig, and cynomolgus monkey.

[0229] 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 and a second arm, the first arm comprising a heavy chain / light chain combination that binds a first antigen, and the second arm comprising a heavy chain / light chain combination that binds a second antigen. A further non-limiting exemplary multispecific antibody is a bispecific variable domain antibody. 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 CD3. In some embodiments, the first arm binds Gal9.

[0230] In certain embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the present invention, including humanized monoclonal antibodies or antigen-binding fragments thereof, include one or more point mutations in the amino acid sequence, which are designed to improve the developability of the antibody. For example, Raybould et al. (Five computational developability guidelines for therapeutic antibody profiling, PNAS 116(10):4025-4030, 2019) describe the Therapeutic Antibody Profiler (TAP), which is 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 TAP, which is freely available at opig.stats.ox.ac.uk / webapps / sabdab-sabpred / TAP.php.

[0231] In addition to achieving the desired affinity for the antigen, there are many obstacles in therapeutic mAb development. These include innate 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 implicated in aggregation, viscosity, and multispecificity. Asymmetry in the net charge of the heavy and light chain variable domains is also associated with self-association and viscosity at high concentrations. Positive and negative charge patches in the CDRs are related to high clearance rates and poor expression levels. Product heterogeneity (e.g., by oxidation, isomerization, or glycosylation) is typically generated by specific sequence motifs that are prone to post-translational or co-translational modifications. Computational tools can be used to facilitate the identification of sequence defects. 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) also described methods for optimizing antibody affinity and stability by the automated design of the variable light-heavy chain interfaces. Additional methods can be used to identify potential developability issues with candidate antibodies, and in preferred embodiments of the present invention, one or more point mutations can be introduced into the candidate antibody by conventional methods to address these issues, thereby generating the optimized therapeutic antibodies of the present invention.

[0232] The sequences of certain representative antibodies are listed below, including the light chain (LC) and heavy chain (HC) variable regions, CDR regions, and framework regions (FR).

[0233] HFB9-1hz1-hG1AA

[0234] VH-CDR1: GYTFTDYTIH (SEQ ID NO: 2)

[0235] VH-CDR2: WFYPGSHSIKYAQKFQGR (SEQ ID NO: 4)

[0236] VH-CDR3: HGGYDGFDY (SEQ ID NO: 6)

[0237] HCVR:

[0238] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYTIHWVRQAPGQGLEWMGWFYPGSHSIKYAQKFQGRVTMTADTSISTAYMELSRLRSDDTAVYFCTRHGGYDGFDYWGQGTLVTVSS(SEQ ID NO:8)

[0239] VL-CDR1:KSSQSLFYSTNQKNYLA(SEQ ID NO:10)

[0240] VL-CDR2:WASTRES(SEQ ID NO:12)

[0241] VL-CDR3:QQYYYFPYT(SEQ ID NO:14)

[0242] LCVR:

[0243] DIVMTQSPDSLAVSLGERATINCKSSQSLFYSTNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYYFPYTFGQGTKLEIK(SEQ ID NO:16)

[0244] For all antibody heavy chain sequences, the framework region sequences HFR1 - HFR4 are defined by the VH - CDR sequences. For example, HFR1 is the sequence of the HCVR at the N - terminus of VH - CDR1. HFR2 is the sequence of the HCVR between VH - CDR1 and VH - CDR2. HFR3 is the sequence of the HCVR between VH - CDR2 and VH - CDR3. HFR4 is the most C - terminal sequence of the HCVR.

[0245] Similarly, for all antibody light chain sequences, the framework region sequences LFR1 - LFR4 are defined by the VL - CDR sequences. For example, LFR1 is the sequence of the LCVR at the N - terminus of VL - CDR1. LFR2 is the sequence of the LCVR between VL - CDR1 and VL - CDR2. LFR3 is the sequence of the LCVR between VL - CDR2 and VL - CDR3. LFR4 is the most C - terminal sequence of the LCVR.

[0246] The HFR1 - HFR4 sequences of HFB9 - 1Hz1 - hG1AA are SEQ ID NO: 1, 3, 5, and 7. The LFR1 - LFR4 sequences of HFB9 - 1Hz1 - hG1AA are SEQ ID NO: 9, 11, 13, and 15.

[0247] HFB9-1hz2-hG1AA

[0248] VH-CDR1: GYTFTDYTIH (SEQ ID NO: 18)

[0249] VH-CDR2: WFYPGSHSIKYAQKFQGR (SEQ ID NO: 20)

[0250] VH-CDR3: HGGYDGFDY (SEQ ID NO: 22)

[0251] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYTIHWVRQAPGQGLEWMGWFYPGSHSIKYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYFCTRHGGYDGFDYWGQGTLVTVSS (SEQ ID NO: 24)

[0252] VL-CDR1: KSSQSLFYSTNQKNYLA (SEQ ID NO: 26)

[0253] VL-CDR2: WASTRES (SEQ ID NO: 28)

[0254] VL-CDR3: QQYYYFPYT (SEQ ID NO: 30)

[0255] DIVMTQSPDSLAVSLGERATINCKSSQSLFYSTNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYYFPYTFGQGTKLEIK (SEQ ID NO: 32)

[0256] The HFR1-HFR4 sequences of HFB9-1Hz2-hG1AA are SEQ ID NO: 17, 19, 21 and 23. The LFR1-LFR4 sequences of HFB9-1Hz2-hG1AA are SEQ ID NO: 25, 27, 29 and 31.

[0257] HFB9-1hz3-hG1AA

[0258] VH-CDR1: GYTFTDYTIH (SEQ ID NO: 34)

[0259] VH-CDR2: WFYPGSHSIKYAQKFQGR (SEQ ID NO: 36)

[0260] VH-CDR3: HGGYDGFDY (SEQ ID NO: 38)

[0261] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYTIHWVRQAPGQGLEWMGWFYPGSHSIKYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCTRHGGYDGFDYWGQGTLVTVSS (SEQ ID NO: 40)

[0262] VL-CDR1: KSSQSLFYSTNQKNYLA (SEQ ID NO: 42)

[0263] VL-CDR2: WASTRES (SEQ ID NO: 44)

[0264] VL-CDR3: QQYYYFPYT (SEQ ID NO: 46)

[0265] DIVMTQSPDSLAVSLGERATINCKSSQSLFYSTNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYYFPYTFGQGTKLEIK (SEQ ID NO: 48)

[0266] The HFR1 - HFR4 sequences of HFB9 - 1Hz3 - hG1AA are SEQ ID NO: 33, 35, 37 and 39. The LFR1 - LFR4 sequences of HFB9 - 1Hz3 - hG1AA are SEQ ID NO: 41, 43, 45 and 47.

[0267] HFB9 - 1hz4 - hG1AA

[0268] VH-CDR1: GYTFTDYTIH (SEQ ID NO: 50)

[0269] VH-CDR2: WFYPGSHSIKYAQKFQGR (SEQ ID NO: 52)

[0270] VH-CDR3: HGGYDGFDY (SEQ ID NO: 54)

[0271] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYTIHWVRQAPGQGLEWMGWFYPGSHSIKYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARHGGYDGFDYWGQGTLVTVSS(SEQ ID NO:56)

[0272] VL-CDR1: KSSQSLFYSTNQKNYLA(SEQ ID NO:58)

[0273] VL-CDR2: WASTRES(SEQ ID NO:60)

[0274] VL-CDR3: QQYYYFPYT(SEQ ID NO:62)

[0275] DIVMTQSPDSLAVSLGERATINCKSSQSLFYSTNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYYFPYTFGQGTKLEIK(SEQ ID NO:64)

[0276] The HFR1 - HFR4 sequences of HFB9 - 1Hz4 - hG1AA are SEQ ID NO:49, 51, 53 and 55. The LFR1 - LFR4 sequences of HFB9 - 1Hz4 - hG1AA are SEQ ID NO:57, 59, 61 and 63.

[0277] HFB9 - 2hz11 - hG1AA

[0278] VH-CDR1: GYTFTEYTIH(SEQ ID NO:66)

[0279] VH-CDR2: WFYPGSGSTEYAQKFQG(SEQ ID NO:68)

[0280] VH-CDR3: HGGYDGFDY(SEQ ID NO:70)

[0281] QVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGQGLEWMGWFYPGSGSTEYAQKFQGRVTMTADTSISTAYMELSRLRSDDTAVYFCERHGGYDGFDYWGQGTTVTVSS(SEQ ID NO:72)

[0282] VL-CDR1: KSSQSLLYSNNQKNYLA (SEQ ID NO: 74)

[0283] VL-CDR2: WASTRGS (SEQ ID NO: 76)

[0284] VL-CDR3: QQYYSYPFT (SEQ ID NO: 78)

[0285] DIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNQKNYLAWYQQKPGQPPKLLIYWASTRGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPFTFGGGTKVEIK (SEQ ID NO: 80)

[0286] The HFR1 - HFR4 sequences of HFB9 - 2Hz11 - hG1AA are SEQ ID NO: 65, 67, 69 and 71. The LFR1 - LFR4 sequences of HFB9 - 2Hz11 - hG1AA are SEQ ID NO: 73, 75, 77 and 79.

[0287] HFB9 - 2hz12 - hG1AA

[0288] VH-CDR1: GYTFTEYTIH (SEQ ID NO: 82)

[0289] VH-CDR2: WFYPGSGSAEYAQKFQG (SEQ ID NO: 84)

[0290] VH-CDR3: HGGYDGFDY (SEQ ID NO: 86)

[0291] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTEYTIHWVRQAPGQGLEWMGWFYPGSGSAEYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCERHGGYDGFDYWGQGTTVTVSS (SEQ ID NO: 88)

[0292] VL-CDR1: KSSQSLLYSNNQKNYLA (SEQ ID NO: 90)

[0293] VL-CDR2: WASTRGS (SEQ ID NO: 92)

[0294] VL-CDR3: QQYYSYPFT (SEQ ID NO: 94)

[0295] DIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNQKNYLAWYQQKPGQPPKLLIYWASTRGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPFTFGGGTKVEIK (SEQ ID NO: 96)

[0296] The HFR1 - HFR4 sequences of HFB9 - 2hz12 - hG1AA are SEQ ID NO: 81, 83, 85, and 87. The LFR1 - LFR4 sequences of HFB9 - 2hz12 - hG1AA are SEQ ID NO: 89, 91, 93, and 95.

[0297] HFB9 - 2hz13 - hG1AA

[0298] VH - CDR1: GYTFTEYTIH (SEQ ID NO: 98)

[0299] VH - CDR2: WFYPGSGSTEYAQKFQG (SEQ ID NO: 100)

[0300] VH - CDR3: HGGYDGFDY (SEQ ID NO: 102)

[0301] QVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGQGLEWMGWFYPGSGSTEYAQKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYYCERHGGYDGFDYWGQGTTVTVSS (SEQ ID NO: 104)

[0302] VL - CDR1: KSSQSLLYSNNQKNYLA (SEQ ID NO: 106)

[0303] VL - CDR2: WASTRGS (SEQ ID NO: 108)

[0304] VL - CDR3: QQYYSYPFT (SEQ ID NO: 110)

[0305] DIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNQKNYLAWYQQKPGQPPKLLIYWASTRGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPFTFGGGTKVEIK (SEQ ID NO: 112)

[0306] The HFR1 - HFR4 sequences of HFB9 - 2Hz13 - hG1AA are SEQ ID NO: 97, 99, 101 and 103, and the LFR1 - LFR4 sequences of hFB9 - 2Hz13 - hG1AA are SEQ ID NO: 105, 107, 109 and 111.

[0307] HFB9 - 2hz14 - hG1AA

[0308] VH - CDR1: GYTFTEYTIH (SEQ ID NO: 114)

[0309] VH - CDR2: WFYPGSGSTEYSPSFQG (SEQ ID NO: 116)

[0310] VH - CDR3: HGGYDGFDY (SEQ ID NO: 118)

[0311] EVQLVQSGAEVKKPGESLKISCKGSGYTFTEYTIHWVRQMPGKGLEWMGWFYPGSGSTEYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCERHGGYDGFDYWGQGTTVTVSS (SEQ ID NO: 120)

[0312] VL - CDR1: KSSQSLLYSNNQKNYLA (SEQ ID NO: 122)

[0313] VL - CDR2: WASTRGS (SEQ ID NO: 124)

[0314] VL - CDR3: QQYYSYPFT (SEQ ID NO: 126)

[0315] DIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNQKNYLAWYQQKPGQPPKLLIYWASTRGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPFTFGGGTKVEIK (SEQ ID NO: 128)

[0316] The HFR1 - HFR4 sequences of HFB9 - 2hz14 - hG1AA are SEQ ID NO: 113, 115, 117 and 119. The LFR1 - LFR4 sequences of HFB9 - 2hz14 - hG1AA are SEQ ID NO: 121, 123, 125 and 127.

[0317] 7. Humanized Antibodies

[0318] In some embodiments, the Gal9 antibody is a humanized antibody. Humanized antibodies can be used as therapeutic molecules because they reduce or eliminate the human immune response to non-human antibodies (e.g., human anti-mouse antibody (HAMA) response), which can lead to an immune response against the antibody therapeutic agent and reduce the effectiveness of the therapeutic agent.

[0319] Antibodies can be humanized by any standard method. Non-limiting exemplary methods of humanization include, for example, the methods described in U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; 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. Publication No. US 2009 / 0136500. All are incorporated by reference.

[0320] A humanized antibody is an antibody in which at least one amino acid in the framework region of the non-human variable region has been replaced with an amino acid from the corresponding position in a human framework region. In 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 10, at least 11, at least 12, at least 15, or at least 20 amino acids in the framework region of the non-human variable region are replaced with amino acids from one or more corresponding positions in one or more human framework regions.

[0321] In some embodiments, some of the corresponding human amino acids used for replacement are from the framework regions of different human immunoglobulin genes. That is, in some such embodiments, one or more non-human amino acids can be replaced with the corresponding amino acids from the human framework region of a first human antibody or encoded by a first human immunoglobulin gene, one or more non-human amino acids can be replaced with the corresponding amino acids from the human framework region of a second human antibody or encoded by a second human immunoglobulin gene, one or more non-human amino acids can be replaced with the corresponding amino acids from the human framework region of a third human antibody or encoded by a third human immunoglobulin gene, and so on. In addition, in some embodiments, all of the corresponding human amino acids used for replacement in a single framework region (e.g., FR2) do not need to be from the same human framework. However, in some embodiments, all of the corresponding human amino acids used for replacement are from the same human antibody or encoded by the same human immunoglobulin gene.

[0322] In some embodiments, an antibody is humanized by replacing one or more entire framework regions with the corresponding human framework regions. In some embodiments, the human framework regions are selected that have the highest level of homology to the non-human framework regions being replaced. In some embodiments, such humanized antibodies are CDR-grafted antibodies.

[0323] In some embodiments, after CDR grafting, one or more framework amino acids are changed back to the corresponding amino acids in the murine framework region. In some embodiments, such "back mutations" are made to preserve one or more murine framework amino acids that appear to contribute to the structure of one or more CDRs and / or that may be involved in antigen contact and / or that appear to be involved in the overall structural integrity of the antibody. In some embodiments, after CDR grafting, the framework region of the antibody is subjected to ten or fewer, nine or fewer, eight or fewer, seven or fewer, six or fewer, five or fewer, four or fewer, three or fewer, two or fewer, one or zero back mutations.

[0324] In some embodiments, the humanized antibody also comprises a human heavy chain constant region and / or a human light chain constant region.

[0325] 8. Human Antibodies

[0326] In some embodiments, the Gal9 antibody is a human antibody. Human antibodies can be prepared by any suitable method. Non-limiting exemplary methods include preparing human antibodies in transgenic mice comprising human immunoglobulin loci. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551-55 (1993); Jakobovits et al, Nature 362:255-8 (1993); Lonberg 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.

[0327] Non-limiting exemplary methods also include using phage display libraries to prepare human antibodies. See, e.g., Hoogenboom et al., J. Mol. Biol. 227:381-8 (1992); Marks et al, J. Mol. Biol. 222:581-97 (1991); and PCT Publication No. WO 99 / 10494.

[0328] Human Antibody Constant Regions

[0329] 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 has an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region has an isotype selected from κ 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 an S241P mutation in the human IgG4 constant region. See, e.g., 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 κ light chain.

[0330] The choice of heavy chain constant region can determine whether the antibody will have effector functions in vivo. In some embodiments, such effector functions include antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), and can result in the killing of cells bound by the antibody. Generally, antibodies comprising human IgG1 or IgG3 heavy chains have effector functions.

[0331] In some embodiments, effector functions are not desired. For example, in some embodiments, effector functions may be undesirable in the treatment of inflammatory 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 an S241P mutation.

[0332] Any of the antibodies described herein can be purified by any suitable method. Such methods include, but are not limited to, using an affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include the antigen and / or epitope to which the antibody binds, and ligands that bind to the antibody constant region. For example, protein A, protein G, protein A / G, or an antibody affinity column can be used to bind the constant region and purify the antibody.

[0333] In some embodiments, hydrophobic interaction chromatography (HIC), such as a butyl or phenyl column, is also used to purify some polypeptides. Many methods for purifying polypeptides are known in the art.

[0334] Alternatively, 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).

[0335] 9. Nucleic acid molecule encoding a Gal9 antagonist

[0336] The invention also provides nucleic acid molecules comprising polynucleotides encoding one or more chains of the antibodies described herein (e.g., gal9 antibodies). In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding the heavy or light chain of the antibody described herein. In some embodiments, the nucleic acid molecule comprises both a polynucleotide encoding the heavy chain of the antibody described herein and a polynucleotide encoding the light chain of the antibody described herein. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide encoding a heavy chain, and a second nucleic acid molecule comprises a second polynucleotide encoding a light chain.

[0337] 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 a scFv, a single polynucleotide encodes a single polypeptide comprising both the heavy and light chains linked together.

[0338] In some embodiments, the polynucleotide encoding the heavy or light chain of the antibody described herein comprises a nucleotide sequence encoding a leader sequence that, when translated, is located at the N-terminus of the heavy or light chain. As described above, the leader sequence can be the native heavy or light chain leader sequence, or it can be another heterologous leader sequence.

[0339] The nucleic acid molecules can be constructed using conventional recombinant DNA techniques in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell (e.g., a mammalian cell).

[0340] 10. Vector

[0341] Provide a vector comprising a polynucleotide encoding a heavy chain and / or a light chain of an antibody described herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. 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 chain and the light chain are expressed from the vector as two separate polypeptides. In some embodiments, the heavy chain and the light chain are expressed as part of a single polypeptide, such as when the antibody is a scFv.

[0342] 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 a host cell in similar amounts (e.g., similar molar amounts or similar mass amounts). In some embodiments, the first vector and the second vector with a molar ratio or mass ratio between 5:1 and 1:5 are transfected into a host cell. In some embodiments, a vector encoding a heavy chain and a vector encoding a light chain with a mass ratio of 1:1 to 1:5 are used. In some embodiments, a vector encoding a heavy chain and a vector encoding a light chain with a mass ratio of 1:2 are used.

[0343] In some embodiments, a vector optimized for expressing a polypeptide in CHO or CHO-derived cells or NSO cells is selected. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004). In some embodiments, a vector for in vivo expressing a Gal9 antagonist in an animal (including a human) is selected. In some such embodiments, the expression of one or more polypeptides is under the control of one or more promoters that act in a tissue-specific manner. For example, liver-specific promoters are described, for example, in PCT Publication No. WO 2006 / 076288.

[0344] 11. Host Cell

[0345] In various embodiments, the heavy chain and / or the light chain of an antibody described herein can be expressed in a prokaryotic cell, such as a bacterial cell; or a eukaryotic cell, such as a fungal cell (e.g., yeast), a plant cell, an insect cell, and a mammalian cell. Such expression can be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells useful for expressing a polypeptide include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; Cells (Crucell); and NS0 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. Publication No. US 2006 / 0270045 A1. In some embodiments, a particular eukaryotic host cell is selected based on the ability to effect desired post-translational modifications to the heavy and / or light chains of the Gal9 antibody. For example, in some embodiments, polypeptides produced by CHO cells have a higher level of sialylation than the same polypeptide produced in 293 cells.

[0346] Introduction of one or more nucleic acids into a desired host cell can be accomplished by any method, including but not limited to calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc., and non-limiting exemplary methods are described in, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected into a desired host cell according to any suitable method.

[0347] In some embodiments, one or more polypeptides can be produced in vivo in an animal that has been engineered or transfected with one or more nucleic acid molecules encoding the polypeptide according to any suitable method. Examples

[0348] Example 1 Humanized Anti-Gal-9 Antibodies Have High Binding Affinity for Human and Mouse Galectin-9

[0349] This example demonstrates that the various anti-human Gal-9 humanized antibodies of the invention have high binding affinity for both human and mouse Gal-9.

[0350] The antibody binding affinity was measured using the commercially available Octet system from ForteBio (Creative Biolabs). According to the description of Creative Biolabs, the Octet platform is based on biolayer interferometry (BLI) technology, which utilizes a complete set of systems including an instrument, biosensors, reagents, and assay kits to support the evaluation of biomolecular interactions in 96-well or 384-well microtiter plates. The Octet system uses a Dip-and-Read assay mode to avoid the need for microfluidics and enables real-time, label-free analysis of affinity and kinetics. There are three biosensor-based assay orientations available for exploring antibody interactions: tandem blockade, premix blockade, and classical sandwich. Compared with Biacore, the Dip-and-Read assay of Octet allows for a longer analyte binding step and facilitates the rebinding of the analyte to the ligand-coated sensor. At the same time, the faster association time consumes less sample, which saves your precious protein.

[0351] Also according to the description of Creative Biolabs, the principle of BLI technology is based on the optical interference pattern of white light reflected from two surfaces (the immobilized protein layer and the internal reference layer). The binding between the ligand immobilized on the surface of the biosensor tip and the analyte in solution produces an increase in the optical thickness at the biosensor tip, which results in a shift in the interference pattern measured in nanometers. The wavelength shift (Δλ) is a direct measure of the change in the optical thickness of the biolayer. When this shift is measured over a period of time and its magnitude is plotted as a function of time, a classical association / dissociation curve is obtained. This interaction is measured in real time, providing the ability to monitor binding specificity, association rate, dissociation rate, and concentration with significant precision and accuracy.

[0352] Using this system, the antibody affinities of the selected humanized antibodies of the present invention against recombinant human and mouse Gal-9 were measured, and the results are summarized in the following table. The data show that the tested humanized antibodies have high affinities in the mid- to low nM range for human and mouse Gal-9 proteins / antigens.

[0353]

[0354] Sequence alignments of the selected humanized antibodies of the present invention were performed using standard sequence alignment software, and the results are shown in Figure 1 and 2 Specifically, in Figure 1 the V H and V L regions of 4 humanized antibodies were aligned to show their respective V H and VL Amino acid residue changes compared to the [region]. Humanization mainly altered the amino acid sequences in the framework regions of the heavy and light chain variable regions (HCVR and LCVR). However, extensive changes also occurred within the heavy chain CDR2 sequence (see Figure 1 ).

[0355] Similarly, in Figure 2 , the V H and V L regions of 6 humanized antibodies were aligned to show amino acid residue changes compared to the original V H and V L regions of the human-mouse chimeric antibody HFB9-2, respectively. Humanization mainly altered the amino acid sequences in the framework regions of the heavy and light chain variable regions (HCVR and LCVR). However, for one humanized antibody, extensive changes also occurred within the heavy chain CDR2 sequence and within one residue in the heavy chain CDR1 sequence (see Figure 2 ).

[0356] Example 2 Anti-Gal9 antibodies showed sub-nanomolar (nM) affinity for Gal9

[0357] This experiment confirmed that the humanized antibodies of the present invention showed very high (sub-nanomolar) affinity for recombinant human GAL9 and cross-reacted with recombinant mouse Gal9 and simian Gal9 (data not shown). At increasing concentrations of each antibody, the EC50 values of each tested humanized antibody were measured, and the results were listed in the table in Figure 3 for binding to recombinant human Gal9, and the results were listed in the table in Figure 4 for binding to recombinant mouse Gal9.

[0358] Obviously, except for the 1hz4 antibody, all three humanized variants of HFB9-1 showed sub-nM level affinity for human and mouse Gal-9 (see Figure 3 and 4 ). Meanwhile, 5 out of 6 humanized HFB9-2 antibodies (except 2hz12) showed sub-nM level affinity for human Gal9, but only 4 out of 5 (except 2h14) maintained sub-nM level affinity for mouse Gal9.

[0359] Strong cross-reactivity against the simian ortholog Gal9 was also observed (data not shown).

[0360] The sequences of these representative antibodies are listed below, including the light chain (LC) and heavy chain (HC) variable regions, CDR regions, and framework regions (FR).

[0361] HFB9-1hz1-hG1AA

[0362] VH-CDR1: GYTFTDYTIH (SEQ ID NO: 2)

[0363] VH-CDR2: WFYPGSHSIKYAQKFQGR (SEQ ID NO: 4)

[0364] VH-CDR3: HGGYDGFDY (SEQ ID NO: 6)

[0365] HCVR:

[0366] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYTIHWVRQAPGQGLEWMGWFYPGSHSIKYAQKFQGRVTMTADTSISTAYMELSRLRSDDTAVYFCTRHGGYDGFDYWGQGTLVTVSS (SEQ ID NO: 8)

[0367] VL-CDR1: KSSQSLFYSTNQKNYLA (SEQ ID NO: 10)

[0368] VL-CDR2: WASTRES (SEQ ID NO: 12)

[0369] VL-CDR3: QQYYYFPYT (SEQ ID NO: 14)

[0370] LCVR:

[0371] DIVMTQSPDSLAVSLGERATINCKSSQSLFYSTNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYYFPYTFGQGTKLEIK (SEQ ID NO: 16)

[0372] For all antibody heavy chain sequences, the framework region sequences HFR1 - HFR4 are defined by the VH-CDR sequences. For example, HFR1 is the sequence of HCVR located at the N-terminus of VH-CDR1. HFR2 is the sequence of HCVR located between VH-CDR1 and VH-CDR2. HFR3 is the sequence of HCVR located between VH-CDR2 and VH-CDR3. HFR4 is the most C-terminal sequence of HCVR.

[0373] Similarly, for all antibody light chain sequences, the framework region sequences LFR1 - LFR4 are defined by the VL - CDR sequences. For example, LFR1 is the sequence of LCVR at the N - terminus of VL - CDR1. LFR2 is the sequence of LCVR between VL - CDR1 and VL - CDR2. LFR3 is the sequence of LCVR between VL - CDR2 and VL - CDR3. LFR4 is the most C - terminal sequence of LCVR.

[0374] The HFR1 - HFR4 sequences of HFB9 - 1Hz1 - hG1AA are SEQ ID NO: 1, 3, 5, and 7. The LFR1 - LFR4 sequences of HFB9 - 1Hz1 - hG1AA are SEQ ID NO: 9, 11, 13, and 15.

[0375] HFB9 - 1hz2 - hG1AA

[0376] VH - CDR1: GYTFTDYTIH (SEQ ID NO: 18)

[0377] VH - CDR2: WFYPGSHSIKYAQKFQGR (SEQ ID NO: 20)

[0378] VH - CDR3: HGGYDGFDY (SEQ ID NO: 22)

[0379] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYTIHWVRQAPGQGLEWMGWFYPGSHSIKYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYFCTRHGGYDGFDYWGQGTLVTVSS (SEQ ID NO: 24)

[0380] VL - CDR1: KSSQSLFYSTNQKNYLA (SEQ ID NO: 26)

[0381] VL - CDR2: WASTRES (SEQ ID NO: 28)

[0382] VL - CDR3: QQYYYFPYT (SEQ ID NO: 30)

[0383] DIVMTQSPDSLAVSLGERATINCKSSQSLFYSTNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYYFPYTFGQGTKLEIK (SEQ ID NO: 32)

[0384] The HFR1 - HFR4 sequences of HFB9 - 1Hz2 - hG1AA are SEQ ID NO: 17, 19, 21, and 23. The LFR1 - LFR4 sequences of HFB9 - 1Hz2 - hG1AA are SEQ ID NO: 25, 27, 29, and 31.

[0385] HFB9 - 1hz3 - hG1AA

[0386] VH - CDR1: GYTFTDYTIH (SEQ ID NO: 34)

[0387] VH - CDR2: WFYPGSHSIKYAQKFQGR (SEQ ID NO: 36)

[0388] VH - CDR3: HGGYDGFDY (SEQ ID NO: 38)

[0389] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYTIHWVRQAPGQGLEWMGWFYPGSHSIKYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCTRHGGYDGFDYWGQGTLVTVSS (SEQ ID NO: 40)

[0390] VL - CDR1: KSSQSLFYSTNQKNYLA (SEQ ID NO: 42)

[0391] VL - CDR2: WASTRES (SEQ ID NO: 44)

[0392] VL - CDR3: QQYYYFPYT (SEQ ID NO: 46)

[0393] DIVMTQSPDSLAVSLGERATINCKSSQSLFYSTNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYYFPYTFGQGTKLEIK (SEQ ID NO: 48)

[0394] The HFR1 - HFR4 sequences of HFB9 - 1Hz3 - hG1AA are SEQ ID NO: 33, 35, 37, and 39. The LFR1 - LFR4 sequences of HFB9 - 1Hz3 - hG1AA are SEQ ID NO: 41, 43, 45, and 47.

[0395] HFB9 - 1hz4 - hG1AA

[0396] VH-CDR1: GYTFTDYTIH (SEQ ID NO: 50)

[0397] VH-CDR2: WFYPGSHSIKYAQKFQGR (SEQ ID NO: 52)

[0398] VH-CDR3: HGGYDGFDY (SEQ ID NO: 54)

[0399] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYTIHWVRQAPGQGLEWMGWFYPGSHSIKYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARHGGYDGFDYWGQGTLVTVSS (SEQ ID NO: 56)

[0400] VL-CDR1: KSSQSLFYSTNQKNYLA (SEQ ID NO: 58)

[0401] VL-CDR2: WASTRES (SEQ ID NO: 60)

[0402] VL-CDR3: QQYYYFPYT (SEQ ID NO: 62)

[0403] DIVMTQSPDSLAVSLGERATINCKSSQSLFYSTNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYYFPYTFGQGTKLEIK (SEQ ID NO: 64)

[0404] The HFR1 - HFR4 sequences of HFB9 - 1Hz4 - hG1AA are SEQ ID NO: 49, 51, 53 and 55. The LFR1 - LFR4 sequences of HFB9 - 1Hz4 - hG1AA are SEQ ID NO: 57, 59, 61 and 63.

[0405] HFB9 - 2hz11 - hG1AA

[0406] VH-CDR1: GYTFTEYTIH (SEQ ID NO: 66)

[0407] VH-CDR2: WFYPGSGSTEYAQKFQG (SEQ ID NO: 68)

[0408] VH-CDR3: HGGYDGFDY (SEQ ID NO: 70)

[0409] QVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGQGLEWMGWFYPGSGSTEYAQKFQGRVTMTADTSISTAYMELSRLRSDDTAVYFCERHGGYDGFDYWGQGTTVTVSS (SEQ ID NO: 72)

[0410] VL-CDR1: KSSQSLLYSNNQKNYLA (SEQ ID NO: 74)

[0411] VL-CDR2: WASTRGS (SEQ ID NO: 76)

[0412] VL-CDR3: QQYYSYPFT (SEQ ID NO: 78)

[0413] DIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNQKNYLAWYQQKPGQPPKLLIYWASTRGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPFTFGGGTKVEIK (SEQ ID NO: 80)

[0414] The HFR1 - HFR4 sequences of HFB9 - 2Hz11 - hG1AA are SEQ ID NO: 65, 67, 69 and 71. The LFR1 - LFR4 sequences of HFB9 - 2Hz11 - hG1AA are SEQ ID NO: 73, 75, 77 and 79.

[0415] HFB9 - 2hz12 - hG1AA

[0416] VH-CDR1: GYTFTEYTIH (SEQ ID NO: 82)

[0417] VH-CDR2: WFYPGSGSAEYAQKFQG (SEQ ID NO: 84)

[0418] VH-CDR3: HGGYDGFDY (SEQ ID NO: 86)

[0419] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTEYTIHWVRQAPGQGLEWMGWFYPGSGSAEYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCERHGGYDGFDYWGQGTTVTVSS(SEQ ID NO: 88)

[0420] VL-CDR1: KSSQSLLYSNNQKNYLA(SEQ ID NO: 90)

[0421] VL-CDR2: WASTRGS(SEQ ID NO: 92)

[0422] VL-CDR3: QQYYSYPFT(SEQ ID NO: 94)

[0423] DIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNQKNYLAWYQQKPGQPPKLLIYWASTRGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPFTFGGGTKVEIK(SEQ ID NO: 96)

[0424] The HFR1 - HFR4 sequences of HFB9 - 2hz12 - hG1AA are SEQ ID NO: 81, 83, 85 and 87. The LFR1 - LFR4 sequences of HFB9 - 2hz12 - hG1AA are SEQ ID NO: 89, 91, 93 and 95.

[0425] HFB9 - 2hz13 - hG1AA

[0426] VH-CDR1: GYTFTEYTIH(SEQ ID NO: 98)

[0427] VH-CDR2: WFYPGSGSTEYAQKFQG(SEQ ID NO: 100)

[0428] VH-CDR3: HGGYDGFDY(SEQ ID NO: 102)

[0429] QVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTIHWVRQAPGQGLE

[0430] WMGWFYPGSGSTEYAQKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYYCERHGGYDGFDYWGQGTTVTVSS(SEQ ID NO: 104)

[0431] VL-CDR1: KSSQSLLYSNNQKNYLA (SEQ ID NO: 106)

[0432] VL-CDR2: WASTRGS (SEQ ID NO: 108)

[0433] VL-CDR3: QQYYSYPFT (SEQ ID NO: 110)

[0434] DIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNQKNYLAWYQQKPGQPPKLLIYWASTRGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPFTFGGGTKVEIK (SEQ ID NO: 112)

[0435] The HFR1-HFR4 sequences of HFB9-2Hz13-hG1AA are SEQ ID NO: 97, 99, 101 and 103, and the LFR1-LFR4 sequences of hFB9-2Hz13-hG1AA are SEQ ID NO: 105, 107, 109 and 111.

[0436] HFB9-2hz14-hG1AA

[0437] VH-CDR1: GYTFTEYTIH (SEQ ID NO: 114)

[0438] VH-CDR2: WFYPGSGSTEYSPSFQG (SEQ ID NO: 116)

[0439] VH-CDR3: HGGYDGFDY (SEQ ID NO: 118)

[0440] EVQLVQSGAEVKKPGESLKISCKGSGYTFTEYTIHWVRQMPGKGLE

[0441] WMGWFYPGSGSTEYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCERHGGYDGFDYWGQGTTVTVSS (SEQ ID NO: 120)

[0442] VL-CDR1: KSSQSLLYSNNQKNYLA (SEQ ID NO: 122)

[0443] VL-CDR2: WASTRGS (SEQ ID NO: 124)

[0444] VL-CDR3: QQYYSYPFT (SEQ ID NO: 126)

[0445] DIVMTQSPDSLAVSLGERATINCKSSQSLLYSNNQKNYLAWYQQKPGQPPKLLIYWASTRGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPFTFGGGTKVEIK (SEQ ID NO: 128)

[0446] The HFR1 - HFR4 sequences of HFB9 - 2hz14 - hG1AA are SEQ ID NO: 113, 115, 117, and 119. The LFR1 - LFR4 sequences of HFB9 - 2hz14 - hG1AA are SEQ ID NO: 121, 123, 125, and 127.

[0447] Example 3 blocks the binding of Gal9 to the receptors TIM3 and CD44 through the binding of anti - Gal9 antibody

[0448] This experiment confirmed that the anti - Gal9 antibody of the present invention can block Gal - 9 from binding to its receptors TIM3 and CD44, thereby antagonizing the downstream signal transduction of Gal - 9.

[0449] Figure 5 The data in [reference] clearly show that the humanized antibody of the present invention blocks the binding of Gal9 to both TIM3 and CD44 receptors in a dose - dependent manner.

[0450] Example 4 Anti - Gal9 antibody neutralizes Gal9 - induced Th1 cell apoptosis

[0451] Yang et al. (Inflammation 40(3):1062 - 1071, 2017) reported that elevated galectin - 9 inhibits TH1 effector function and induces apoptosis of activated CD4 + T cells in osteoarthritis. This experiment confirmed that the anti - GAL9 antibody of the present invention neutralizes Gal9 - induced TH1 cell apoptosis.

[0452] Specifically, human PBMCs were isolated from healthy donors and incubated with increasing concentrations of the antibody of the present invention in the presence of a certain amount of Gal9 that induces T cell apoptosis in the absence of the antibody. The percentage of apoptotic CD4 + T cells was measured within the antibody concentration range to determine the EC50 value of the antibody. The results are summarized in Figure 6In. The data clearly show that treating human PBMCs from healthy donors with the antibodies of the present invention prevents Gal-9-induced apoptosis of Th1 cells in a dose-dependent manner.

[0453] Example 5 Anti-Gal9 Antibodies Inhibit Gal9-Induced Treg Expansion

[0454] As discussed above, galectin-9 is directly expressed by Tregs, and its activation is associated with increased expression of Gal9. This experiment confirmed that the inhibition of galectin-9 by the anti-Gal9 antibodies of the present invention inhibits Treg expansion.

[0455] Specifically, human PBMCs were isolated from healthy donors and incubated with increasing concentrations of the antibodies of the present invention in the presence of a certain amount of Gal9 that stimulates Treg expansion in the absence of antibodies. Foxp3 + Ki67 高 The percentage of T cells was determined to measure the EC50 value of the antibody. The results are summarized in Figure 7 In. The data clearly show that treating human PBMCs from healthy donors with the antibodies of the present invention inhibits Gal-9-induced Treg expansion in a dose-dependent manner, where higher antibody concentrations are associated with lower percentages of expanded Tregs based on Foxp3 and Ki67 marker gene expression.

[0456] Example 6 Treatment with a Combination of Anti-Gal99 Antibody and Anti-PD-1 Antibody Shows Synergistic Effects in Inhibiting Tumor Growth and Prolonging Survival in Vivo

[0457] This experiment confirmed that in a xenograft mouse model, the anti-Gal9 monoclonal antibody and anti-PD-1 antibody of the present invention have a synergistic effect in inhibiting tumor growth in vivo.

[0458] Specifically, approximately 500,000 cancer cells were inoculated into experimental mice, and the tumor masses were allowed to grow to a predetermined size. Then, the mice were randomized and intraperitoneally (i.p.) injected 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) the anti-mPD-1 antibody at a dose of 10 mg / kg, or (4) a combination of 10 mg / kg of the anti-mPD-1 antibody and 10 mg / kg of the anti-HFB9-2 antibody.

[0459] The first dose of antibody for each group was administered on day 1, and subsequent doses were administered every 3 days, with a total of four doses for any group using the anti-mPD-1 antibody and a total of seven doses for any group using the anti-hFB9-2 antibody and the control antibody. Data are presented as mean ± s.e.m. (n = 8 mice per group)( Figure 8 ).

[0460] Apparently, the anti-Gal9 antibody and anti-mPD-1 antibody of the present invention showed a synergistic effect in inhibiting tumor growth in vivo, wherein during the 7-week study period, the combination therapy substantially completely inhibited tumor growth to no more than 500 mm 3 . Meanwhile, tumor growth in the control group and the anti-HFB9-2 group exceeded this level as early as 2 weeks, and the anti-mPD-1 group exceeded this level as early as 4 weeks.

[0461] In addition, in terms of survival( Figure 9 ), all mice in the control group died at about the end of the third week, all mice in the anti-HFB9-2 antibody group died at about the end of the fifth week, and only 25% of the mice (2 out of 8) in the anti-mPD-1 group survived without tumors at the end of the seventh week. However, at the same time, the combination treatment group had a 75% survival rate, including 5 out of 8 mice without tumors and one with a tumor of about 100 mm 3 .

[0462] This surprising finding strongly indicates that simultaneous inhibition of Gal-9 function and the PD-1 / PD-L1 immune checkpoint can synergistically inhibit tumor growth in vivo and prolong survival.

[0463] Example 7 The anti-Gal9 antibody is stable

[0464] To confirm that the humanized anti-Gal9 antibody of the present invention is stable in storage and thus suitable for further development as a therapeutic agent, various developability assays were performed on selected humanized antibodies.

[0465] In the first experiment, 1 - 2.75 mg / mL of the present inventors' 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 various antibodies was measured on days 0, 3, 7, and 14. The results (not shown) indicated that all tested antibodies were stable under the test conditions.

[0466] In a second experiment, the stability of the same antibodies was tested at 0, 3, and 6 hours under low pH conditions (100 mM AcH, pH 3.5, 25 °C). The results (not shown) again confirmed that all the antibodies tested were stable under the test conditions.

[0467] In a third experiment, the same antibodies were subjected to 1, 2, or 3 freeze-thaw cycles. The results (not shown) again confirmed that all the antibodies tested were stable under the test conditions.

[0468] Example 8 Levels of Galectin-9 in Plasma and Serum from AML Patients

[0469] 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 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 of AML patients was prepared according to standard procedures. For healthy donors, plasma or serum samples were obtained from commercial sources.

[0470] By ELISA, using " ELISA Human Galectin-9" from R&D, the levels of Galectin-9 protein in plasma or serum were evaluated, and statistical analysis (unpaired two-tailed t-test) was performed using 5 for Windows software.

[0471] As Figure 10 shown, the levels of Galectin-9 protein in plasma from AML patients were significantly higher at the time of disease diagnosis or relapse / refractory phase (R / R) than those observed in plasma from healthy individuals. The levels of Galectin-9 protein in plasma from AML patients in complete remission after chemotherapy were close to the normal physiological range.

[0472] As Figure 11 shown, at the time of diagnosis, the levels of Galectin-9 protein in plasma from Fab M2 or Fab M3 AML patients were significantly lower than those observed in plasma from Fab M0, M1, M4, or M5 AML patients. At the time of diagnosis, the levels of Galectin-9 protein in plasma from Fab M3 AML patients were within the normal physiological range. ​

[0473] In addition, LGALS9 mRNA expression levels were examined in AML patients and healthy individuals. The LGALS9 mRNA expression levels were extracted from the publicly available "AML_Ohsu_Nature2018" dataset deposited by Tyner et al (for a complete description of the study, see PMID 30333627). The data are Figure 12 presented as normalized log2 RPKM. The dashed lines represent the median LGALS9 levels in BM-derived MNCs from healthy individuals or AML patients, respectively.

[0474] As Figure 12 shown, at diagnosis, the galectin-9-encoding mRNA levels in BM-derived MNCs from AML patients (considering all Fabs) were higher than those observed in BM-derived MNCs or CD34+ cells from healthy individuals. In addition, at diagnosis, the galectin-9-encoding mRNA levels in BM-derived MNCs from Fab M3 AML patients were significantly lower than those observed in BM-derived MNCs from Fab M0, M1, M4, or M5 AML patients or BM-derived MNCs from healthy individuals or CD34+ cells.

Claims

1. An isolated monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof is specific for galectin-9, and wherein the monoclonal antibody or antigen-binding fragment thereof comprises: (1a) 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 (1b) 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 (2a) 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 (2b) 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 (3a) 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 (3b) 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 (4a) 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 (4b) 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:

126.

2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein: The (1a) heavy chain variable region (HCVR) further comprises the HFR3 sequence of SEQ ID NO: 69; or The (2a) heavy chain variable region (HCVR) further comprises the HFR3 sequence of SEQ ID NO: 85; or The (3a) heavy chain variable region (HCVR) further comprises the HFR3 sequence of SEQ ID NO: 101; or The heavy chain variable region (HCVR) of (4a) further comprises the HFR3 sequence of SEQ ID NO:

117.

3. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein: The heavy chain variable region (HCVR) of (1a) further comprises the HFR3 sequence of SEQ ID NO: 69 and further comprises the HFR1 sequence of SEQ ID NO: 65; or The heavy chain variable region (HCVR) of (2a) further comprises the HFR3 sequence of SEQ ID NO: 85 and further comprises the HFR1 sequence of SEQ ID NO: 81; or The heavy chain variable region (HCVR) of (3a) further comprises the HFR3 sequence of SEQ ID NO: 101 and further comprises the HFR1 sequence of SEQ ID NO: 97; or The heavy chain variable region (HCVR) of (4a) further comprises the HFR3 sequence of SEQ ID NO: 117 and further comprises the HFR1 sequence of SEQ ID NO:

113.

4. The isolated monoclonal antibody or antigen-binding fragment thereof of claims 1-3, wherein: (1A) The HCVR sequence is SEQ ID NO: 72; and (1B) The LCVR sequence is SEQ ID NO: 80, or (2A) The HCVR sequence is SEQ ID NO: 88; and (2B) The LCVR sequence is SEQ ID NO: 96, or (3A) The HCVR sequence is SEQ ID NO: 104; and (3B) The LCVR sequence is SEQ ID NO: 112, or (4A) The HCVR sequence is SEQ ID NO: 120; and (4B) The LCVR sequence is SEQ ID NO:

128.

5. The isolated monoclonal antibody or antigen-binding fragment thereof of claims 1-3, which is a humanized antibody and comprises: The HCVR sequence of SEQ ID NO: 72 and the LCVR sequence of SEQ ID NO:

80.

6. The isolated monoclonal antibody or antigen-binding fragment thereof according to claims 1-3, wherein the antigen-binding fragment is Fab, Fab', F(ab')2, single-chain Fv, disulfide-linked F v , intracellular antibody or scFv-Fc.

7. Use of the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6 and an antagonist of the PD-1 / PD-L1 immune checkpoint in the manufacture of a medicament for the treatment of a hematological cancer in a patient in need thereof, wherein the hematological cancer is AML, the method comprising administering to the patient an effective amount of the medicament.

8. Use according to claim 7, wherein the antagonist of the PD-1 / PD-L1 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.

9. Use according to claim 7 or 8, wherein the method further comprises administering to the patient a chemotherapeutic agent, an anti-angiogenic agent, a growth inhibitory agent, an immuno-oncology agent and / or an anti-tumor composition.

10. Use according to claim 7 or 8, wherein the monoclonal antibody or antigen-binding fragment thereof cross-reacts with murine Gal9.

11. Use according to claim 7 or 8, wherein the monoclonal antibody or antigen-binding fragment thereof binds human Gal9 with an EC of 0.1 - 0.2 nM, and / or binds mouse Gal9 with an EC of 0.5 - 1.0 nM 50 and binds human Gal9, and / or binds mouse Gal9 with an EC of 0.5 - 1.0 nM 50 ​ 12. Use according to claim 7 or 8, wherein the monoclonal antibody or antigen-binding fragment thereof binds to human Gal9 with a K of less than 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 2 nM or 1 nM d bind to human Gal9.

13. Use according to claim 7 or 8, wherein the monoclonal antibody or antigen-binding fragment thereof binds Gal9 and inhibits the binding of Gal9 to the Gal9 receptor.

14. Use according to claim 7 or 8, wherein the monoclonal antibody or antigen-binding fragment thereof neutralizes Th1 cell apoptosis of Gal-9-induced T cells.

15. Use according to claim 7 or 8, wherein the monoclonal antibody or antigen-binding fragment thereof inhibits Gal9-induced Treg expansion.

16. Use according to claim 7 or 8, wherein the monoclonal antibody or antigen-binding fragment thereof synergistically inhibits in vivo tumor growth and / or prolongs survival in mice with xenograft tumors in combination with an antagonist of an immune checkpoint.

17. A polynucleotide encoding the isolated monoclonal antibody or antibody-binding fragment thereof according to any one of claims 1-6.

18. The polynucleotide of claim 17, which is codon-optimized for expression in human cells.

19. A vector comprising the polynucleotide of claim 17 or 18. Use of the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-6 for the preparation of a medicament for a method of 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 having a risk of cancer recurrence, or a method of identifying and treating a patient with cancer, the method comprising: When a patient is identified as having a level of galectin-9 in a sample from the patient that is higher than the reference level of galectin-9 in a healthy or control individual, administering to the patient an effective amount of the medicament, wherein the cancer is a hematological cancer and the hematological cancer is AML.

21. Use according to claim 20, 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.

22. Use according to claim 20 or 21, wherein the method further comprises administering to the patient an antagonist of an immune checkpoint.

23. Use according to claim 22, wherein the immune checkpoint is the PD-1 / PD-L1 immune checkpoint.

24. Use according to claim 22, 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.

25. Use according to claim 20, wherein the patient is a Fab M0, M1, M4 or M5 AML patient, or wherein the patient is not a Fab M2 or M3 AML patient.

26. Use according to claim 20 or 21, wherein the sample is a blood sample, a plasma sample or a serum sample.

27. Use of a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1-6 in the preparation of a medicament for a method of rescuing or promoting the proliferation of effector T cells and / or enhancing the activity of effector T cells in diagnosing a patient suffering from AML, being in the process of developing AML or at risk of AML recurrence, or for a method of identifying and treating a patient suffering from AML, said method comprising: When it is determined that the level of galectin-9-encoding mRNA in a sample of mononuclear cells (MNCs) derived from the bone marrow (BM) of the patient is statistically significantly higher or lower than the reference level in BM-derived MNCs or CD34 cells in healthy or control individuals, an effective amount of the drug is administered to the patient. + ​ 28. Use according to claim 27, wherein (1) when the patient is a patient with Fab M0, M1, M2, M4 or M5 AML, 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) when the patient is a patient with Fab M3 AML, the level of galectin-9-encoding mRNA in the BM-derived MNC sample from the patient is significantly lower than the reference level.

29. Use of an isolated monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1-6 in the preparation of a medicament for the treatment of cancer, wherein the cancer is a hematological cancer, the hematological cancer is AML, and wherein the antibody or its antigen-binding portion rescues effector T cell proliferation and / or enhances effector T cell activity.

30. Use according to claim 29, wherein the effector T cell is a Th1 cell.

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