Modified anti-galectin-9 antibodies and uses thereof

Abstract

Provided herein are anti-galectin-9 (Gal9) antibodies that are matured in affinity. The antibodies have improved binding affinity to Gal9. Also provided herein are methods of using the affinity mature anti-Gal9 antibodies, including methods of treating cancer, methods of rescuing or promoting effector T cell proliferation, methods of enhancing effector T cell activity, and / or methods of identifying and treating cancer in a subject, comprising administering the anti-Gal9 antibodies described herein. Also provided are polynucleotides encoding the heavy or light chain or antigen-binding portion thereof described herein, as well as vectors, particularly expression vectors, comprising the polynucleotides described herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 389,607, filed on July 15, 2022, and U.S. Provisional Application No. 63 / 389,610, filed on July 15, 2022. The disclosure of the prior application is considered part of the disclosure of the present application, and the disclosure of the prior application is incorporated by reference into the disclosure of the present application in its entirety.

[0003] Incorporation of Sequence Listing

[0004] The material in the attached sequence listing is hereby incorporated into this application by reference.The attached sequence listing xml file named 148640-004302_SL.xml was created on July 13, 2023 and is 80,112 bytes. Summary of the invention

[0005] The present invention relates generally to anti-galectin-9 antibodies, and more particularly to anti-galectin-9 antibodies for use in treating cancer.

[0006] Galectin-9 (or Gal9) is a member of the galectin (or S-type lectin) protein family with at least 15 members in vertebrates (including 10 members in humans). Gal9 is a soluble 34-39 kDa protein without a leader peptide, but is still secreted via a non-classical mechanism. It preferentially interacts with β-galactosidic residues of glycoproteins and glycolipids. In humans, Gal9 exists in three isoforms (long, medium and short).

[0007] Gal9 (a member of the lectin family of β-galactoside binding proteins) contains 2 different carbohydrate recognition domains (CRDs) connected by a flexible linker (tandem repeat galectins). Gal9 can bind / link two ligands simultaneously, promoting the association or organization of two or more monomeric receptors, leading to altered signal transduction and possibly altered rates of receptor endocytosis. Several cell surface binding partners have been identified, including Tim3, PD-1, VISTA, Dectin-1, CD44, CD206, 4-1BB (CD137), TNFRSF25, PDI, etc. Gal9 plays an important role in regulating the activity of several immune cell types, thereby creating an immunosuppressive environment in the tumor microenvironment (TME). Notably, Gal9 (i) induces apoptosis of CD4+ and CD8+ T cells, (ii) inhibits Th17 cell development, (iii) induces suppressive Treg expansion, (iv) promotes the expansion of myeloid-derived suppressor cells, (v) promotes the M2-polarized tumor-associated macrophage (TAM) phenotype, and (vi) impairs natural killer (NK) cell function. Several of these Gal9 activities have been demonstrated using Gal9 knockout mouse studies.

[0008] Gal9 is one of the most studied ligands for T-cell immunoglobulin and mucin domain-containing molecule-3 (TIM-3), also known as hepatitis A virus cellular receptor 2 (HAVCR2), and is expressed on a variety of hematological malignancies, such as chronic lymphocytic leukemia (CLL), myelodysplastic syndrome (MDS), Hodgkin and non-Hodgkin lymphomas, acute myeloid leukemia (AML), as well as solid tumors, such as lung cancer, breast cancer, and hepatocellular carcinoma.

[0009] The interaction of Gal9 with TIM-3 / HAVCR2 has been found to attenuate T cell expansion and effector function in the tumor microenvironment and chronic infection. In addition, Gal9 promotes tumorigenesis through tumor cell transformation, cell cycle regulation, angiogenesis, and cell adhesion.

[0010] Gal9 is also directly expressed by regulatory T lymphocytes (or Tregs), and its expression increases during Treg activation. On the other hand, Gal9 is expressed by effector T lymphocytes such as CD8 + It is very weakly expressed by cytotoxic T lymphocytes, and this expression disappears during the activation of effector T lymphocytes. It has been found that inhibition of Gal9 by anti-Gal9 antibodies can inhibit the suppressive activity of Tregs.

[0011] Wu et al. (Immunity [Immunity] 41 (2): 270-282, 2014) reported that Gal9 is critical in regulating immune responses. There are two subsets of Treg cells, namely "natural" Treg (nTreg) cells and "induced" Treg (iTreg) cells. Gal9 is highly expressed by iTreg cells and is crucial for the generation and function of iTreg cells rather than nTreg cells. Gal9 expression in iTreg cells is driven by the transcription factor Smad3, forming a feedforward loop, which further promotes Foxp3 expression. Gal9 increases iTreg cell stability and function by directly binding to its receptor CD44, which forms a complex with transforming growth factor-β (TGF-β) receptor I (TGF-βRI) and activated Smad3. It was further found that Gal9 signaling can regulate iTreg cell induction by acting mainly through the CNS1 region of the Foxp3 locus. In addition to being an effector molecule of Treg cells, exogenous Gal9 also synergizes with TGF-β to enhance iTreg cell differentiation and maintenance.

[0012] The various types of T lymphocytes usually develop into, for example, "effector" cells or effector T lymphocytes, which will carry out specialized immune functions to protect the host organism. + T lymphocytes or helper T lymphocytes secrete major cytokines that assist the humoral function of B lymphocytes (production of specific antibodies) and CD8 + Cytotoxic activity of T lymphocytes.

[0013] Tregs constitutively overexpress CD4 and CD25 molecules (hence they are also called “CD4 + CD25 + ”) and Foxp3 transcription factor. This small percentage of CD4 + CD25 + T lymphocytes are participants of negatively regulating immune responses that will recognize various self-antigens through their T cell receptors (TCR). Therefore, Tregs play a major role in the physiology of the immune system by protecting the organism from the emergence of autoimmune diseases.

[0014] Treg exerts immunosuppressive activity on effector T lymphocytes. Once activated, this activity promotes tumor growth under pathological conditions (such as tumors). Therefore, the suppressive activity of Treg can be understood as reducing the activity of anti-tumor immune response by inhibiting the function of effector T lymphocytes.

[0015] Gal9 is mainly associated with tumor immunosuppression caused by interaction with different immune receptors. For example, Gal9 inhibits Th1 responses and induces peripheral tolerance, as confirmed by reduced Th1 apoptosis after Gal9 blockade, increased susceptibility of Gal9 knockout mice to collagen-induced arthritis (CIA), and prolonged graft survival and autoimmune disease (AID) inhibition after Gal9 administration. Gal9 also regulates peripheral NK cell function to promote maternal-fetal tolerance, promotes the expansion of myeloid-derived suppressor cells (MDSC), and cooperates with TGF-β to promote Treg expansion.

[0016] High expression of Gal9 is evident in both tumors and blood of patients in several solid tumors and hematological malignancies and is associated with aggressive disease and poor survival outcomes. Recently, several published reports have provided support for a combination therapy approach using anti-Gal9 antibodies with currently used immunotherapies, including immune checkpoint inhibitor therapy.

[0017] Yang et al. (Nature Commun. [Nature · Communication] 12, 832, 2021) demonstrated that (i) PD-1 binds to Gal9C-CRD, and Gal9 and PD-L1 bind to different sites on PD-1; (ii) Pembrolizumab does not block the binding of Gal9 to PD-1; (iii) the complex of Tim-3 and PD-1 / gal9 C-CRD contributes to the persistence of exhausted T cells by limiting Tim3-Gal9-driven apoptosis; and (iv) combined treatment with anti-PD-1 and anti-Gal9 antibodies can provide better anti-tumor effects and general outcomes (e.g., survival) compared to each therapy used alone. In addition, Yang et al., above, generated 16,291 immune cell transcriptomes from 48 tumor samples collected from 32 melanoma patients treated with anti-PD1 therapy, and evaluated their Gal9 expression and its association with treatment outcomes. Tumor-infiltrating lymphocytes (TILs) from anti-PD1 nonresponders expressed much higher levels of gal9 compared to patients who responded to therapy.

[0018] Limagne et al. (Oncoimmunology 8:e1564505, 2019) evaluated a patient population with metastatic non-small cell lung cancer (mNSCLC) and found that accumulation of Tim3+ lymphoid cells and Gal9+ monocytic myeloid-derived suppressor cells (mMDSCs) was associated with resistance to PD-1 blockade.

[0019] Li et al. (Immunotherapy 15, 135, 2023) have shown that Gal9 and PD-L1 levels are higher in pancreatic ductal adenocarcinoma (PDAC) tumors compared with normal tissues, and levels are significantly correlated with tumor invasion of lymph nodes and tumor lymph node metastasis stage. In the murine KPC model (an established and clinically relevant PDAC model), significant tumor growth inhibition was observed in the anti-PD-L1 and anti-Gal9 combination therapy group compared with anti-PD-L1 or anti-Gal9 alone or the control group.

[0020] Together, these findings suggest that in certain human cancers, combined anti-Gal9 and anti-PD1 therapy may be an effective treatment option.

[0021] Circulating levels of Gal9 are significantly higher in certain cancer patients compared to healthy controls. Therefore, there remains an unmet need for anti-Gal9 antibodies for use in the treatment of cancer.

[0022] Aspects of the invention described herein relate to antibodies against Gal9 and their use for treating diseases associated with the suppressive activity of regulatory T lymphocytes (Tregs).

[0023] In one aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, comprising (a) a heavy chain variable region (HCVR) comprising (i) a HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), wherein X1 is selected from T, E, P or A; X2 is selected from T, G or S; and X3 is selected from E, S or D; (ii) a HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), wherein X4 is selected from E, W or V; and (iii) a HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) a light chain variable region (LCVR) comprising (i) a HCVR CDR3 sequence having the amino acid sequence KSSX5X6X7LX8X9X 10 X11X 12 X 13 NX 14 LA (SEQ ID NO: 13) LCVR CDR1 sequence, wherein X5 is selected from Q or R; X6 is selected from S or N; X7 is selected from L, V or I; X8 is selected from Y or W; X9 is selected from S or P; X 10 Selected from N, S, P or A; X 11 Selected from N or H; X 12 Selected from Q, N or Y; X 13 Selected from K or R; and X14 Selected from Y or H; (ii) having an amino acid sequence WX 15 SX 16 RX 17 X 18 LCVR CDR2 sequence, where X 15 Selected from A or G; X 16 Selected from T, N, M or A; X 17 Selected from G or E; and X 18 is selected from the group consisting of S, E, Y, P or T; and (iii) has an amino acid sequence of QQYYX 19 X 20 The LCVR CDR3 sequence of PFT (SEQ ID NO: 30), wherein X 19 Selected from S or F; and X 20 selected from Y or F, with the proviso that the antibody does not comprise the HCVR CDR1 sequence GYTFTEYTIH (SEQ ID NO:2), the HCVR CDR2 sequence WFYPGSGSTEYAQKFQG (SEQ ID NO:9), the HCVR CDR3 sequence HGGYDGFDY (SEQ ID NO:12), the LCVR CDR1 sequence KSSQSLLYSNNQKNYLA (SEQ ID NO:14), the LCVR CDR2 sequence WASTRGS (SEQ ID NO:22) and the LCVR CDR3 sequence QQYYSYPFT (SEQ ID NO:31), wherein the antibody or antigen-binding fragment thereof specifically binds Gal9.

[0024] In one embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises (a) a HCVR comprising (i) a HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), wherein X1 is selected from T or E, and X2 is selected from T or G, and X3 is E; (ii) a HCVRCDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), wherein X4 is selected from W or V; and (iii) a HCVRCDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) a LCVR comprising (i) a LCVR CDR1 sequence having the amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13), wherein X5 is Q, X6 is S, X7 is L, X8 is Y, X9 is selected from S or P, X10 It is N, X 11 It is N, X 12 It's Q, X 13 is K; and X 14 is Y; (ii) has the amino acid sequence WX 15 SX 16 RX 17 X 18 LCVR CDR2 sequence, where X 15 It's A, X 16 It's T, X 17 is G, and X 18 selected from S or E; and (iii) having the amino acid sequence QQYYX 19 X 20 The LCVR CDR3 sequence of PFT (SEQ ID NO: 30), wherein X 19 It is S, and X 20 Selected from Y or F, wherein the antibody or antigen-binding fragment thereof specifically binds Gal9.

[0025] In a specific embodiment thereof, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, comprising (A) a HCVR comprising a HCVRCDR1 sequence of SEQ ID NO:3, a HCVR CDR2 sequence of SEQ ID NO:10, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:15, a LCVR CDR2 sequence of SEQ ID NO:23, and a LCVR CDR3 sequence of SEQ ID NO:32; or (B) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:4, a HCVR CDR2 sequence of SEQ ID NO:11, and a HCVR CDR3 sequence of SEQ ID NO:12; and a light chain variable region (LCVR) comprising a LCVR CDR1 sequence of SEQ ID NO:14, a LCVR CDR2 sequence of SEQ ID NO:24, and a LCVR CDR3 sequence of SEQ ID NO:31. CDR3 sequence, wherein the antibody or antigen-binding fragment thereof specifically binds to Gal9.

[0026] In one embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises (a) a HCVR comprising (i) a HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), wherein X1 is selected from T, P or A; X2 is selected from T, S or G; and X3 is selected from S or D; (ii) a HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), wherein X4 is E; and (iii) a HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) a LCVR comprising: (i) a HCVR CDR3 sequence having the amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13) LCVR CDR1 sequence, wherein X5 is selected from Q or R; X6 is selected from S or N; X7 is selected from L, V or I; X8 is selected from Y or W; X9 is S, X 10 Selected from S, P or A; X 11 Selected from N or H; X 12 is N or Y; X 13 is K or R; and X 14 is Y or H; (ii) has the amino acid sequence WX 15 SX 16 RX 17 X 18 LCVR CDR2 sequence, where X 15 Selected from A or G; X 16 Selected from T, N, M or A; X 17 Selected from G or E; and X 18 is selected from S, Y, P or T; and (iii) has an amino acid sequence of QQYYX 19 X 20 The LCVR CDR3 sequence of PFT (SEQ ID NO: 30), wherein X 19 is F, and X 20 Y; wherein the antibody or antigen-binding fragment thereof specifically binds Gal9.

[0027] In a specific embodiment thereof, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, comprising (C) a HCVR comprising a HCVRCDR1 sequence of SEQ ID NO:5, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a light chain variable region (LCVR) comprising a LCVR CDR1 sequence of SEQ ID NO:16, a LCVR CDR2 sequence of SEQ ID NO:25, and a LCVR CDR3 sequence of SEQ ID NO:33; (D) a HCVR comprising a HCVRCDR1 sequence of SEQ ID NO:6, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:17, a LCVR CDR2 sequence of SEQ ID NO:26, and a LCVR CDR3 sequence of SEQ ID NO:33. (E) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:7, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:18, a LCVR CDR2 sequence of SEQ ID NO:27, and a LCVR CDR3 sequence of SEQ ID NO:33; (F) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:7, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:19, a LCVR CDR2 sequence of SEQ ID NO:28, and a LCVR CDR3 sequence of SEQ ID NO:33; or (G) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:7, a HCVR CDR2 sequence of SEQ ID NO:9, and a LCVR CDR3 sequence of SEQ ID NO:33. NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:20, a LCVRCDR2 sequence of SEQ ID NO:29, and a LCVR CDR3 sequence of SEQ ID NO:33, wherein the antibody or antigen-binding fragment thereof specifically binds to Gal9.

[0028] In another embodiment, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, comprising (a) a HCVR comprising (i) a HCVR CDR1 sequence having an amino acid sequence of GYX1FX2X3YTIH (SEQ ID NO: 1), wherein X1 is selected from E or P; X2 is selected from T or S; and X3 is selected from E or S; (ii) a HCVR CDR2 sequence having an amino acid sequence of WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), wherein X4 is selected from W or E; and (iii) a HCVR CDR3 sequence having an amino acid sequence of HGGYDGFDY (SEQ ID NO: 12); and (b) a light chain variable region (LCVR) comprising: (i) a HCVR CDR1 sequence having an amino acid sequence of KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13) LCVR CDR1 sequence, wherein X5 is Q, X6 is selected from S or N, X7 is selected from L or V, X8 is selected from Y or W, X9 is selected from P or S, X 10 Select from N or S, X 11 It is N, X 12 Select from Q or N, X 13 is K; and X 14 is Y; (ii) has the amino acid sequence WX 15 SX 16 RX 17 X 18 LCVR CDR2 sequence, where X 15 Selected from A or G; X 16 Select from T or N; X 17 Select from G or E, and X 18 is S; and (iii) has the amino acid sequence QQYYX 19 X 20 The LCVR CDR3 sequence of PFT (SEQ ID NO: 30), wherein X 19 Selected from S or F; and X 20 Selected from Y or F, wherein the antibody or antigen-binding fragment thereof specifically binds Gal9.

[0029] In a specific embodiment thereof, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, comprising (A) a HCVR comprising a HCVRCDR1 sequence of SEQ ID NO:3, a HCVR CDR2 sequence of SEQ ID NO:10, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:15, a LCVR CDR2 sequence of SEQ ID NO:23, and a LCVR CDR3 sequence of SEQ ID NO:32; or (C) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:5, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a light chain variable region (LCVR) comprising a LCVR CDR1 sequence of SEQ ID NO:16, a LCVR CDR2 sequence of SEQ ID NO:25, and a LCVR CDR3 sequence of SEQ ID NO:33. CDR3 sequence, wherein the antibody or antigen-binding fragment thereof specifically binds to Gal9.

[0030] In one aspect, the HCVR sequence of the antibody or antigen-binding fragment thereof of (A), (B), (C), (D), (E), (F) or (G) further comprises the HFR3 sequence of SEQ ID NO: 68, and optionally further comprises the HFR1 sequence of SEQ ID NO: 66, the HFR2 sequence of SEQ ID NO: 67 and / or the HFR4 sequence of SEQ ID NO: 71. In another aspect, the HCVR sequence of the antibody or antigen-binding fragment thereof of (A), (B), (C), (D), (E), (F) or (G) further comprises the HFR3 sequence of SEQ ID NO: 69, and optionally further comprises the HFR1 sequence of SEQ ID NO: 66, the HFR2 sequence of SEQ ID NO: 67 and / or the HFR4 sequence of SEQ ID NO: 71. In another aspect, the HCVR sequence of the antibody or antigen-binding fragment thereof of (A), (B), (C), (D), (E), (F) or (G) further comprises the HFR3 sequence of SEQ ID NO: 70, and optionally further comprises the HFR1 sequence of SEQ ID NO: 66, the HFR2 sequence of SEQ ID NO: 67 and / or the HFR4 sequence of SEQ ID NO: 71. In another aspect, the HCVR sequence of the antibody or antigen-binding fragment thereof of (A), (B), (C), (D), (E), (F) or (G) further comprises the HFR3 sequence of SEQ ID NO: 76, and optionally further comprises the HFR1 sequence of SEQ ID NO: 66, the HFR2 sequence of SEQ ID NO: 67 and / or the HFR4 sequence of SEQ ID NO: 71. In another aspect, the HCVR sequence of the antibody or antigen-binding fragment thereof of (A), (B), (C), (D), (E), (F) or (G) further comprises the HFR3 sequence of SEQ ID NO: 77, and optionally further comprises the HFR1 sequence of SEQ ID NO: 66, the HFR2 sequence of SEQ ID NO: 67 and / or the HFR4 sequence of SEQ ID NO: 71. In one aspect, the LCVR sequence of the antibody or antigen-binding fragment thereof of (A), (B), (C), (D), (E), (F) or (G) optionally further comprises the LFR1 sequence of SEQ ID NO: 72, the LFR2 sequence of SEQ ID NO: 73, the LFR3 sequence of SEQ ID NO: 74 and / or the LFR4 sequence of SEQ ID NO: 75.

[0031] In another aspect, the HCVR sequence is SEQ ID NO:39 and / or the LCVR sequence is SEQ ID NO:41; or the HCVR sequence is SEQ ID NO:43 and / or the LCVR sequence is SEQ ID NO:45; or the HCVR sequence is SEQ ID NO:47 and / or the LCVR sequence is SEQ ID NO:49; or the HCVR sequence is SEQ ID NO:51 and / or the LCVR sequence is SEQ ID NO:53; or the HCVR sequence is SEQ ID NO:55 and / or the LCVR sequence is SEQ ID NO:57; or the HCVR sequence is SEQ ID NO:59 and / or the LCVR sequence is SEQ ID NO:61; or the HCVR sequence is SEQ ID NO:63 and / or the LCVR sequence is SEQ ID NO:65. In some aspects, the antibody comprises: a HCVR sequence of SEQ ID NO:39 and a LCVR sequence of SEQ ID NO:41; or a HCVR sequence of SEQ ID NO:43 and a LCVR sequence of SEQ ID NO:45; or a HCVR sequence of SEQ ID NO:47 and a LCVR sequence of SEQ ID NO:49; or a HCVR sequence of SEQ ID NO:51 and a LCVR sequence of SEQ ID NO:53; or a HCVR sequence of SEQ ID NO:55 and a LCVR sequence of SEQ ID NO:57; or a HCVR sequence of SEQ ID NO:59 and a LCVR sequence of SEQ ID NO:61; or a HCVR sequence of SEQ ID NO:63 and a LCVR sequence of SEQ ID NO:65.

[0032] In one aspect, the antibody is a humanized antibody. In another aspect, the antigen-binding fragment thereof is a Fab, Fab', F(ab')2, F d , single-chain Fv or scFv, disulfide-linked F v , V-NAR domain, IgNar, intracellular antibody, IgGACH2, mini antibody, F(ab')3, tetrabodies, tribodies, diabodies, single domain antibodies, DVD-Ig, Fcab, mAb2, (scFv)2, tandem scFv, TandAb, nanobody or scFv-Fc. In other aspects, the monoclonal antibody or antigen-binding fragment thereof has a K of less than about 5nM, 2nM, 1nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM, 0.05nM or 0.01nM. dBinds to human Gal9. In one aspect, the antibody binds to Gal9 and inhibits the binding of Gal9 to a Gal9 receptor (e.g., TIM3 or CD44). In another aspect, the antibody binds to Gal9 and does not inhibit the binding of Gal9 to a Gal9 receptor (e.g., TIM3 or CD44). In one aspect, the antibody reverses the expression of Gal9-induced T cells (e.g., CD4 + In another aspect, the antibody inhibits Gal9-induced Treg expansion.

[0033] In another embodiment, the invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the isolated monoclonal antibodies or antigen-binding fragments described herein, thereby treating the cancer in the subject.

[0034] In one aspect, the cancer is a blood cancer or a solid tumor. In some aspects, the blood cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML) and hairy cell leukemia; AIDS-related lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, mycosis fungoides, non-Hodgkin's lymphoma, primary central nervous system lymphoma, Sezary syndrome, cutaneous T-cell lymphoma, Waldenstrom's macroglobulinemia, diffuse large B-cell lymphoma (DLBCL) and multiple myeloma. In other aspects, solid tumors are selected from the group consisting of: breast cancer, head and neck cancer, lung cancer, melanoma, uveal melanoma, colorectal cancer, renal cancer, urothelial carcinoma, ovarian cancer, endometrial cancer, liver cancer, pancreatic cancer, bile duct cancer, gastric cancer, gastroesophageal cancer, esophageal cancer, cervical cancer, head and neck squamous cell carcinoma and prostate cancer. On the one hand, the method further includes administering chemotherapeutic agents, anti-angiogenic agents, growth inhibitors, immuno-oncology agents, checkpoint inhibitors and / or anti-tumor compositions to the patient. In some aspects, the checkpoint inhibitor is anti-PD-1. In other aspects, the immuno-oncology agent is an anti-glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) antibody.

[0035] In additional embodiments, the present invention provides polynucleotides encoding the heavy chain or light chain, or antigen binding portion thereof, of any of the antibodies or antigen binding fragments thereof described herein.

[0036] In one embodiment, the present invention provides a vector comprising a polynucleotide described herein, wherein the vector is an expression vector selected from the group consisting of a mammalian expression vector, a yeast expression vector, an insect expression vector, and a bacterial expression vector.

[0037] In another embodiment, the present invention provides a method for rescuing or promoting effector T cell proliferation, enhancing effector T cell activity and / or identifying and treating cancer in a subject, the method comprising: (i) determining the level of Gal9 in a sample from the subject, and (ii) administering an effective amount of any one of the isolated monoclonal antibodies or antigen-binding fragments thereof described herein to the subject, the subject having a Gal9 level higher than a reference level of Gal9, thereby rescuing or promoting effector T cell proliferation, enhancing effector T cell activity and / or identifying and treating the subject.

[0038] On the one hand, the subject is diagnosed with cancer, has a risk of developing cancer, or has cancer recurrence. On the other hand, the reference level of Gal9 is the level of Gal9 measured in a sample from a healthy or control individual. In some aspects, determining includes comparing the level of Gal9 in the sample with the reference level. On the one hand, the cancer is a blood cancer or a solid tumor. In some aspects, the blood cancer is selected from the group consisting of: ALL, AML, CLL, CML and hairy cell leukemia; AIDS-related lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, mycosis fungoides, non-Hodgkin's lymphoma, primary central nervous system lymphoma, Sezary syndrome, cutaneous T-cell lymphoma, Waldenstrom's macroglobulinemia, DLBCL and multiple myeloma. In other aspects, solid tumors are selected from the group consisting of: breast cancer, head and neck cancer, lung cancer, melanoma, uveal melanoma, colorectal cancer, renal cancer, urothelial carcinoma, ovarian cancer, endometrial cancer, liver cancer, pancreatic cancer, bile duct cancer, gastric cancer, gastroesophageal cancer, esophageal cancer, cervical cancer, head and neck squamous cell carcinoma and prostate cancer. On the one hand, the sample is a blood sample, a plasma sample, a serum sample, a tumor biopsy sample or a bone marrow (BM) derived mononuclear cell (MNC) sample. On the one hand, the method further includes administering chemotherapeutic agents, anti-angiogenic agents, growth inhibitors, immuno-oncology agents, checkpoint inhibitors and / or anti-tumor compositions to the patient. In some aspects, the checkpoint inhibitor is an anti-PD-1 antibody. In other aspects, the immuno-oncology agent is an anti-GITR antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1A-Figure 1D Sequence alignments are shown. Figure 1A An alignment of the sequences of the heavy chains of Antibody A (Ab A), Antibody 001 (Ab 001), and Antibody 002 (Ab 002) anti-Gal9 antibodies is shown. Figure 1B An alignment of the sequences of the light chains of Ab A, Ab 001 and Ab 002 anti-Gal9 antibodies is shown. Figure 1CShown is an alignment of the sequences of the heavy chains of Antibody A (Ab A), Antibody 003 (Ab003), Antibody 004 (Ab 004), Antibody 005 (Ab 005), Antibody 006 (Ab 006), and Antibody 007 (Ab007) anti-Gal9 antibodies. Figure 1D An alignment of the sequences of the light chains of Ab A, Ab 003, Ab 004, Ab 005, Ab 006 and Ab 007 anti-Gal9 antibodies is shown.

[0040] Figure 2A-2D Shown is the effect of anti-Gal9 antibody on Gal9-induced apoptosis. Figure 2A Shown is the effect of Ab A anti-Gal9 antibody on T cell apoptosis. Figure 2B Shown is the effect of affinity matured Ab 001 anti-Gal9 antibody on T cell apoptosis. Figure 2C Shown is the effect of Ab 001 on Gal9-induced CD4+ T cell apoptosis. Figure 2D Shown is the effect of Ab 001 on Gal9-induced CD8+ T cell apoptosis.

[0041] Figure 3 Comparable effects of Ab 001 and Ab 003 on TIM3 dimerization are shown. DETAILED DESCRIPTION

[0042] Aspects of the invention described herein relate to antibodies against Gal9 and their use for treating diseases associated with the suppressive activity of regulatory T lymphocytes (Tregs).

[0043] Before describing the compositions and methods of the present invention, it should be understood that the present invention is not limited to the specific compositions, methods and experimental conditions described, because such compositions, methods and conditions may vary. It should also be understood that the terms used herein are only used for the purpose of describing specific embodiments and are not intended to be limiting, because the scope of the present invention will only be limited in the appended claims.

[0044] As used in this specification and the appended claims, quantifiers are not used and the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods and / or steps of the type described herein that will become apparent to those skilled in the art upon reading this disclosure.

[0045] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, it will be understood that modifications and variations are encompassed within the spirit and scope of the present disclosure. Preferred methods and materials are now described.

[0047] Provided herein are affinity matured antibodies that specifically bind to Gal9. Affinity maturation is a process used to generate increased affinity, avidity, and anti-antigen activity from antibodies (see, e.g., Teixeira et al., mAbs 14(1)e2115200, 2022).

[0048] The antibody HFB9-2hz11 (Ab A) disclosed in International Publication No. WO WO2021 / 139682 was subjected to affinity maturation. Seven antibodies were identified that had significantly improved Gal9 binding affinity. These antibodies exhibited variability in the CDR regions, and these antibodies maintained and improved Gal9 binding compared to the parent antibody. Thus, in one aspect, the invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, comprising (a) a heavy chain variable region (HCVR) comprising (i) a HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), wherein X1 is selected from T, E, P or A; X2 is selected from T, G or S; and X3 is selected from E, S or D; (ii) a HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), wherein X4 is selected from E, W or V; and (iii) a HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) a light chain variable region (LCVR) comprising (i) a HCVR CDR3 sequence having the amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13) LCVR CDR1 sequence, wherein X5 is selected from Q or R; X6 is selected from S or N; X7 is selected from L, V or I; X8 is selected from Y or W; X9 is selected from S or P; X 10 Selected from N, S, P or A; X 11 Selected from N or H; X 12 Selected from Q, N or Y; X 13 Selected from K or R; and X 14Selected from Y or H; (ii) having an amino acid sequence WX 15 SX 16 RX 17 X 18 LCVR CDR2 sequence, where X 15 Selected from A or G; X 16 Selected from T, N, M or A; X 17 Selected from G or E; and X 18 is selected from the group consisting of S, E, Y, P or T; and (iii) has an amino acid sequence of QQYYX 19 X 20 The LCVR CDR3 sequence of PFT (SEQ ID NO: 30), wherein X 19 Selected from S or F; and X 20 selected from Y or F, with the proviso that the antibody does not comprise the HCVR CDR1 sequence GYTFTEYTIH (SEQ ID NO:2), the HCVR CDR2 sequence WFYPGSGSTEYAQKFQG (SEQ ID NO:9), the HCVRCDR3 sequence HGGYDGFDY (SEQ ID NO:12), the LCVR CDR1 sequence KSSQSLLYSNNQKNYLA (SEQ ID NO:14), the LCVR CDR2 sequence WASTRGS (SEQ ID NO:22) and the LCVR CDR3 sequence QQYYSYPFT (SEQ ID NO:31), wherein the antibody or antigen-binding fragment thereof specifically binds Gal9.

[0049] In one embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises (a) a HCVR comprising (i) a HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), wherein X1 is selected from T or E, and X2 is selected from T or G, and X3 is E; (ii) a HCVRCDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), wherein X4 is selected from W or V; and (iii) a HCVRCDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) a LCVR comprising (i) a LCVR CDR1 sequence having the amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13), wherein X5 is Q, X6 is S, X7 is L, X8 is Y, X9 is selected from S or P, X 10It is N, X 11 It is N, X 12 It's Q, X 13 is K; and X 14 is Y; (ii) has the amino acid sequence WX 15 SX 16 RX 17 X 18 LCVR CDR2 sequence, where X 15 It's A, X 16 It's T, X 17 is G, and X 18 selected from S or E; and (iii) having the amino acid sequence QQYYX 19 X 20 The LCVR CDR3 sequence of PFT (SEQ ID NO: 30), wherein X 19 It is S, and X 20 Selected from Y or F, wherein the antibody or antigen-binding fragment thereof specifically binds Gal9.

[0050] Specific embodiments include an isolated monoclonal antibody or antigen-binding fragment thereof comprising (A) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:3, a HCVR CDR2 sequence of SEQ ID NO:10, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:15, a LCVR CDR2 sequence of SEQ ID NO:23, and a LCVR CDR3 sequence of SEQ ID NO:32; or (B) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:4, a HCVR CDR2 sequence of SEQ ID NO:11, and a HCVR CDR3 sequence of SEQ ID NO:12; and a light chain variable region (LCVR) comprising a LCVR CDR1 sequence of SEQ ID NO:14, a LCVR CDR2 sequence of SEQ ID NO:24, and a LCVR CDR3 sequence of SEQ ID NO:31. CDR3 sequence, wherein the antibody or antigen-binding fragment thereof specifically binds to Gal9.

[0051] In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises (a) a HCVR comprising (i) a HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), wherein X1 is selected from T, P or A; X2 is selected from T, S or G; and X3 is selected from S or D; (ii) a HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), wherein X4 is E; and (iii) a HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) a LCVR comprising: (i) a HCVR CDR3 sequence having the amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13) LCVR CDR1 sequence, wherein X5 is selected from Q or R; X6 is selected from S or N; X7 is selected from L, V or I; X8 is selected from Y or W; X9 is S, X 10 Selected from S, P or A; X 11 Selected from N or H; X 12 is N or Y; X 13 is K or R; and X 14 is Y or H; (ii) has the amino acid sequence WX 15 SX 16 RX 17 X 18 LCVR CDR2 sequence, where X 15 Selected from A or G; X 16 Selected from T, N, M or A; X 17 Selected from G or E; and X 18 is selected from S, Y, P or T; and (iii) has an amino acid sequence of QQYYX 19 X 20 The LCVR CDR3 sequence of PFT (SEQ ID NO: 30), wherein X 19 is F, and X 20 Y; wherein the antibody or antigen-binding fragment thereof specifically binds Gal9.

[0052] Specific embodiments include an isolated monoclonal antibody or antigen-binding fragment thereof comprising (C) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:5, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a light chain variable region (LCVR) comprising a LCVR CDR1 sequence of SEQ ID NO:16, a LCVR CDR2 sequence of SEQ ID NO:25, and a LCVR CDR3 sequence of SEQ ID NO:33; (D) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:6, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:17, a LCVR CDR2 sequence of SEQ ID NO:26, and a LCVR CDR3 sequence of SEQ ID NO:33; (E) a HCVR comprising a LCVR CDR1 sequence of SEQ ID NO:16, a LCVR CDR2 sequence of SEQ ID NO:25, and a LCVR CDR3 sequence of SEQ ID NO:33. NO:7, a HCVR CDR1 sequence, a HCVR CDR2 sequence, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:18, a LCVR CDR2 sequence of SEQ ID NO:27, and a LCVR CDR3 sequence of SEQ ID NO:33; (F) a HCVR comprising a HCVR CDR1 sequence, a HCVR CDR2 sequence, and a HCVR CDR3 sequence of SEQ ID NO:7, a HCVR CDR2 sequence of SEQ ID NO:9, and a LCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence, a LCVR CDR2 sequence of SEQ ID NO:28, and a LCVR CDR3 sequence of SEQ ID NO:33; or (G) a HCVR comprising a HCVR CDR1 sequence, a HCVR CDR2 sequence, and a HCVR CDR3 sequence of SEQ ID NO:12 CDR3 sequence; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO: 20, a LCVR CDR2 sequence of SEQ ID NO: 29, and a LCVR CDR3 sequence of SEQ ID NO: 33, wherein the antibody or antigen-binding fragment thereof specifically binds Gal9.

[0053] In another embodiment, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, comprising (a) a HCVR comprising (i) a HCVR CDR1 sequence having an amino acid sequence of GYX1FX2X3YTIH (SEQ ID NO: 1), wherein X1 is selected from E or P; X2 is selected from T or S; and X3 is selected from E or S; (ii) a HCVR CDR2 sequence having an amino acid sequence of WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), wherein X4 is selected from W or E; and (iii) a HCVR CDR3 sequence having an amino acid sequence of HGGYDGFDY (SEQ ID NO: 12); and (b) a light chain variable region (LCVR) comprising: (i) a HCVR CDR1 sequence having an amino acid sequence of KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13) LCVR CDR1 sequence, wherein X5 is Q, X6 is selected from S or N, X7 is selected from L or V, X8 is selected from Y or W, X9 is selected from P or S, X 10 Select from N or S, X 11 It is N, X 12 Select from Q or N, X 13 is K; and X 14 is Y; (ii) has the amino acid sequence WX 15 SX 16 RX 17 X 18 LCVR CDR2 sequence, where X 15 Selected from A or G; X 16 Select from T or N; X 17 Select from G or E, and X 18 is S; and (iii) has the amino acid sequence QQYYX 19 X 20 The LCVR CDR3 sequence of PFT (SEQ ID NO: 30), wherein X 19 Selected from S or F; and X 20 Selected from Y or F, wherein the antibody or antigen-binding fragment thereof specifically binds Gal9.

[0054] Specific embodiments include an isolated monoclonal antibody or antigen-binding fragment thereof comprising (A) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:3, a HCVR CDR2 sequence of SEQ ID NO:10, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:15, a LCVR CDR2 sequence of SEQ ID NO:23, and a LCVR CDR3 sequence of SEQ ID NO:32; or (C) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:5, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a light chain variable region (LCVR) comprising a LCVR CDR1 sequence of SEQ ID NO:16, a LCVR CDR2 sequence of SEQ ID NO:25, and a LCVR CDR3 sequence of SEQ ID NO:33. CDR3 sequence, wherein the antibody or antigen-binding fragment thereof specifically binds to Gal9.

[0055] The terms "antibody", "ab", "immunoglobulin" or "Ig" refer in the broadest sense to glycoproteins with the same structural features (i.e., molecules containing an antigen binding site that immunospecifically binds to an antigen), and encompass various antibody structures, including natural or artificial, monovalent or multivalent antibodies, including but not limited to monoclonal antibodies (including chimeric monoclonal antibodies, humanized monoclonal antibodies and human monoclonal antibodies, particularly humanized monoclonal antibodies), polyclonal antibodies, single-chain antibodies, antibody fragments and multispecific antibodies (e.g., bispecific antibodies). Although antibodies exhibit binding specificity to specific antigens, immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. "Antibodies" encompass any polypeptide comprising an antigen binding site, regardless of source, species of origin, method of production and characteristics. The term "antibody" can also broadly refer to molecules comprising complementary determining regions (CDR) 1, CDR2 and CDR3 of a heavy chain and CDR1, CDR2 and CDR3 of a light chain, wherein the molecule is capable of binding to an antigen.

[0056] 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, wherein each light chain comprises a light chain variable region and at least a portion of a light chain constant region.

[0057] "Native antibodies" and "complete immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. The light chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. Depending on the amino acid sequence of the constant domain of its heavy chain, immunoglobulins can be assigned to different classes. There are five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes). For example, IgG antibodies include, but are not limited to, IgG1 (containing a γ1 constant region), IgG2 (containing a γ2 constant region), IgG3 (containing a γ3 constant region), and IgG4 (containing a γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (containing an α1 constant region) and IgA2 (containing an α2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 (containing a μ1 constant region) and IgM2 (containing a μ2 constant region). The heavy chain constant domains corresponding to different immunoglobulin classes are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different immunoglobulin classes are well known.

[0058] An intact antibody may have one or more "effector functions," which refer to those biological activities attributable to the Fc region (a native sequence Fc region or an amino acid sequence variant Fc region or any other modified Fc region) of an antibody. Examples of antibody effector functions include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor (BCR)); and cross-presentation of antigens by antigen-presenting cells or dendritic cells.

[0059] Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V H), followed by multiple constant domains. Each light chain has a variable domain at one end (V L ) and have a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form an interface between the light chain and the heavy chain variable domain. Each variable region contains three segments called complementary determining regions (CDRs) or hypervariable regions, and the more highly conserved parts of the variable domains are called framework regions (FRs). The variable domains of the heavy and light chains each contain four FR regions, mainly using a β-folded configuration, connected by three CDRs, which form a loop connecting the β-folded structure and forming a part of the β-folded structure in some cases. The CDRs in each chain are closely held together by FRs, and together with the CDRs from the other chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al., NIH Publication No. 91-3242, Vol. I, pp. 647-669

[1991] ). The constant domains do not directly participate in the binding of antibodies to antigens, but exhibit various effector functions, such as antibody participation in antibody-dependent cellular toxicity.

[0060] As used herein, the term "heavy chain variable region (HCVR)" refers to at least a region comprising 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 comprises at least a portion (e.g., all) of FR1 (HFR1) at the N-terminus of CDR-H1 and / or at least a portion (e.g., all) of FR4 (HFR4) at the C-terminus of CDR-H3.

[0061] As used herein, the term "heavy chain constant region" refers to a region comprising at least three heavy chain constant domains, CH1, CH2, and CH3. Non-limiting exemplary heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody comprising a γ constant region is an IgG antibody, an antibody comprising a δ constant region is an IgD antibody, an antibody comprising an α constant region is an IgA antibody, an antibody comprising an ε constant region is an IgE antibody, and an antibody comprising a μ constant region is an IgM antibody.

[0062] As used herein, the term "light chain variable region (LCVR)" refers to a region comprising a 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 comprises at least a portion (e.g., all) of FR1 (LFR1) at the N-terminus of CDR-L1 and / or at least a portion (e.g., all) of FR4 (LFR4) at the C-terminus of CDR-L3.

[0063] As used herein, the term "light chain constant region" refers to the region comprising the light chain constant domain CL. Non-limiting exemplary light chain constant regions include λ and κ.

[0064] As used herein, the term "light chain" refers to a polypeptide comprising at least a light chain variable region and 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 and with or without a leader sequence.

[0065] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for the possible naturally occurring mutations that may be present in a small amount, the individual antibodies constituting the population are identical. Monoclonal antibodies are highly specific and are directed to a single antigenic site. In addition, in contrast to polyclonal antibody preparations that typically include different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on an antigen. In addition to its specificity, the advantage of monoclonal antibodies is that they are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibodies to be produced by any particular method. For example, the monoclonal antibody to be used according to the present invention can be prepared by the hybridoma method first described by Kohler et al., Nature [Natural], 256:495 (1975), or can be prepared by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). For example, "monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), from yeast displays, and from transgenic animals.

[0066] The terms "amino acid" and "naturally occurring amino acid" refer in the broadest sense to α-amino acids, which are organic compounds containing both an amino functional group and a carboxylic acid functional group from which proteins are produced. Each α-amino acid contains a central carbon atom attached to an amino group, a carboxylic acid, a hydrogen, and an "R" group. The "R" group varies between each amino acid and is the defining difference that distinguishes each amino acid. Twenty amino acids are encoded by the eukaryotic genetic code and are naturally incorporated into polypeptide chains during protein construction, and are therefore referred to as "naturally occurring" amino acids. Other non-naturally occurring amino acids are known that are not found in proteins or are not produced naturally in eukaryotic cells by standard cellular mechanisms (e.g., synthesized from intermediates produced during glycolysis). The twenty naturally occurring amino acids are classified by the chemical properties of their different "R" group side chains. These properties include positively or negatively charged side chains, polar uncharged side chains, hydrophobic side chains, and special case side chains of glycine, cysteine, and proline (which contain side chains that are not easily classified by the above properties).

[0067] The 20 naturally occurring amino acids include: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).

[0068] Of the 20 naturally occurring amino acids, five have side chains that can be charged. At pH = 7, the following three are positively charged: lysine (Lys, K), arginine (Arg, R), and histidine (His, H) (basic side chains); and the following two are negatively charged: aspartic acid (Asp, D) and glutamic acid (Glu, E) (acidic side chains). Polar uncharged amino acids include serine (Ser, S), threonine (Thr, T), asparagine (Asn, N), and glutamine (Gln, Q), which readily form hydrogen bonds with water and other amino acids. They do not ionize under normal conditions, with the notable exception of the catalytic serine in serine proteases. The non-polar or hydrophobic amino acids are alanine (Ala, A), valine (Val, V), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F), tryptophan (Trp, W), and tyrosine (Tyr, Y), and their interactions are the main driving force behind the process of folding proteins into their functional three-dimensional structures. None of the side chains of these amino acids are easily ionized and therefore have no pKa, with the exception of tyrosine, where the hydroxyl group can be deprotonated at high pH to form a negatively charged phenolate. Due to this, tyrosine can be placed in the category of polar uncharged amino acids, but its very low solubility in water matches well with the characteristics of hydrophobic amino acids.

[0069] Several side chains are not well described by the charge, polarity, and hydrophobicity categories. Glycine (Gly, G) can be considered a polar amino acid because its small size means that its solubility depends primarily on the amino and carboxylic acid groups. However, the lack of any side chains provides glycine with unique flexibility among amino acids and has a great impact on protein folding. Cysteine ​​(Cys, C) can also easily form hydrogen bonds, which would put it in the polar amino acid category, but it can often be found in protein structures forming covalent bonds (called disulfide bonds) with other cysteines. These bonds affect the folding and stability of proteins and are essential in antibody formation. Proline (Pro, P) has an alkyl side chain and can be considered hydrophobic, but because the side chain is connected back to the alpha amino group, it becomes particularly inflexible when incorporated into proteins. Like glycine, it affects protein structure in a unique way among amino acids. G, C, and P are generally classified as "special case side chain" amino acids.

[0070] The antibodies or antigen-binding fragments thereof described herein specifically bind to Gal9.

[0071] Galectins, members of the animal lectin family, are defined by shared consensus amino acid sequences that confer specific binding to glycoconjugates containing β-galactosides. The galectin family is ubiquitously expressed from lower organisms such as nematodes and sponges to higher mammalian species such as humans. The presence of such proteins in many species coupled with the presence of highly conserved amino acid residues critical for ligand recognition of the carbohydrate recognition domain (CRD) suggests that galectins participate in key conserved biological processes. The galectin CRD, which is typically composed of approximately 130 amino acid residues tightly folded into a sandwich structure of five- and six-stranded β-sheets, recognizes the basic structure of N-acetyllactosamine (LacNAc). To date, fifteen members of the mammalian galectin family have been identified. Hirabayashi and Kasai proposed designating galectin subfamilies as prototype, chimeric, or tandem repeat based on their domain organization. The prototype galectins (galectins-1, 2, 5, 7, 10, 11, 13, 14 and 15) contain a single CRD with a short N-terminal sequence, while the tandem repeat galectins (galectins-4, 6, 8, 9 and 12) contain two non-identical CRDs connected by a short linker peptide sequence. The single chimeric galectin (galectin-3) has a CRD with an extended N-terminus containing several repeats of a proline-tyrosine-glycine-rich motif. Gal9 (tandem repeat galectin) was originally isolated from mouse embryonic kidney cells and was later found to be widely distributed in rat and mouse tissues. In contrast, the expression of human Gal9 is limited to peripheral blood leukocytes and lymphoid tissues. It is reported that the potent eosinophil chemoattractant ecalectin, originally cloned from a human T cell line, is identical to human Gal9. There are several mammalian Gal9 isoforms, each with a different length of linker.

[0072] Gal9 exhibits multiple biological functions, including cell aggregation, eosinophil chemoattraction, and apoptosis of mouse thymocytes and T cells and human melanoma cells. Mouse Gal9 induces thymocyte apoptosis in a lactose-inhibitable manner. The chemoattractant activity of Gal9 depends on its carbohydrate binding activity and requires two CRDs. Therefore, the physiological function of Gal9 may depend on its carbohydrate recognition ability. Two physiological targets of mouse Gal9 have been reported, namely Tim-3 and glucose transporter (GLUT)-2. Tim-3 is specifically expressed on the surface of type 1 T helper (Th1) cells. Gal9 recognizes one or more carbohydrates covalently bound to Tim-3; the Gal9-Tim3 pathway induces cell death of Th1 cells, indicating that Gal9 plays a vital role in downregulating Th1 responses.

[0073] However, Gal9 also interacts with GLUT-2 on the extracellular surface, which is a glucose transporter expressed on the surface of pancreatic β cells and is essential for glucose-stimulated insulin secretion. Gal9 recognizes GLUT-2 by recognizing carbohydrate moieties as required for GLUT-2 to reside on the cell surface. Loss of glycosylation or addition of glycans reduces the half-life of GLUT-2 on the cell surface and causes receptor endocytosis and redistribution to endosomes and lysosomes.

[0074] Although the exact target carbohydrate structure recognized by Gal9 in vivo has not yet been identified, in vitro analysis has demonstrated that human Gal9 has a high affinity for a variety of oligosaccharides containing β-galactosides. In vitro analysis revealed that the N-terminal CRD (NCRD) and C-terminal CRD (CCRD) of human Gal9 have different oligosaccharide binding affinities. Compared with the CCRD of human Gal9, the NCRD exhibits a significant affinity for complex glycoconjugates such as Forsmann pentasaccharide and polymerized N-acetyllactosamine.

[0075] Since the biological activity of human Gal9 will depend on the ligand specificity of each CRD and the subsequent multivalent binding conferred by the two CRDs, structural analysis of the specific carbohydrate recognition properties of the human Gal9 NCRD should provide insights into the physiological functions of Gal9.

[0076] In one aspect, the HCVR sequence of the antibody or antigen-binding fragment thereof of (A), (B), (C), (D), (E), (F) or (G) further comprises the HFR3 sequence of SEQ ID NO: 68, and optionally further comprises the HFR1 sequence of SEQ ID NO: 66, the HFR2 sequence of SEQ ID NO: 67 and / or the HFR4 sequence of SEQ ID NO: 71. In another aspect, the HCVR sequence of the antibody or antigen-binding fragment thereof of (A), (B), (C), (D), (E), (F) or (G) further comprises the HFR3 sequence of SEQ ID NO: 69, and optionally further comprises the HFR1 sequence of SEQ ID NO: 66, the HFR2 sequence of SEQ ID NO: 67 and / or the HFR4 sequence of SEQ ID NO: 71. In another aspect, the HCVR sequence of the antibody or antigen-binding fragment thereof of (A), (B), (C), (D), (E), (F) or (G) further comprises the HFR3 sequence of SEQ ID NO: 70, and optionally further comprises the HFR1 sequence of SEQ ID NO: 66, the HFR2 sequence of SEQ ID NO: 67 and / or the HFR4 sequence of SEQ ID NO: 71. In another aspect, the HCVR sequence of the antibody or antigen-binding fragment thereof of (A), (B), (C), (D), (E), (F) or (G) further comprises the HFR3 sequence of SEQ ID NO: 76, and optionally further comprises the HFR1 sequence of SEQ ID NO: 66, the HFR2 sequence of SEQ ID NO: 67 and / or the HFR4 sequence of SEQ ID NO: 71. In another aspect, the HCVR sequence of the antibody or antigen-binding fragment thereof of (A), (B), (C), (D), (E), (F) or (G) further comprises the HFR3 sequence of SEQ ID NO: 77, and optionally further comprises the HFR1 sequence of SEQ ID NO: 66, the HFR2 sequence of SEQ ID NO: 67 and / or the HFR4 sequence of SEQ ID NO: 71. In one aspect, the LCVR sequence of the antibody or antigen-binding fragment thereof of (A), (B), (C), (D), (E), (F) or (G) optionally further comprises the LFR1 sequence of SEQ ID NO: 72, the LFR2 sequence of SEQ ID NO: 73, the LFR3 sequence of SEQ ID NO: 74 and / or the LFR4 sequence of SEQ ID NO: 75.

[0077] In another aspect, the HCVR sequence is SEQ ID NO:39 and / or the LCVR sequence is SEQ ID NO:41; or the HCVR sequence is SEQ ID NO:43 and / or the LCVR sequence is SEQ ID NO:45; or the HCVR sequence is SEQ ID NO:47 and / or the LCVR sequence is SEQ ID NO:49; or the HCVR sequence is SEQ ID NO:51 and / or the LCVR sequence is SEQ ID NO:53; or the HCVR sequence is SEQ ID NO:55 and / or the LCVR sequence is SEQ ID NO:57; or the HCVR sequence is SEQ ID NO:59 and / or the LCVR sequence is SEQ ID NO:61; or the HCVR sequence is SEQ ID NO:63 and / or the LCVR sequence is SEQ ID NO:65. In some aspects, the antibody comprises: a HCVR sequence of SEQ ID NO:39 and a LCVR sequence of SEQ ID NO:41; or a HCVR sequence of SEQ ID NO:43 and a LCVR sequence of SEQ ID NO:45; or a HCVR sequence of SEQ ID NO:47 and a LCVR sequence of SEQ ID NO:49; or a HCVR sequence of SEQ ID NO:51 and a LCVR sequence of SEQ ID NO:53; or a HCVR sequence of SEQ ID NO:55 and a LCVR sequence of SEQ ID NO:57; or a HCVR sequence of SEQ ID NO:59 and a LCVR sequence of SEQ ID NO:61; or a HCVR sequence of SEQ ID NO:63 and a LCVR sequence of SEQ ID NO:65.

[0078] In a specific embodiment, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, comprising a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO: 3, a HCVR CDR2 sequence of SEQ ID NO: 10, and a HCVR CDR3 sequence of SEQ ID NO: 12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO: 15, a LCVR CDR2 sequence of SEQ ID NO: 23, and a LCVR CDR3 sequence of SEQ ID NO: 32, wherein the antibody or antigen-binding fragment thereof specifically binds Gal9. In one aspect, the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises a HFR3 sequence of SEQ ID NO: 68, and optionally further comprises a HFR1 sequence of SEQ ID NO: 66, a HFR2 sequence of SEQ ID NO: 67, and / or a HFR4 sequence of SEQ ID NO: 71. In one embodiment, the HCVR sequence is SEQ ID NO: 39. In other aspects, the LCVR sequence of the antibody or antigen-binding fragment thereof optionally further comprises an LFR1 sequence of SEQ ID NO: 72, an LFR2 sequence of SEQ ID NO: 73, an LFR3 sequence of SEQ ID NO: 74, and / or an LFR4 sequence of SEQ ID NO: 75. In one embodiment, the LCVR sequence is SEQ ID NO: 41. An antibody is specifically exemplified herein as having a heavy chain sequence of SEQ ID NO: 38 and a light chain sequence of SEQ ID NO: 40 (Ab001).

[0079] In another specific embodiment, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, comprising a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO: 5, a HCVR CDR2 sequence of SEQ ID NO: 9, and a HCVR CDR3 sequence of SEQ ID NO: 12; and a light chain variable region (LCVR) comprising a LCVR CDR1 sequence of SEQ ID NO: 16, a LCVR CDR2 sequence of SEQ ID NO: 25, and a LCVR CDR3 sequence of SEQ ID NO: 33, wherein the antibody or antigen-binding fragment thereof specifically binds Gal9. In one aspect, the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises a HFR3 sequence of SEQ ID NO: 69, and optionally further comprises a HFR1 sequence of SEQ ID NO: 66, a HFR2 sequence of SEQ ID NO: 67, and / or a HFR4 sequence of SEQ ID NO: 71. In one embodiment, the HCVR sequence is SEQ ID NO: 47. In other aspects, the LCVR sequence of the antibody or antigen-binding fragment thereof optionally further comprises an LFR1 sequence of SEQ ID NO: 72, an LFR2 sequence of SEQ ID NO: 73, an LFR3 sequence of SEQ ID NO: 74, and / or an LFR4 sequence of SEQ ID NO: 75. In one embodiment, the LCVR sequence is SEQ ID NO: 49. An antibody is specifically exemplified herein as having a heavy chain sequence of SEQ ID NO: 46 and a light chain sequence of SEQ ID NO: 48 (Ab 003).

[0080] In one aspect, the antibody is a humanized antibody.

[0081] The "humanized" form of non-human (e.g., mouse) antibodies is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) containing the minimum sequence derived from non-human immunoglobulins. In most cases, humanized antibodies are such human immunoglobulins (receptor antibodies) in which the residues from the complementary determining regions (CDRs) of the receptor are replaced by residues from CDRs with desired specificity, affinity, and ability from non-human species (donor antibodies) (such as mice, rats, or rabbits). In some cases, the framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized antibodies may include residues that are neither found in the CDRs or framework sequences of the receptor antibodies nor in the input. These modifications are carried out to further improve and maximize antibody manufacturability, stability, and / or performance. In general, a humanized antibody will comprise substantially all of at least one and typically two variable domains, in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. A humanized antibody will also optimally comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. For further details, see Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992). Humanized antibodies include PRIMATIZED TM An antibody, wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing a macaque with an antigen of interest (see Weber, J et al., From rabbit antibody repertoires to rabbit monoclonal antibodies. Exp Mol Med 49, e305 (2017)).

[0082] Methods for humanizing non-human antibodies are well known in the art (see Yaghoub Safdari et al., (2013) Antibody humanization methods-a review and update [antibody humanization methods-review and update], Biotechnology and Genetic Engineering Reviews [biotechnology and gene engineering review], 29:2, 175-186, DOI: 10.1080 / 02648725.2013.801235). Preferably, the humanized antibody has one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are generally referred to as "import" residues, which are typically taken from the "import" variable domain. Humanization can be performed essentially by replacing rodent CDRs or CDR sequences with the corresponding sequences of human antibodies according to the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536

[1988] ). Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), in which far less than the entire human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0083] The selection of human variable domains (both light and heavy chains) to be used for the preparation of humanized antibodies is very important for reducing antigenicity. According to the so-called "best fit" method, the sequence of the variable domains of rodent antibodies is screened for the entire library of known human variable domain sequences. The human sequence closest to the rodent sequence is then accepted as the human framework region (FR) of the humanized antibody (Sims et al., J. Immunol. [Journal of Immunology], 151: 2296 (1993); Chothia et al., J. Mol. Biol. [Journal of Molecular Biology], 196: 901

[1987] ). Another method uses a specific framework region derived from the consensus sequence of all human antibodies of a specific subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623

[1993] ).

[0084] It is further important to humanize antibodies while retaining high affinity to antigen and other favorable biological properties. In order to achieve this goal, humanized antibodies are prepared according to a preferred method by using the three-dimensional model analysis of the parental sequence and the process of various conceptual humanized products of the parental and humanized sequences. The three-dimensional immunoglobulin model is usually available and is familiar to those skilled in the art. It is available to illustrate and display the computer program of the possible three-dimensional conformational structure of the selected candidate immunoglobulin sequence. Check that these displays allow the possible role of the analysis residue in the function of the candidate immunoglobulin sequence, i.e., analyze the residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from receptor and input sequence to achieve desired antibody characteristics, such as the increased affinity to one or more target antigens. Usually, CDR residues directly and most substantially participate in affecting antigen binding.

[0085] Alternatively, it is now possible to produce transgenic animals (e.g., mice) that are capable of producing a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production after immunization. For example, homozygous deletions of antibody heavy chain joining region (JH) genes in chimeric and germline mutant mice have been described to result in complete inhibition of endogenous antibody production. Transferring human germline immunoglobulin gene arrays in such germline mutant mice will result in the production of human antibodies after antigen challenge. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States], 90: 2551 (1993); Jakobovits et al., Nature [Nature], 362: 255-258 (1993); Bruggermann et al., Year in Immuno. [Year of Immunology], 7: 33 (1993). Human antibodies can also be derived from antibody display libraries, such as phage display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597

[1991] and Valldorf, Bernhard et al., "Antibody display technologies: selecting the cream of the crop" Biological Chemistry, Vol. 403, No. 5-6, 2022, pp. 455-477).

[0086] Humanized antibodies can be made by replacing sequences of Fv variable regions that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. A general review of humanized chimeric antibodies is provided by Morrison et al. (Science [Science] 229: 1202-1207 (1985)) and Oi et al. (BioTechniques [Biotechnology] 4: 214 (1986)). These methods include separating, manipulating and expressing nucleic acid sequences encoding all or part of the immunoglobulin Fv variable region from at least one of the heavy chain or light chain. The source of such nucleic acid is well known to those skilled in the art, and can be obtained, for example, from hybridomas that produce antibodies. The recombinant DNA encoding the humanized or chimeric antibody or its fragment can then be cloned into an appropriate expression vector.

[0087] Alternatively, humanized antibodies can be generated by CDR substitution (U.S. Pat. No. 5,225,539; Jones, Nature 321:552-525 (1986); Verhoeyan et al., Science 239:1534 (1988); and Beidler, J. Immunol. 141:4053-4060 (1988)).

[0088] In another aspect, the antigen-binding fragment thereof is Fab, Fab', F(ab')2, F d , single-chain Fv or scFv, disulfide-linked F v , V-NAR domain, IgNar, intracellular antibody, IgGACH2, mini antibody, F(ab')3, tetrabodies, tribodies, diabodies, single domain antibodies, DVD-Ig, Fcab, mAb2, (scFv)2, tandem scFv, TandAb, nanobody (see mdpi.com / 2073-4468 / 8 / 2 / 28 / pdf) or scFv-Fc.

[0089] Experimentally, antibodies can be cleaved with the proteolytic enzyme papain, which results in the breakage of each heavy chain, producing three separate antibody fragments. An "antibody fragment" comprises a portion of an intact antibody, preferably an antigen-binding region or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab' and F(ab')2, Fc fragments or Fc fusion products, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), and fragments comprising VL or VH domains; diabodies, triabodies, etc. (Zapata et al. Protein Eng. [Protein Engineering] 8(10): 1057-1062

[1995] ). The term "antibody fragment" or (antibody) "antigen-binding portion" includes, but is not limited to, fragments that are capable of binding to an antigen. Two units consisting of a light chain and a heavy chain fragment that is approximately equal in mass to the light chain are referred to as Fab fragments (i.e., "antigen-binding" fragments). A third unit consisting of two equal segments of a heavy chain is referred to as an Fc fragment. The Fc fragment is typically not involved in antigen-antibody binding, but is important in later processes involving clearance of antigens in the body.

[0090] Fab fragment contains the constant domain of light chain and the first constant domain (CH1) of heavy chain. "Fab" refers to an antibody fragment with a molecular weight of about 50,000 daltons and has the activity of binding to antigen. It comprises about half of the N-terminal side of the heavy chain connected by a disulfide bridge and the whole light chain. Fab can be obtained in particular by treating immunoglobulin with a protease (papain). The difference between Fab' fragment and Fab fragment is that several residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the hinge region of the antibody. Fab'-SH is the name of such Fab' in this article, in which one or more cysteine ​​residues of the constant domain carry free sulfhydryl groups.

[0091] F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The term "F(ab')2" refers to a fragment of about 100,000 daltons and having the activity of binding to an antigen. This fragment is slightly larger than two Fab fragments connected via a disulfide bridge in the hinge region. These fragments are obtained by treating immunoglobulins with a protease (pepsin). Fab fragments can be obtained from F(ab')2 fragments by cleaving the disulfide bridges in the hinge region.

[0092] The Fc region of an antibody is a tail region of an antibody that interacts with cell surface receptors and some proteins of the complement system. This property allows antibodies to activate the immune system. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments of the second and third constant domains of the two heavy chains of the antibody; IgM and IgEFc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. The Fc region of IgG carries highly conserved N-glycosylation sites. Glycosylation of the Fc fragment is essential for Fc receptor-mediated activity. The N-glycans attached to this site are mainly complex types of core fucosylated diantennary structures. In addition, a small amount of these N-glycans also carry bisecting GlcNAc and α-2,6-linked sialic acid residues.

[0093] Fc fusion proteins (also referred to as Fc chimeric fusion proteins, Fc-Ig, Ig-based chimeric fusion proteins, and Fc-tagged proteins) consist of the Fc domain of an IgG genetically linked to a peptide or protein of interest. Fc fusion proteins have become valuable reagents for in vivo and in vitro studies. The scope of the binding partner of an Fc fusion can be a single peptide, a ligand activated after binding to a cell surface receptor, a signaling molecule, an extracellular domain of a receptor activated after dimerization, or a bait protein for identifying a binding partner in a protein microarray. One of the most valuable features of the Fc domain in vivo is that it can significantly prolong the plasma half-life of the protein of interest, which leads to improved therapeutic efficacy for biotherapeutics; this attribute makes Fc fusion proteins attractive biotherapeutics.

[0094] The Fc fusion protein can be part of a pharmaceutical composition comprising the Fc fusion protein and a pharmaceutically acceptable carrier excipient or carrier. Pharmaceutically acceptable carriers, excipients or stabilizers are well known in the art (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides such as XTEN (see Haeckel A et al., XTEN as Biological Alternative to PEGylation Allows Complete Expression of a Protease-Activatable Killin-Based Cytostatic [XTEN as a biological alternative to PEGylation allows complete expression of cell inhibitors based on protease-activatable Killin]. PLoS One. [Public Library of Science ONE] 2016 Jun 13;11(6):e 0157193. doi:10.1371 / journal.pone.0157193. PMID:27295081; PMCID:PMC4905650); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0095] "Fv" is the smallest antibody fragment containing complete antigen recognition and antigen binding sites. The dimer of "scFv" corresponds to two scFv molecules connected together by a peptide bond. The Fv chain is often the result of fusion gene expression, which contains genes encoding VH and VL connected by a linker sequence encoding a peptide. The human scFv fragment can include a CDR region preferably maintained in an appropriate conformation by means of genetic recombination technology. This region is composed of a dimer of a heavy chain variable domain and a light chain variable domain that are tightly non-covalently associated. It is in this configuration that the three CDRs of each variable domain interact to limit the antigen binding site on the surface of the VH-VL dimer. Together, six CDRs give antibodies antigen binding specificity. However, even a single variable domain (or half of an Fv containing only three CDRs specific to an antigen) can also have the ability to recognize and bind to an antigen, but the affinity is lower than the entire binding site.

[0096] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0097] A "dsFv" fragment is a VH-VL heterodimer stabilized by disulfide bridges; it can be bivalent (dsFv2). A bivalent sc(Fv)2 or multivalent antibody fragment can be formed spontaneously by association of monovalent scFvs, or by linking scFv fragments via peptide binding sequences.

[0098] The Fc fragment supports the biological properties of the antibody, in particular its ability to be recognized by immune effectors or to activate complement. It consists of the constant segments outside the hinge region of the heavy chain.

[0099] The term "diabodies" means small antibody fragments with two antigen-fixing sites. These fragments contain a variable heavy chain domain VH connected to a variable light chain domain VL in the same VH-VL polypeptide chain. Using binding sequences that are too short to allow the two domains of the same chain to match, matching with the two complementary domains of the other chain must occur, and thus two antigen-fixing sites are generated.

[0100] Various techniques for producing antibody fragments have been developed. Traditionally, these fragments are obtained via proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods [Journal of Biochemical and Biophysical Methods] 24: 107-117 (1992) and Brennan et al., Science [Science], 229: 81

[1985] ). However, these fragments can now be directly produced by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage library discussed above. Alternatively, Fab'-SH fragments can be directly recovered from Escherichia coli, and chemically coupled to form F (ab') 2 fragments (Carter et al., Bio / Technology [Biology / Technology] 10: 163-167

[1992] ). According to another method, F (ab') 2 fragments can be directly isolated from recombinant host cell cultures. Other techniques for producing antibody fragments will be clear to skilled practitioners. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93 / 16185.

[0101] In other aspects, the monoclonal antibody or antigen-binding fragment thereof has a K of less than about 5 nM, 2 nM, 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.05 nM, or 0.01 nM. d Binds human Gal9.

[0102] In some aspects, the affinity matured monoclonal antibody or antigen-binding fragment thereof has an affinity for Gal9 that is about 2-10 times greater than that of the non-affinity matured Ab A. For example, the affinity matured monoclonal antibody or antigen-binding fragment thereof has an affinity for Gal9 that is about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more times greater than that of the non-affinity matured Ab A.

[0103] The term "antigen binding domain" refers to the portion of an antibody molecule that contains a region that specifically binds or is complementary to a portion or all of an antigen. In the case of a larger antigen, an antibody may bind only to a specific portion of the antigen. An "epitope" or "antigenic determinant" is the portion of an antigen molecule that is responsible for interacting with the antigen binding domain of an antibody. The antigen binding domain may be provided by one or more antibody variable domains (e.g., a so-called Fd antibody fragment consisting of a VH domain). The antigen binding domain may include an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0104] "Antigen" according to the present invention covers any substance that will cause an immune response. In particular, "antigen" refers to any substance that specifically reacts with an antibody or T lymphocyte (T cell), preferably a peptide or protein. According to the present invention, the term "antigen" includes any molecule comprising at least one epitope. Preferably, the antigen in the context of the present invention is a molecule that optionally induces an immune response after treatment. According to the present invention, any suitable antigen can be used, which is a candidate for an immune response, wherein the immune response is preferably a cellular immune response. In the context of an embodiment of the present invention, the antigen is preferably presented by a cell, preferably by an antigen presenting cell, and the antigen presenting cell includes a diseased cell, particularly a cancer cell (in the case of an MHC molecule), which results in an immune response against the antigen. Antigen is preferably a product corresponding to a naturally occurring antigen, or is derived from a naturally occurring antigen. Such naturally occurring antigens include tumor antigens.

[0105] The term "epitope" refers to an antigenic determinant in a molecule (such as an antigen), ie, a part or fragment of a molecule that is recognized by the immune system. An epitope of a protein (such as a tumor antigen) preferably comprises a continuous or discontinuous part of the protein.

[0106] The terms "epitope", "antigenic peptide", "antigenic epitope", "immunogenic peptide" and "MHC binding peptide" may be used interchangeably herein and preferably relate to an incomplete representation of an antigen that is preferably capable of eliciting an immune response against the antigen or a cell that expresses or contains and preferably presents the antigen.

[0107] The term "binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner. A variety of methods for measuring binding affinity or binding activity are known in the art, any of which can be used for the purposes of the present invention. Specific illustrative embodiments are described below.

[0108] As used herein, "specific binding" refers to the binding of an antibody to a predetermined antigen. Typically, the antibody binds to a specific antigen in a manner corresponding to about 10 -8 M or smaller K D The antibody binds with such affinity (as determined by K D -dissociation constant) binds to the predetermined antigen with an affinity that is at least 1 / 10, and preferably at least 1 / 100, of the affinity of the antibody to a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). Alternatively, the antibody may be expressed as a dissociation constant corresponding to about 10 6 M -1 , or about 10 7 M -1 , or about 10 8 M -1, or 10 9 M -1 or higher K A The antibody binds with such affinity (as determined by K A -association constant) binds to the predetermined antigen with an affinity that is at least 10 times, and preferably at least 100 times, greater than the affinity with which the antibody binds to a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein).

[0109] As used herein, the term “k d ”(sec -1 ) is intended to refer to the dissociation rate constant for a particular antibody-antigen interaction. This value is also referred to as the off value. As used herein, the term "K D ”(M -1 ) is intended to refer to the equilibrium dissociation constant for a specific antibody-antigen interaction.

[0110] In some embodiments, the dissociation constant (K) of the Gal9 antibody of the present invention for Gal9 (eg, for human Gal9) is d )≤5 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM or ≤0.001 nM (e.g., 10 -8 M or smaller, for example, from 10 -8 M to 10 -13 M, for example, from 10 -9 M to 10 -13 In certain embodiments, the dissociation constant (K) of a Gal9 antibody for Gal9 (eg, for human Gal9) is d )≤5 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM or ≤0.001 nM (e.g., 10 -8 M or smaller, for example, from 10 - 8 M to 10 -13 M, for example, from 10 -9 M to 10 -13 M).

[0111] As used herein, the term “k a ”(M -1 sec -1 ) is intended to refer to the association rate constant for a particular antibody-antigen interaction. As used herein, the term "K A “(M) is intended to refer to the equilibrium association constant for a particular antibody-antigen interaction.

[0112] The antibodies described herein can be characterized using a surface plasmon resonance (SPR) assay or using a solution equilibrium assay (SEA).

[0113] Surface plasmon resonance (SPR) is a physical phenomenon that occurs at the thin gold surface on the SPR chip. In the SPR assay, the binding target of the analyte (e.g., antibody) is covalently attached to the surface by a dextran linker. When the analyte flows through the surface, the binding that occurs between it and the surface can be directly measured as a change in the local refractive index. This produces binding and dissociation curves and binding maximum data. The SPR assay is typically more sensitive than plate-based assays, and detects antibodies with a much wider range. This is mainly due to the lack of a washing step, which typically removes most low-affinity target populations. The label-free nature of SPR also eliminates the confusing effects of cross-linked labels, simplifies analysis and significantly reduces false negatives. The SPR assay also realizes online isotype typing by adding anti-isotype antibodies after the association period.

[0114] SPR assays or label-free interaction measurements can be applied to determine antibody specificity, study the thermodynamics of the interaction, investigate the mechanism of action or for epitope characterization or affinity screening.

[0115] The ability to reliably estimate affinity using unpurified samples is a prerequisite for effective screening. The use of high-throughput affinity screening processes enables the reduction of overall project costs and duration by avoiding time-consuming purification steps and enabling initial characterization of thousands of antibody candidates in a short period of time. The essential features of such affinity screening tools are (1) reliable identification of candidates with improved affinity; (2) reasonable estimation of the equilibrium dissociation constant (KD) of the corresponding clones; (3) compatibility with crude production matrices (such as bacterial extracts or cell culture supernatants); and (4) high-throughput processing (e.g., by automation of all sample processing steps). Although state-of-the-art technologies like surface plasmon resonance (SPR; e.g., Biacore) and biolayer interferometry (BLI; e.g., Octet) partially meet these criteria, 4,5, it remains a challenge to distinguish affinities in the picomolar (pM) range with reasonable throughput.

[0116] Solution equilibrium titration (SET) or solution equilibrium assay (SEA) uses highly sensitive electrochemiluminescence as a readout system. Because the binding partner is not labeled, the resulting KD represents a reliable approximation of the true affinity. For screening, diluted bacterial lysates or cell culture supernatants are balanced with four different concentrations of soluble target molecules, and unbound antibodies are then quantified on 384-well Meso Scale Discovery (MSD) plates coated with the corresponding antigen. In order to determine the KD value based on the resulting titration curve, a fitting model derived from the law of mass action for 1: 1 and 2: 1 binding modes is applied to simultaneously evaluate hundreds of interactions.

[0117] On the one hand, the antibody binds to Gal9 and inhibits Gal9 from binding to a Gal9 receptor (e.g., TIM3 or CD44). On the other hand, the antibody binds to Gal9 and does not inhibit Gal9 from binding to a Gal9 receptor (e.g., TIM3 or CD44). In some embodiments, the Gal9 antibody having any of the features provided herein inhibits at least 25%, 50%, 75%, 80%, 90% or 100% of the signal transduction of Gal9.

[0118] On the other hand, antibodies reversed Gal9-induced T cell (such as CD4 + Th1 apoptosis of T cells).

[0119] In one aspect, the antibody inhibits Gal9-induced Treg expansion.

[0120] In another embodiment, the invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the isolated monoclonal antibodies or antigen-binding fragments described herein, thereby treating the cancer in the subject.

[0121] The term "subject" as used herein refers to any individual or patient to whom the subject method is performed. Typically, the subject is a human, but as will be appreciated by those skilled in the art, the subject may be an animal. Thus, other animals (including vertebrates such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals (including cattle, horses, goats, sheep, pigs, chickens, etc.) and primates (including monkeys, chimpanzees, orangutans and gorillas)) are included within the definition of a subject.

[0122] The term "treatment" is used interchangeably with the term "treatment method" herein and refers to 1) a therapeutic treatment or measure that cures, slows down, alleviates the symptoms of a diagnosed pathological condition or disorder, and / or stops the progression of a diagnosed pathological condition or disorder, and 2) preventive / preventative measures. Those individuals in need of treatment may include individuals who already have a specific medical disorder and those individuals who may eventually develop a disorder (i.e., those individuals in need of preventive measures). "Treatment" covers any administration or application of therapeutic agents for diseases (also referred to herein as "disorders" or "disorders") in mammals (including humans), and includes inhibiting disease or disease progression, inhibiting or slowing a disease or its progression, preventing its development, partially or completely alleviating a disease, partially or completely alleviating one or more symptoms of a 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 feature and / or reducing the incidence, degree or likelihood of the feature.

[0123] The terms "therapeutically effective amount", "effective dose", "therapeutically effective dose", "effective amount" and the like refer to the amount of the subject compound that will elicit a biological or medical response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, physician or other clinician. Typically, the response is an improvement in a patient's symptoms or a desired biological outcome (e.g., treatment of cancer). The therapeutically effective amount of the Gal9 antibody and antigen-binding fragments thereof of the present invention can vary depending on factors such as the disease state, age, sex and weight of the individual and the ability of the antibody and antigen-binding fragments thereof to elicit a desired response in the individual. The therapeutically effective amount encompasses an amount in which the therapeutically beneficial effects outweigh any toxic or deleterious effects of the Gal9 antibody and antigen-binding fragments thereof.

[0124] The terms "administration of" and / or "administering" should be understood as meaning to provide a therapeutically effective amount of a pharmaceutical composition to a subject in need of treatment. The route of administration can be enteral, topical or parenteral. Therefore, the route of administration includes, but is not limited to, intradermal, subcutaneous, intravenous, intramuscular, intravitreal, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal, oral, sublingual, buccal, rectal, vaginal, nasal, ocular administration and infusion, inhalation and atomization. As used herein, the phrases "parenteral administration" and "administered parenterally" mean administration methods other than enteral and topical administration. In various embodiments, the Gal9 antibody can be administered subcutaneously or intravenously.

[0125] The antibodies and antigen-binding fragments thereof described herein can be prepared as pharmaceutical compositions comprising the antibodies or their antigen-binding fragments and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid or carrier conventional in the art for use with a therapeutic agent, which together with the therapeutic agent constitute a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dose and concentration employed and is compatible with the other ingredients of the formulation. A 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 gel capsule. If the therapeutic agent is to be administered subcutaneously, the carrier is ideally non-allergic to the skin and does not cause injection site reactions.

[0126] The pharmaceutical composition can be administered in a variety of unit dosage forms according to the administration method. Suitable unit dosage forms include, but are not limited to, powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injections, implantable sustained-release formulations, lipid complexes, and the like.

[0127] The subject compositions may be formulated into preparations in solid, semisolid, liquid or gaseous form; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols.

[0128] In various embodiments, compositions comprising Gal9 antibodies and antigen-binding fragments thereof 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); Strickley RG, Lambert WJ. A review of Formulations of Commercially Available Antibodies. J Pharm Sci. [Journal of Pharmaceutical Sciences] July 2021; 110(7):2590-2608.e56.doi:10.1016 / j.xphs.2021.03.017.Epub 2021 March 28.PMID:33789155). Various pharmaceutically acceptable carriers (including vehicles, adjuvants and diluents) are available. In addition, various pharmaceutically acceptable auxiliary substances (such as pH adjusters and buffers, tension adjusters, stabilizers, wetting agents, etc.) are also available. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof.

[0129] In various embodiments, compositions comprising Gal9 antibodies and antigen-binding fragments thereof 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 oil, synthetic fatty acid glyceride, higher fatty acid ester or propylene glycol); and if necessary, together with traditional additives (such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives).

[0130] In various embodiments, the compositions can be formulated for inhalation, for example, using pressurized acceptable propellants (eg, dichlorodifluoromethane, propane, nitrogen, etc.).

[0131] In various embodiments, compositions can also be formulated into sustained-release microcapsules, such as with biodegradable or non-biodegradable polymers. Non-limiting exemplary biodegradable formulations include polylactic-glycolic acid (PLGA) polymers. Non-limiting exemplary non-biodegradable formulations include polyglycerol fatty acid esters. Some methods of preparing such formulations are described in, for example, EP 1125584 A1.

[0132] Also provided is a drug dosage package comprising one or more containers, each container containing one or more doses of Gal9 antibodies and antigen-binding fragments thereof. In some embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising a Gal9 antibody and an antigen-binding fragment thereof, and has or does not have one or more additional agents. In some embodiments, this unit dose is provided in a disposable prefilled syringe for injection. In various embodiments, the composition contained in the unit dose may include saline, sucrose, etc.; a buffer such as phosphate, etc.; and / or may be formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder, which may be reconstituted after adding an appropriate 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 composition of the present invention comprises heparin and / or proteoglycans.

[0133] The pharmaceutical composition is administered in an amount effective to treat or prevent a particular indication. The therapeutically effective amount typically depends on the weight of the subject being treated, his or her physical or health condition, the extensiveness of the condition to be treated, or the age of the subject being treated.

[0134] In some embodiments, the Gal9 antibody and its antigen-binding fragment can be administered in an amount ranging from about 50 μg / kg body weight to about 50 mg / kg body weight per dose. In some embodiments, the Gal9 antibody and its antigen-binding fragment can be administered in an amount ranging from about 100 μg / kg body weight to about 50 mg / kg body weight per dose. In some embodiments, the Gal9 antibody and its antigen-binding fragment can be administered in an amount ranging from about 100 μg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the Gal9 antibody and its antigen-binding fragment can be administered in an amount ranging from about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose.

[0135] In some embodiments, the Gal9 antibody and its antigen-binding fragments can be administered in an amount ranging from about 10 mg to about 1,000 mg per dose. In some embodiments, the Gal9 antibody and its antigen-binding fragments can be administered in an amount ranging from about 20 mg to about 500 mg per dose. In some embodiments, the Gal9 antibody and its antigen-binding fragments can be administered in an amount ranging from about 20 mg to about 300 mg per dose. In some embodiments, the Gal9 antibody and its antigen-binding fragments can be administered in an amount ranging from about 20 mg to about 200 mg per dose.

[0136] The Gal9 antibody and its antigen binding fragment composition can be administered to the subject as needed. In some embodiments, an effective dose of the Gal9 antibody and its antigen binding fragment is administered to the subject once or multiple times. In various embodiments, an effective dose of the Gal9 antibody and its antigen binding fragment is administered to the subject once a month, less than once a month (e.g., like every two months, every three months, or every six months). In other embodiments, an effective dose of the Gal9 antibody and its antigen binding fragment is administered more than once a month (e.g., like every two weeks, every week, twice a week, three times a week, every day, or multiple times a day). An effective dose of the Gal9 antibody and its antigen binding fragment is administered to the subject at least once. In some embodiments, an effective dose of the Gal9 antibody and its antigen binding fragment can be administered multiple times, including a period of at least one month, at least six months, or at least one year. In some embodiments, the Gal9 antibody and its antigen binding fragment are administered to the subject as needed to alleviate one or more symptoms of the disease.

[0137] The antibodies described herein can be used to treat cancer.

[0138] Cancer is a group of diseases that involve abnormal cell growth that has the potential to invade or spread to other parts of the body. In 2015, approximately 90.5 million people were living with cancer, with approximately 14.1 million new cases occurring each year and causing approximately 8.8 million deaths (15.7% of all deaths). The most common types of cancer in men are lung, prostate, colorectal, and stomach cancers. In women, the most common types are breast, colorectal, lung, and cervical cancers.

[0139] The term "cancer" refers to a group of diseases characterized by abnormal and uncontrolled cell proliferation starting from one site (primary site) with the potential to invade and spread to other sites (secondary sites, metastases), which distinguishes cancer (malignant tumors) from benign tumors. Virtually all organs can be affected, resulting in more than 100 types of cancer that may affect humans. Cancer can arise from many causes, including genetic predisposition, viral infection, exposure to ionizing radiation, exposure to environmental pollutants, tobacco and / or alcohol use, obesity, poor diet, lack of physical activity, or any combination thereof.

[0140] As used herein, "neoplasm" or "tumor" (including grammatical variations thereof) means a new and abnormal growth of tissue, which may be benign or cancerous. In related aspects, a neoplasm is indicative of a neoplastic disease or disorder, including, but not limited to, various cancers. For example, such cancers may include prostate cancer, pancreatic cancer, bile duct cancer, colon cancer, rectal cancer, liver cancer, kidney cancer, lung cancer, testicular cancer, breast cancer, ovarian cancer, pancreatic cancer, brain cancer and head and neck cancer, melanoma, sarcoma, multiple myeloma, leukemia, lymphoma, etc.

[0141] In one aspect, the cancer is a hematological cancer or a solid tumor.

[0142] Cancers that begin in cells of blood-forming tissues (such as the bone marrow) or the immune system are called blood cancers or blood cancers. Blood cancers affect the production and function of blood cells and are classified into three main types: leukemias, lymphomas, and multiple myeloma.

[0143] As used herein, "leukemia" refers to a blood cancer caused by the rapid production of abnormal white blood cells. Examples of leukemias include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia, chronic myeloid leukemia, and hairy cell leukemia.

[0144] As used herein, "lymphoma" refers to a type of blood cancer that affects the lymphatic system. Examples of lymphomas include AIDS-related lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, Hodgkin's lymphoma, mycosis fungoides, non-Hodgkin's lymphoma, primary central nervous system lymphoma, Sezary syndrome, cutaneous T-cell lymphoma, and Waldenstrom's macroglobulinemia.

[0145] As used herein, "myeloma" is a cancer of plasma cells. Examples of myelomas include chronic myeloproliferative neoplasm, Langerhans cell histiocytosis, multiple myeloma, plasma cell neoplasm, myelodysplastic syndrome, and myelodysplastic / myeloproliferative neoplasm.

[0146] In some embodiments, the Gal9 antibodies and antigen-binding fragments thereof of the present invention can be used alone, or alternatively in combination with any other suitable compound known to be able to treat a disease or indication.

[0147] In some aspects, administration can be combined with one or more additional therapeutic agents. The phrases "combination therapy", "in combination with", etc. refer to the use of more than one drug or treatment at the same time to increase the response. The composition of the present invention can be used in combination with other drugs or treatments for the treatment of cancer, for example. In particular, administration of the composition of the present invention to a subject can be combined with any anticancer therapy. Such therapy can be administered before, simultaneously with, or after administration of the composition of the present invention.

[0148] In certain embodiments, Gal9 antibody and another treatment are administered to a subject (e.g., a subject suffering from cancer) simultaneously or continuously. For example, Gal9 antibody can be administered together with one or more of the following: radiotherapy, surgery or chemotherapy, such as targeted chemotherapy or immunotherapy. The administration of two medicaments can be, 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 a week or weeks apart, or the administration of the second medicament can be, 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 a week or weeks after the first medicament has been administered.

[0149] In some aspects, the blood cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia, chronic myeloid leukemia and hairy cell leukemia, AIDS-related lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, Hodgkin lymphoma, mycosis fungoides, non-Hodgkin lymphoma, primary central nervous system lymphoma, Sezary syndrome, cutaneous T-cell lymphoma, Waldenstrom's macroglobulinemia, diffuse large B-cell lymphoma (DLBCL) and multiple myeloma.

[0150] The French, American, and British (FAB) classification of acute myeloid leukemia (AML) classifies AML into different stages of the disease. The FAB subtypes are shown in Table 1.

[0151] Table 1: FAB classification

[0152] FAB subtype describe M0 Undifferentiated acute myeloblastic leukemia M1 Minimally mature acute myeloblastic leukemia M2 Mature acute myeloblastic leukemia M3 Acute promyelocytic leukemia (APL) M4 Acute myelomonocytic leukemia M5 Acute monocytic leukemia M6 Acute erythroblastic leukemia M7 Acute megakaryoblastic leukemia

[0153] Examples of solid cancers include, but are not limited to, carcinoma, sarcoma, squamous cell carcinoma, small cell lung cancer, pituitary cancer, 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 or uterine cancer, salivary gland cancer, kidney cancer, renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, brain cancer, endometrial cancer, testicular cancer, bile duct cancer, gallbladder cancer, gastric cancer, melanoma, gastroesophageal cancer, esophageal cancer, cervical cancer, and various types of head and neck cancer.

[0154] In other aspects, the solid tumor is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, melanoma, uveal melanoma, colorectal cancer, renal cancer, urothelial carcinoma, ovarian cancer, endometrial cancer, liver cancer, pancreatic cancer, bile duct cancer, gastric cancer, gastroesophageal cancer, esophageal cancer, cervical cancer, head and neck squamous cell carcinoma, and prostate cancer.

[0155] In one aspect, the method further comprises administering to the patient a chemotherapeutic agent, an anti-angiogenic agent, a growth inhibitory agent, an immuno-oncology agent, a checkpoint inhibitor, and / or an anti-tumor composition.

[0156] A "chemotherapeutic agent" is 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 cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including hexamethylmelamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; polyacetylamines (particularly bullatacin and bullatacinone); camptothecins (including synthetic analogs of topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogs of adolesin, carzelesin and biszelesin); scutellarin (particularly scutellarin 1 and scutellarin 8); caudate tin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, naphthyl mustard, cholophosphamide, estramustine, ifosfamide, mechlorethamine, nitrogen oxide mustard hydrochloride, melphalan, new mustard, phenylephrine, prednimustine, trofosfamide, uracil mustard mustard); nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma ll and calicheamicin omega ll (see, e.g., Agnew, Chem lntl. Ed. Engl [German Angewandte Chemie International English Edition], 33:183-186 (1994); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarcinogenin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, actinomycin C, carabicin, carminamicin, carmoamicin, chromomycin, dactinomycin, daunomycin, detoxorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (such as mitomycin C), mycophenolic acid, nogamycin, olivetomycin, peplomycin, potycin (pot firomycin), puromycin, quelamycin, rhodorubicin, streptozotocin, streptozotocin, tuberculocin, ubenimex, netastatin, doxycycline; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as folinic acid, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-thiol Purines, thioimidazole, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as caprotestosterone, drostanolone propionate, cyclothiodine, melastosane, testolactone; antiadreners, such as aminoglutethimide, mitotane, and triptostane; folic acid supplements, such as folinic acid (folinic acid acid; acetoglucuronide; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demeclofenac; diazocine; elfomithine; elliptonium acetate; epothilone; etoglucone; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansine alkaloids, such as maytansine and ansamitocin; mitoguanidine; mitoxantrone; mopidanmol; nitraerine; pentostatin; methamine mustard; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; polysaccharide complex (JHS Natural Products Company (JHS Natural Products, Eugene, Oregon); razoxane; lisoxin; sizoran; spirogermanamine; tenuisporic acid; triazoquinone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, mycloracin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulanol; piperobroman;gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), albumin-engineered nanoparticle formulations of paclitaxel without cremophor (American Pharmaceutical Partners, Schaumburg, Ill.), nab-paclitaxel (Celgene), and doxetaxel (Rhone-Poulenc Rorer, Antoni, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunorubicin; aminopterin; xeloda; ibandronate; irinotecan (CPT-11) (including irinotecan with 5-FU and leucovorin) topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; Compretin; leucovorin (LV); oxaliplatin, including oxaliplatin treatment regimen (FOLFOX); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib) and VEGF-A that reduce cell proliferation, and pharmaceutically acceptable salts, acids or derivatives of any of the above. ;

[0157] Other non-limiting exemplary chemotherapeutic agents include anti-hormonal agents that act to modulate or inhibit the effects of hormones on cancer, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, troxifene, keoxifene, LY117018, onapristone and toremifene; aromatase inhibitors that inhibit aromatase (which regulates estrogen production in the adrenal glands), such as, for example, 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, and anastrozole; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation (e.g., 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, such as vaccines, vaccines and vaccines; rIL-2; topoisomerase 1 inhibitors; rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0158] "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 conjugate or fusion protein thereof, which directly or indirectly inhibits angiogenesis, vasculogenesis or undesirable vascular permeability. It is understood that anti-angiogenic agents include those agents that bind to angiogenic factors or their receptors and block the angiogenic activity of the angiogenic factors or their receptors. For example, anti-angiogenic agents are antibodies or other antagonists to angiogenic agents, such as antibodies to VEGF-A (e.g., bevacizumab) or VEGF-A receptors (e.g., KDR receptors or Flt-1 receptors), anti-PDGFR inhibitors such as (imatinib mesylate), small molecules that block VEGF receptor signaling (e.g., PTK787 / ZK2284, SU6668, / SUI 1248 (sunitinib malate), AMG706 or 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., Table 3 lists anti-angiogenic therapies for malignant melanoma); Ferrara and Alitalo (1999) Nature 54:317-3179 (e.g., Table 3 lists anti-angiogenic therapies for malignant melanoma); Medicine [Nature Medicine] 5(12):1359-1364; Tonini et al. (2003) Oncogene [Cancer Gene] 22:6549-6556 (for example, Table 2 lists known anti-angiogenic factors); and Sato (2003) Int. J. Clin. Oncol. [International Journal of Clinical Oncology] 8:200-206 (for example, Table 1 lists anti-angiogenic agents used in clinical trials).

[0159] As used herein, "growth inhibitor" refers to a compound or composition that inhibits the growth of cells (such as cells expressing VEGF) in vitro or in vivo. Therefore, a growth inhibitor can be an agent that significantly reduces the percentage of cells in the S phase (such as cells expressing VEGF). Examples of growth inhibitors include, but are not limited to, agents that block cell cycle progression (outside the S phase), such as agents that induce G1 arrest and M phase arrest. Classical M phase blockers include vinca (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that cause G1 arrest also cause S phase arrest, such as DNA alkylating agents, such as tamoxifen, prednisone, dacarbazine, nitrogen mustard, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, by Murakami et al., entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" (WB Saunders, Philadelphia, 1995), e.g., p. 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (Rhone Brown Roller Company), derived from the European yew, is a semisynthetic analog of paclitaxel (Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, thereby inhibiting mitosis in cells.

[0160] The term "anti-tumor composition" refers to a composition containing at least one active therapeutic agent that can be used to treat cancer. Examples of therapeutic agents include, but are not limited to, for example, chemotherapeutic agents, growth inhibitors, cytotoxic agents, agents used in radiation therapy, 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, CTLA4 inhibitors (e.g., anti-CTLA antibody ipilimumab), PD-1 inhibitors 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, CTLA4, VISTA or VEGF receptors), TRAIL / Apo2 and other biologically active and organic chemical agents, etc. Combinations thereof are also included in the present invention.

[0161] "Checkpoint inhibitor therapy" is a current form of cancer treatment that uses immune checkpoints that affect the function of the immune system. Immune checkpoints can be stimulatory or inhibitory. Tumors can use these checkpoints to protect themselves from immune system attack. Checkpoint therapy can block inhibitory checkpoints and restore immune system function. Checkpoint proteins include programmed cell death 1 protein (PDCD1, PD-1; also known as CD279) and its ligand PD-1 ligand 1 (PD-L1, CD274), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), A2AR (adenosine A2A receptor), B7-H3 (or CD276), B7-H4 (or VTCN1), BTLA (B and T lymphocyte attenuation factor or CD272), IDO (indoleamine 2,3-dioxygenase), KIR (killer cell immunoglobulin-like receptor), LAG3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin domain and mucin domain molecule 3), and VISTA (V-domain Ig inhibitor of T cell activation).

[0162] Programmed cell death protein 1, also known as PD-1 and CD279 (cluster of differentiation 279), is a cell surface receptor that plays an important role in downregulating the immune system and promoting self-tolerance by inhibiting T cell inflammatory activity. PD-1 is an immune checkpoint and prevents autoimmunity through a dual mechanism of promoting apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes while reducing apoptosis of regulatory T cells (anti-inflammatory, suppressive T cells).

[0163] PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 protein is upregulated on macrophages and dendritic cells (DCs) in response to LPS and GM-CSF treatment, and is upregulated on T cells and B cells during TCR and B cell receptor signaling, while in resting mice, PD-L1 mRNA can be detected in the heart, lungs, thymus, spleen, and kidneys. After treatment with IFN-γ, PD-L1 is expressed on almost all murine tumor cell lines, including PA1 myeloma, P815 mastocytoma, and B16 melanoma. PD-L2 expression is more restricted and is mainly expressed by DCs and some tumor lines.

[0164] CTLA4 or CTLA-4 (cytotoxic T lymphocyte-associated protein 4), also known as CD152 (differentiation cluster 152), is a protein receptor that acts as an immune checkpoint to downregulate immune responses. CTLA4 is constitutively expressed in regulatory T cells, but is only upregulated in conventional T cells after activation - a phenomenon that is particularly noteworthy in cancer. CTLA4 is a member of the immunoglobulin superfamily, which is expressed by activated T cells and transmits inhibitory signals to T cells. CTLA4 is homologous to the T cell co-stimulatory protein CD28, and both molecules bind to CD80 and CD86 (also known as B7-1 and B7-2, respectively) on antigen-presenting cells. The affinity and avidity of CTLA-4 binding to CD80 and CD86 are greater than the affinity and avidity of binding to CD28, thus enabling it to outcompete CD28 compared to its ligand. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. CTLA4 is also found in regulatory T cells and contributes to their inhibitory function. T cell activation via the T cell receptor and CD28 results in increased expression of CTLA-4.

[0165] There are several checkpoint inhibitors currently used to treat cancer. PD-1 inhibitors include pembrolizumab (Keytruda) and nivolumab (Opdivo). PD-L1 inhibitors include atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi). CTLA-4 inhibitors include iplimumab (Yervoy). There are several other checkpoint inhibitors in development, including anti-B7-H3 antibodies (MGA271), anti-KIR antibodies (Lirilumab), and anti-LAG3 antibodies (BMS-986016).

[0166] As used herein, "immuno-oncology agent" refers to an anti-cancer agent that specifically targets cancer by targeting the immune system; this generally refers to a therapeutic antibody. In some aspects, the immuno-oncology agent is an anti-glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) antibody.

[0167] Tumor necrosis factor receptor superfamily member 18 (TNFRSF18), also known as glucocorticoid-induced TNFR-related protein (GITR) or CD357, also known as activation-inducible TNFR family receptor (AITR), is encoded by the TNFRSF18 gene on chromosome 1.

[0168] GITR is a member of the TNFR superfamily and has a high degree of homology with other members of TNFRSF (such as CD137, OX40 or CD27) in the cytoplasmic domain (characterized by cysteine ​​pseudo-repeats). GITR is constitutively expressed on CD25+CD4+ regulatory T cells, and its expression is upregulated on all T cell subsets after activation. GITR is also expressed on mouse neutrophils and NK cells. GITR interacts with its ligand (GITRL) expressed on antigen presenting cells (APCs) and endothelial cells.

[0169] GITR is an immune checkpoint molecule with potential in cancer treatment. GITR signaling can promote anti-tumor and anti-infection immune responses, but can also be a driver of autoimmune diseases. Different responses to GITR signaling depend on GITR expression on different immune cell types. How GITR signaling is regulated in different cells remains unknown. GITR agonistic antibodies are in clinical trials as activators of effector CD8 T cells while reducing the number of circulating inhibitory regulatory T cells. The limited response to GITR agonistic antibodies has been enhanced in combination with anti-PD-1 or anti-CTLA-4 therapy.

[0170] In additional embodiments, the present invention provides polynucleotides encoding the heavy chain or light chain, or antigen binding portion thereof, of any of the antibodies or antigen binding fragments thereof described herein.

[0171] The present invention also provides nucleic acid molecules, which include polynucleotides encoding one or more chains of antibodies (such as anti-Gal9 antibodies) described herein. In some embodiments, the nucleic acid molecules include polynucleotides encoding the heavy chain or light chain of the antibodies described herein. In some embodiments, the nucleic acid molecules include both polynucleotides encoding the heavy chain of the antibodies described herein and polynucleotides encoding the light chain of the antibodies described herein. In some embodiments, the first nucleic acid molecule includes a first polynucleotide encoding a heavy chain, and the second nucleic acid molecule includes a second polynucleotide encoding a light chain.

[0172] In some such embodiments, the heavy chain and light chain are expressed from one nucleic acid molecule, or are expressed as two separate polypeptides from two separate nucleic acid molecules. In some embodiments, such as when the antibody is a scFv, a single polynucleotide encodes a single polypeptide comprising both the heavy chain and the light chain linked together.

[0173] In some embodiments, the polynucleotide encoding the heavy chain or light chain of the antibody described herein comprises a nucleotide sequence encoding a leader sequence that is located at the N-terminus of the heavy chain or light chain when translated. As discussed above, the leader sequence can be a native heavy chain or light chain leader sequence, or can be another heterologous leader sequence.

[0174] As used herein, the term "nucleic acid" or "oligonucleotide" refers to a polynucleotide, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acids include, but are not limited to, genomic DNA, cDNA, mRNA, iRNA, miRNA, tRNA, ncRNA, rRNA, and recombinantly produced and chemically synthesized molecules, such as aptamers, plasmids, antisense DNA strands, shRNA, ribozymes, conjugated nucleic acids and oligonucleotides. According to the present invention, nucleic acids can exist as single-stranded or double-stranded linear or covalently circular closed molecules. Nucleic acids can be isolated. The term "isolated nucleic acid" means that nucleic acids (i) are amplified in vitro, for example, via polymerase chain reaction (PCR), (ii) are produced by cloning and recombination, (iii) are separated and purified, for example, by cutting and gel electrophoresis, (iv) are synthesized, for example, by chemical synthesis, or (vi) are extracted from a sample. Nucleic acids can be used to introduce into cells, i.e., transfect cells, particularly in the form of RNA, which can be prepared from a DNA template by in vitro transcription. In addition, RNA can be modified by stabilizing sequences, capping, and polyadenylation before application.

[0175] The term "sample" can include whole blood, blood plasma, serum, buffy coat, body fluid, lymphocyte, tissue, amniotic fluid, cultured cells, etc. The term "sample" can also refer to urine sample, saliva sample, blood cell-free DNA, and skin, mucous membrane or other body areas or surfaces or samples thereof to be checked by means of swabs. In various embodiments, the device for collecting the sample can contain a preservative. The preservative can include preservatives such as hydrochloric acid, boric acid, acetic acid, toluene or thymol.

[0176] Nucleic acids can be extracted from samples by any method known in the art, including by using an organic solvent (such as a mixture of phenol and chloroform) followed by precipitation with ethanol. Among other methods for extracting cell-free nucleic acids, one such method includes, for example, the use of polylysine-coated silica particles. Alternatively, commercially available kits (such as Cell-free DNA was extracted using the QIAQUIN DNA Mini Kit (Qiagen, Germantown, MD).

[0177] The extracted nucleic acid is amplified by one of the alternative methods for amplification well known in the art, including, for example, Luminex's technology, which allows up to 500 bioassays to be analyzed simultaneously by reading the bioassays on the surface of tiny polystyrene beads; multiplex PCR, which allows the simultaneous amplification of several DNA sequences; multiplex ligation-dependent probe amplification (MLPA), which is used to amplify multiple targets using a single pair of primers; quantitative PCR (qPCR), which measures and quantifies amplification in real time; ligation chain reaction (LCR), which performs amplification using primers that cover the entire sequence, thus preventing the amplification of sequences with mutations; and rolling circle amplification (RCA), in which a circular DNA is amplified before it is amplified. The two ends of the sequence are joined by a ligase; helicase-dependent amplification (HDA), which relies on a helicase to separate the double-stranded DNA; loop-mediated isothermal amplification (LAMP), which employs a DNA polymerase with high strand displacement activity; nucleic acid sequence-based amplification, which is specifically designed for RNA targets; strand displacement amplification (SDA), which relies on a strand-displacing DNA polymerase to initiate replication at a nick made by a strand restriction endonuclease or nickase at a site contained in the primer; and multiple displacement amplification (MDA), which is based on the use of a DNA polymerase from bacteriophage Amplification methods as used herein have been used and tested and are well known in the art.

[0178] As used herein, "amplified DNA" or "PCR product" refers to an amplified fragment of DNA of a defined size. Various techniques are available and well known in the art for detecting PCR products. PCR product detection methods include, but are not limited to, gel electrophoresis using agarose or polyacrylamide gels and the addition of ethidium bromide stain (DNA intercalator), labeled probes (radioactive or non-radioactive labels, Southern blotting), labeled deoxyribonucleotides (for direct incorporation of radioactive or non-radioactive labels), or silver staining to directly visualize the amplified PCR products; restriction endonuclease digestion, which relies on agarose or polyacrylamide gels or high performance liquid chromatography (HPLC); dot blotting, using hybridization of amplified DNA on specific labeled probes (radioactive or non-radioactive labels); high pressure liquid chromatography using ultraviolet detection; electrochemiluminescence combined with voltage-triggered chemical reactions / photon detection; and direct sequencing using radioactive or fluorescently labeled deoxyribonucleotides to determine the precise order of nucleotides with the target DNA fragment, oligonucleotide ligation assay (OLA), PCR, qPCR, DNA sequencing, fluorescence, gel electrophoresis, magnetic beads, allele-specific primer extension (ASPE) and / or direct hybridization.

[0179] Generally, nucleic acids can be extracted, isolated, amplified or analyzed by a variety of techniques, such as those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed.), Cold Spring Harbor Laboratory Press, Woodbury, NY 2,028 p. (2012); or those described in U.S. Pat. No. 7,957,913; U.S. Pat. No. 7,776,616; U.S. Pat. No. 5,234,809; U.S. Publication No. 2010 / 0285578; and U.S. Publication No. 2002 / 0190663.

[0180] The example of nucleic acid analysis includes but is not limited to order-checking and DNA-protein interaction. Order-checking can be carried out by any method known in the art. DNA sequencing technology includes the classical dideoxy sequencing reaction (Sanger method) separated by gel using labeled terminator or primer and plate or capillary, and next generation sequencing method, such as sequencing by synthesis, pyrophosphate sequencing, 454 sequencing, Illumina / Solexa sequencing, allele-specific hybridization with labeled oligonucleotide probe library, sequencing by synthesis subsequently connected with allele-specific hybridization of labeled clone library, incorporation of real-time monitoring labeled nucleotide during polymerization step, polymerase clone sequencing (polonysequencing) and SOLiD sequencing. Separated molecules can be sequenced by using the order or single extension reaction of polymerase or ligase and by single or sequential differential hybridization with probe library.

[0181] DNA-protein interactions can be analyzed by ChIP sequencing (also known as ChIP-seq). ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify binding sites for DNA-associated proteins. It can be used to accurately locate the overall binding sites of any target protein. Previously, microarray-based chromatin immunoprecipitation (ChIP-on-chip) was the most commonly used technique for studying these protein-DNA relationships. The ChIP method is well known in the art and has been described.

[0182] In one embodiment, the present invention provides a vector comprising a polynucleotide described herein, wherein the vector is an expression vector selected from the group consisting of a mammalian expression vector, a yeast expression vector, an insect expression vector, and a bacterial expression vector.

[0183] Provided are vectors comprising polynucleotides encoding the heavy chain and / or light chain of the antibodies 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 the heavy chain and a second polynucleotide sequence encoding the light chain. In some embodiments, the heavy chain and the light chain are expressed as two separate polypeptides from the vector. In some embodiments, such as when the antibody is a scFv, the heavy chain and the light chain are expressed as part of a single polypeptide.

[0184] In some embodiments, the first vector comprises a polynucleotide encoding a heavy chain, and the 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 (such as similar molar amounts or similar mass amounts). In some embodiments, a first vector and a second vector with a molar ratio or a mass ratio between 5:1 and 1:5 are transfected into a host cell. In some embodiments, for a vector encoding a heavy chain and a vector encoding a light chain, a mass ratio between 1:1 and 1:5 is used. In some embodiments, for a vector encoding a heavy chain and a vector encoding a light chain, a mass ratio of 1:2 is used.

[0185] In some embodiments, a vector optimized for expressing a polypeptide in CHO or CHO-derived cells or in NSO cells is selected. Exemplary vectors are described, for example, in Running Deer et al., Biotechnol. Prog. [Progress in Biotechnology] 20: 880-889 (2004). In some embodiments, a vector is selected for in vivo expression of a Gal9 antibody and its antigen-binding fragment in animals (including humans). In some such embodiments, expression of one or more polypeptides is under the control of one or more promoters that function in a tissue-specific manner. For example, a liver-specific promoter is described, for example, in PCT Publication No. WO 2006 / 076288.

[0186] The terms "vector", "expression vector" or "plasmid DNA" are used herein to refer to a recombinant nucleic acid construct that is manipulated by human intervention. A recombinant nucleic acid construct may contain two or more nucleotide sequences that are linked in a manner such that the product is not found in cells in nature. In particular, two or more nucleotide sequences may be operably linked, such as genes encoding a protein of interest, one or more protein tags, a functional domain, etc.

[0187] Carriers suitable for use in the preparation of proteins and / or protein conjugates include those selected from the following carriers: baculovirus, phage, plasmid, phagemid, cosmid, F cosmid, bacterial artificial chromosome, viral DNA, artificial chromosome based on P1, yeast plasmid and yeast artificial chromosome. For example, viral DNA vectors can be selected from vaccinia, adenovirus, fowl pox virus, pseudorabies virus and SV40 derivatives. One type of vector is a genomic integration vector or an "integration vector" that can be integrated into the chromosomal DNA of a host cell. Another type of vector is an additional vector, such as a nucleic acid capable of extrachromosomal replication. Vectors capable of directing the expression of genes to which they are operably linked are referred to herein as "expression vectors". Viral vectors include adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, vaccinia virus, measles virus, herpes virus and bovine papilloma virus vectors (for a review of viral and non-viral vectors, see Kay et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 94: 12744-12746 (1997)). Viral vectors are modified so that the natural tropism and pathogenicity of the virus have been altered or removed. The genome of the virus can also be modified to increase its infectivity and accommodate the packaging of the nucleic acid encoding the polypeptide of interest.

[0188] The nucleic acid constructs of the invention can be introduced into the cells to be altered, thereby allowing the chimeric protein to be expressed in the cells. A variety of methods are known in the art and are suitable for introducing nucleic acids into cells, including viral and non-viral mediated techniques. Examples of typical non-viral mediated techniques include, but are not limited to, electroporation, calcium phosphate mediated transfer, nuclear transfection, sonoporation, heat shock, magnetofection, liposome mediated transfer, microinjection, microparticle mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethyleneimine, polyethylene glycol (PEG), etc.), or cell fusion. Other transfection methods include proprietary transfection reagents such as Lipofectamine TM 、Dojindo Hilymax TM 、Fugene TM 、jetPEI TM 、Effectene TM and DreamFect TM .

[0189] Nucleic acid construct of the present invention can be introduced into the host cell to be changed, thereby allowing chimeric protein to be expressed in the cell. A variety of host cells are known in the art and are suitable for chimeric protein expression. Examples of typical cells for transfection include, but are not limited to, bacterial cells, eukaryotic cells, yeast cells, insect cells or plant cells. For example, Escherichia coli, Bacillus, Streptomyces, Pichia pastoris, Salmonella typhimurium, Drosophila S2, Spodoptera exigua SJ9, CHO, COS (e.g., COS-7), 3T3-F442A, HeLa, HUVEC, HUAEC, NIH 3T3, Jurkat, 293, 293H or 293F.

[0190] In various embodiments, the heavy and / or light chains of the antibodies described herein can be expressed in prokaryotic cells (such as bacterial cells); or in eukaryotic cells (such as fungal cells (such as yeast), plant cells, insect cells and mammalian cells). Such expression can be performed, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; cells (Crucell); and NSO cells. In some embodiments, the heavy and / or light chains of the antibodies described herein can be expressed in yeast. See, for example, U.S. Publication No. US2006 / 0270045A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform the desired post-translational modification on the heavy and / or light chains of the Gal9 antibody. For example, in some embodiments, the sialylation level of a polypeptide produced by CHO cells is higher than the sialylation level of the same polypeptide produced in 293 cells.

[0191] One or more nucleic acids can be introduced into the desired host cells by any method, including but not limited to calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Edition Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected in the desired host cells according to any suitable method.

[0192] In some embodiments, one or more polypeptides can be produced according to any suitable method in an animal that has been engineered or transfected with one or more nucleic acid molecules encoding the polypeptides.

[0193] The polynucleotide can be delivered to a cell (e.g., a plurality of different cells or cell types comprising a target cell or cell type and / or a non-target cell type) in a vector (e.g., an expression vector). Examples of vectors include, but are not limited to (a) non-viral vectors, such as nucleic acid vectors, including linear oligonucleotides and circular plasmids; artificial chromosomes, such as human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), and bacterial artificial chromosomes (BACs or PACs); episomal vectors; transposons (e.g., PiggyBac); and (b) viral vectors, such as retroviral vectors, lentiviral vectors, adenoviral vectors, and AAV vectors. Viral vectors have several advantages for delivering nucleic acids, including high infectivity and / or tropism for certain target cells or tissues. In some cases, viral vectors can be used to deliver the polynucleotides described herein.

[0194] In another embodiment, the present invention provides a method for rescuing or promoting effector T cell proliferation, enhancing effector T cell activity and / or identifying and treating a subject, the method comprising: (i) determining the level of Gal9 in a sample from a subject, and (ii) administering an effective amount of any one of the isolated monoclonal antibodies or antigen-binding fragments thereof described herein to the subject, the subject having a Gal9 level higher than a reference level of Gal9, thereby rescuing or promoting effector T cell proliferation, enhancing effector T cell activity and / or identifying and treating the subject.

[0195] "Disorder associated with the suppressive activity of regulatory T lymphocytes" means any disorder (non-autoimmune) in which the suppressive activity of regulatory T lymphocytes plays a role, particularly by promoting the development or persistence of the disorder. In particular, it has been demonstrated that the suppressive activity of regulatory T lymphocytes promotes the development of tumors. Therefore, the present invention is more particularly directed to cancers in which the suppressive activity of T lymphocytes plays a role.

[0196] In pathological situations, Tregs may cause inappropriate immunosuppression, which could, for example, promote tumor growth. Tregs have been 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.

[0197] During activation, Gal9 is directly expressed by Tregs, while effector T lymphocytes express it only very weakly, or not at all. Therefore, by targeting Gal9, for example using a Gal9-specific antibody, the suppressive activity of regulatory T lymphocytes can be specifically inhibited without the risk of causing exhaustion of effector T lymphocytes. Therefore, antibodies against Gal9 and suppressing the suppressive activity of regulatory T lymphocytes according to the present invention can be used to treat diseases or disorders associated with the suppressive activity of regulatory T lymphocytes, in particular to treat cancer.

[0198] Cancers treatable by the methods / purposes of the present invention include those cancers in which regulatory T lymphocytes exert their inhibitory activity, such as those cancers in which relatively large amounts of regulatory T lymphocytes are present in tumor tissue or circulation. The amplification of regulatory T lymphocytes (which can be measured by the frequency of Treg) is generally associated with the increase of Treg activation. The frequency of regulatory T lymphocytes can be assessed 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.

[0199] In one aspect, the subject is diagnosed with cancer, is at risk for developing cancer, or has a recurrence of cancer.

[0200] In another aspect, the reference level of Gal9 is the level of Gal9 measured in a sample from a healthy or control individual.

[0201] 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 suffer from the disease or condition that the methods or compositions of the present invention are being used to identify. In one embodiment, the reference sample, reference cell, or reference tissue is obtained from a healthy part of the body of the same subject or patient for whom the disease or condition is being identified using the compositions or methods of the present invention. In one embodiment, the reference sample, reference cell, or reference tissue is obtained from a healthy part of the body of at least one individual who is not the subject or patient for whom the disease or condition is being identified using the compositions or methods of the present invention. In some embodiments, the reference sample, reference cell, or reference tissue was previously obtained from a patient prior to contracting the disease or condition or at a pre-stage of the disease or condition.

[0202] In some aspects, determining comprises comparing the level of Gal9 in the sample to a reference level.

[0203] In one aspect, the cancer is a blood cancer or a solid tumor. In some aspects, the blood cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia, chronic myeloid leukemia and hairy cell leukemia, AIDS-related lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, Hodgkin's lymphoma, mycosis fungoides, non-Hodgkin's lymphoma, primary central nervous system lymphoma, Sezary syndrome, cutaneous T-cell lymphoma, Waldenstrom's macroglobulinemia, diffuse large B-cell lymphoma (DLBCL) and multiple myeloma. In other aspects, the solid tumor is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, melanoma, uveal melanoma, colorectal cancer, renal cancer, urothelial carcinoma, ovarian cancer, endometrial cancer, liver cancer, pancreatic cancer, bile duct cancer, gastric cancer, gastroesophageal cancer, esophageal cancer, cervical cancer, head and neck squamous cell carcinoma, or prostate cancer.

[0204] In one aspect, the sample is a blood sample, a plasma sample, a serum sample, a tumor biopsy sample, or a bone marrow (BM) derived mononuclear cell (MNC) sample.

[0205] In one aspect, the method further comprises administering to the patient a chemotherapeutic agent, an anti-angiogenic agent, a growth inhibitor, an immuno-oncology agent, a checkpoint inhibitor, and / or an anti-tumor composition. In some aspects, the checkpoint inhibitor is anti-PD-1. In other aspects, the immuno-oncology agent is an anti-glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) antibody.

[0206] Other Embodiments

[0207] Embodiment 1. An isolated monoclonal antibody or antigen-binding fragment thereof, the isolated monoclonal antibody or antigen-binding fragment thereof comprising:

[0208] (a) a HCVR comprising (i) a HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), wherein X1 is selected from T, E, P or A; X2 is selected from T, G or S; and X3 is selected from E, S or D; (ii) a HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), wherein X4 is selected from E, W or V; and (iii) a HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and

[0209] (b) a LCVR comprising (i) a molecule having the amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13NX 14 LA (SEQ ID NO: 13) LCVR CDR1 sequence, wherein X5 is selected from Q or R; X6 is selected from S or N; X7 is selected from L, V or I; X8 is selected from Y or W; X9 is selected from S or P; X 10 Selected from N, S, P or A; X 11 Selected from N or H; X 12 Selected from Q, N or Y; X 13 Selected from K or R; and X 14 Selected from Y or H; (ii) having an amino acid sequence WX 15 SX 16 RX 17 X 18 LCVR CDR2 sequence, where X 15 Selected from A or G; X 16 Selected from T, N, M or A; X 17 Selected from G or E; and X 18 is selected from the group consisting of S, E, Y, P or T; and (iii) has an amino acid sequence of QQYYX 19 X 20 The LCVR CDR3 sequence of PFT (SEQ ID NO: 30), wherein X 19 Selected from S or F; and X 20 Select from Y or F,

[0210] wherein the antibody or antigen-binding fragment thereof specifically binds galectin-9 (Gal9), provided that the antibody does not comprise the HCVR CDR1 sequence GYTFTEYTIH (SEQ ID NO: 2), the HCVR CDR2 sequence WFYPGSGSTEYAQKFQG (SEQ ID NO: 9), the HCVR CDR3 sequence HGGYDGFDY (SEQ ID NO: 12), the LCVR CDR1 sequence KSSQSLLYSNNQKNYLA (SEQ ID NO: 14), the LCVR CDR2 sequence WASTRGS (SEQ ID NO: 22) and the LCVR CDR3 sequence QQYYSYPFT (SEQ ID NO: 31).

[0211] Embodiment 2. The isolated monoclonal antibody or antigen-binding fragment thereof according to embodiment 1, wherein the isolated monoclonal antibody or antigen-binding fragment thereof comprises:

[0212] (a) a HCVR comprising (i) a HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), wherein X1 is selected from T or E, and X2 is selected from T or G, and X3 is E; (ii) a HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), wherein X4 is selected from W or V; and (iii) a HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and

[0213] (b) a LCVR comprising (i) a molecule having the amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13), wherein X5 is Q, X6 is S, X7 is L, X8 is Y, X9 is selected from S or P, X 10 It is N, X 11 It is N, X 12 It's Q, X 13 is K; and X 14 is Y; (ii) has the amino acid sequence WX 15 SX 16 RX 17 X 18 LCVR CDR2 sequence, where X 15 It's A, X 16 It's T, X 17 is G, and X 18 selected from S or E; and (iii) having the amino acid sequence QQYYX 19 X 20 The LCVR CDR3 sequence of PFT (SEQ ID NO: 30), wherein X 19 It is S, and X 20 Select from Y or F,

[0214] The antibody or antigen-binding fragment thereof specifically binds to Gal9.

[0215] Embodiment 3. The isolated monoclonal antibody or antigen-binding fragment thereof according to embodiment 1 or 2, wherein the isolated monoclonal antibody or antigen-binding fragment thereof comprises:

[0216] (A) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:3, a HCVR CDR2 sequence of SEQ ID NO:10, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVRCDR1 sequence of SEQ ID NO:15, a LCVR CDR2 sequence of SEQ ID NO:23, and a LCVR CDR3 sequence of SEQ ID NO:32; or

[0217] (B) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:4, a HCVR CDR2 sequence of SEQ ID NO:11, and a HCVR CDR3 sequence of SEQ ID NO:12; and a light chain variable region (LCVR) comprising a LCVR CDR1 sequence of SEQ ID NO:14, a LCVR CDR2 sequence of SEQ ID NO:24, and a LCVR CDR3 sequence of SEQ ID NO:31,

[0218] The antibody or antigen-binding fragment thereof specifically binds to Gal9.

[0219] Embodiment 4. The isolated monoclonal antibody or antigen-binding fragment thereof according to embodiment 1, wherein the isolated monoclonal antibody or antigen-binding fragment thereof comprises:

[0220] (a) a HCVR comprising (i) a HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), wherein X1 is selected from T, P or A; X2 is selected from T, S or G; and X3 is selected from S or D; (ii) a HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), wherein X4 is E; and (iii) a HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and

[0221] (b) a LCVR comprising: (i) a LCVR having an amino acid sequence of KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13) LCVR CDR1 sequence, wherein X5 is selected from Q or R; X6 is selected from S or N; X7 is selected from L, V or I; X8 is selected from Y or W; X9 is S, X 10 Selected from S, P or A; X 11Selected from N or H; X 12 is N or Y; X 13 is K or R; and X 14 is Y or H; (ii) has the amino acid sequence WX 15 SX 16 RX 17 X 18 LCVR CDR2 sequence, where X 15 Selected from A or G; X 16 Selected from T, N, M or A; X 17 Selected from G or E; and X 18 is selected from S, Y, P or T; and (iii) has an amino acid sequence of QQYYX 19 X 20 The LCVR CDR3 sequence of PFT (SEQ ID NO: 30), wherein X 19 is F, and X 20 It is Y.

[0222] The antibody or antigen-binding fragment thereof specifically binds to Gal9.

[0223] Embodiment 5. The isolated monoclonal antibody or antigen-binding fragment thereof according to embodiment 1 or 4, wherein the isolated monoclonal antibody or antigen-binding fragment thereof comprises:

[0224] (C) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:5, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a light chain variable region (LCVR) comprising a LCVR CDR1 sequence of SEQ ID NO:16, a LCVR CDR2 sequence of SEQ ID NO:25, and a LCVR CDR3 sequence of SEQ ID NO:33; or

[0225] (D) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:6, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:17, a LCVR CDR2 sequence of SEQ ID NO:26, and a LCVR CDR3 sequence of SEQ ID NO:33; or

[0226] (E) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:7, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:18, a LCVR CDR2 sequence of SEQ ID NO:27, and a LCVR CDR3 sequence of SEQ ID NO:33; or

[0227] (F) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:7, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:19, a LCVR CDR2 sequence of SEQ ID NO:28, and a LCVR CDR3 sequence of SEQ ID NO:33; or

[0228] (G) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:7, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:20, a LCVR CDR2 sequence of SEQ ID NO:29, and a LCVR CDR3 sequence of SEQ ID NO:33,

[0229] The antibody or antigen-binding fragment thereof specifically binds to Gal9.

[0230] Embodiment 6. The isolated monoclonal antibody or antigen-binding fragment thereof according to embodiment 1, wherein the isolated monoclonal antibody or antigen-binding fragment thereof comprises:

[0231] (a) a HCVR comprising (i) a HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), wherein X1 is selected from E or P; X2 is selected from T or S; and X3 is selected from E or S; (ii) a HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), wherein X4 is selected from W or E; and (iii) a HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and

[0232] (b) a light chain variable region (LCVR), the light chain variable region comprising: (i) a polypeptide having an amino acid sequence of KSSX5X6X7LX8X9X10X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13) LCVR CDR1 sequence, wherein X5 is Q, X6 is selected from S or N, X7 is selected from L or V, X8 is selected from Y or W, X9 is selected from P or S, X 10 Select from N or S, X 11 It is N, X 12 Select from Q or N, X 13 is K; and X 14 is Y; (ii) has the amino acid sequence WX 15 SX 16 RX 17 X 18 LCVR CDR2 sequence, where X 15 Selected from A or G; X 16 Select from T or N; X 17 Select from G or E, and X 18 is S; and (iii) has the amino acid sequence QQYYX 19 X 20 The LCVR CDR3 sequence of PFT (SEQ ID NO: 30), wherein X 19 Selected from S or F; and X 20 Select from Y or F,

[0233] The antibody or antigen-binding fragment thereof specifically binds to Gal9.

[0234] Embodiment 7. The isolated monoclonal antibody or antigen-binding fragment thereof according to embodiment 1 or 6, comprising:

[0235] (A) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:3, a HCVR CDR2 sequence of SEQ ID NO:10, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVRCDR1 sequence of SEQ ID NO:15, a LCVR CDR2 sequence of SEQ ID NO:23, and a LCVR CDR3 sequence of SEQ ID NO:32; or

[0236] (C) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:5, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a light chain variable region (LCVR) comprising a LCVR CDR1 sequence of SEQ ID NO:16, a LCVR CDR2 sequence of SEQ ID NO:25, and a LCVR CDR3 sequence of SEQ ID NO:33,

[0237] The antibody or antigen-binding fragment thereof specifically binds to Gal9.

[0238] Embodiment 8. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising: a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO: 3, a HCVRCDR2 sequence of SEQ ID NO: 10, and a HCVR CDR3 sequence of SEQ ID NO: 12; and a LCVR comprising a LCVRCDR1 sequence of SEQ ID NO: 15, a LCVR CDR2 sequence of SEQ ID NO: 23, and a LCVR CDR3 sequence of SEQ ID NO: 32, wherein the antibody or antigen-binding fragment thereof specifically binds Gal9.

[0239] Embodiment 9. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 8, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:68, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0240] Embodiment 10. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 8, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:69, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0241] Embodiment 11. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 8, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:70, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0242] Embodiment 12. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 8, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:76, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0243] Embodiment 13. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 8, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:77, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0244] Embodiment 14. An isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of embodiments 8-13, wherein: the LCVR sequence of the antibody or antigen-binding fragment thereof further comprises an LFR1 sequence of SEQ ID NO:72, an LFR2 sequence of SEQ ID NO:73, an LFR3 sequence of SEQ ID NO:74 and / or an LFR4 sequence of SEQ ID NO:75.

[0245] Embodiment 15. The isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 8, wherein: the HCVR sequence is SEQ ID NO:39 and / or the LCVR sequence is SEQ ID NO:41.

[0246] Embodiment 16. The isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 8 or 5, wherein: the HCVR sequence is SEQ ID NO:39, and the LCVR sequence is SEQ ID NO:41.

[0247] Embodiment 17. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising: a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO: 4, a HCVR CDR2 sequence of SEQ ID NO: 11, and a HCVR CDR3 sequence of SEQ ID NO: 12; and a light chain variable region (LCVR) comprising a LCVR CDR1 sequence of SEQ ID NO: 14, a LCVR CDR2 sequence of SEQ ID NO: 24, and a LCVR CDR3 sequence of SEQ ID NO: 31, wherein the antibody or antigen-binding fragment thereof specifically binds to galectin-9 (Gal9).

[0248] Embodiment 18. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 17, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:68, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0249] Embodiment 19. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 17, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:69, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0250] Embodiment 20. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 17, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:70, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0251] Embodiment 21. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 17, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:76, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0252] Embodiment 22. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 17, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:77, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0253] Embodiment 23. An isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of embodiments 17-22, wherein: the LCVR sequence of the antibody or antigen-binding fragment thereof further comprises an LFR1 sequence of SEQ ID NO:72, an LFR2 sequence of SEQ ID NO:73, an LFR3 sequence of SEQ ID NO:74 and / or an LFR4 sequence of SEQ ID NO:75.

[0254] Embodiment 24. The isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 17, wherein the HCVR sequence is SEQ ID NO:43 and / or the LCVR sequence is SEQ ID NO:45.

[0255] Embodiment 25. The isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 17 or 24, wherein the HCVR sequence is SEQ ID NO:43, and the LCVR sequence is SEQ ID NO:45.

[0256] Embodiment 26. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising: a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:5, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a light chain variable region (LCVR) comprising a LCVR CDR1 sequence of SEQ ID NO:16, a LCVR CDR2 sequence of SEQ ID NO:25, and a LCVR CDR3 sequence of SEQ ID NO:33, wherein the antibody or antigen-binding fragment thereof specifically binds Gal9.

[0257] Embodiment 27. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 26, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:68, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0258] Embodiment 28. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 26, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:69, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0259] Embodiment 29. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 26, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:70, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0260] Embodiment 30. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 26, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:76, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0261] Embodiment 31. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 26, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:77, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0262] Embodiment 32. An isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of embodiments 26-31, wherein: the LCVR sequence of the antibody or antigen-binding fragment thereof further comprises an LFR1 sequence of SEQ ID NO:72, an LFR2 sequence of SEQ ID NO:73, an LFR3 sequence of SEQ ID NO:74 and / or an LFR4 sequence of SEQ ID NO:75.

[0263] Embodiment 33. The isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 26, wherein the HCVR sequence is SEQ ID NO:47 and / or the LCVR sequence is SEQ ID NO:49.

[0264] Embodiment 34. The isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 26 or 33, wherein the HCVR sequence is SEQ ID NO:47 and the LCVR sequence is SEQ ID NO:49.

[0265] Embodiment 35. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising: a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:7, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:18, a LCVR CDR2 sequence of SEQ ID NO:27, and a LCVR CDR3 sequence of SEQ ID NO:33, wherein the antibody or antigen-binding fragment thereof specifically binds to galectin-9 (Gal9).

[0266] Embodiment 36. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 35, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:68, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0267] Embodiment 37. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 35, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:69, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0268] Embodiment 38. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 35, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:70, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0269] Embodiment 39. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 35, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:76, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0270] Embodiment 40. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 35, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:77, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0271] Embodiment 41. An isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of embodiments 35-40, wherein: the LCVR sequence of the antibody or antigen-binding fragment thereof further comprises an LFR1 sequence of SEQ ID NO:72, an LFR2 sequence of SEQ ID NO:73, an LFR3 sequence of SEQ ID NO:74 and / or an LFR4 sequence of SEQ ID NO:75.

[0272] Embodiment 42. The isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 35, wherein the HCVR sequence is SEQ ID NO:51 and / or the LCVR sequence is SEQ ID NO:53.

[0273] Embodiment 43. The isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 35 or 42, wherein the HCVR sequence is SEQ ID NO:51, and the LCVR sequence is SEQ ID NO:53.

[0274] Embodiment 44. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising: a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:7, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:18, a LCVR CDR2 sequence of SEQ ID NO:27, and a LCVR CDR3 sequence of SEQ ID NO:33, wherein the antibody or antigen-binding fragment thereof specifically binds to galectin-9 (Gal9).

[0275] Embodiment 45. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 44, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:68, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0276] Embodiment 46. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 44, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:69, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0277] Embodiment 47. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 44, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:70, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0278] Embodiment 48. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 44, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:76, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0279] Embodiment 49. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 44, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:77, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0280] Embodiment 50. An isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of embodiments 44-49, wherein: the LCVR sequence of the antibody or antigen-binding fragment thereof further comprises an LFR1 sequence of SEQ ID NO:72, an LFR2 sequence of SEQ ID NO:73, an LFR3 sequence of SEQ ID NO:74 and / or an LFR4 sequence of SEQ ID NO:75.

[0281] Embodiment 51. The isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 44, wherein the HCVR sequence is SEQ ID NO:55 and / or the LCVR sequence is SEQ ID NO:57.

[0282] Embodiment 52. The isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 44 or 51, wherein the HCVR sequence is SEQ ID NO:55 and the LCVR sequence is SEQ ID NO:57.

[0283] Embodiment 53. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising: a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:7, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:19, a LCVR CDR2 sequence of SEQ ID NO:28, and a LCVR CDR3 sequence of SEQ ID NO:33, wherein the antibody or antigen-binding fragment thereof specifically binds to galectin-9 (Gal9).

[0284] Embodiment 54. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 53, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:68, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0285] Embodiment 55. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 53, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:69, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0286] Embodiment 56. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 53, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:70, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0287] Embodiment 57. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 53, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:76, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0288] Embodiment 58. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 53, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:77, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0289] Embodiment 59. An isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of embodiments 53-58, wherein: the LCVR sequence of the antibody or antigen-binding fragment thereof further comprises an LFR1 sequence of SEQ ID NO:72, an LFR2 sequence of SEQ ID NO:73, an LFR3 sequence of SEQ ID NO:74 and / or an LFR4 sequence of SEQ ID NO:75.

[0290] Embodiment 60. The isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 53, wherein the HCVR sequence is SEQ ID NO:59 and / or the LCVR sequence is SEQ ID NO:61.

[0291] Embodiment 61. The isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 53 or 60, wherein the HCVR sequence is SEQ ID NO:59 and the LCVR sequence is SEQ ID NO:61.

[0292] Embodiment 62. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising: a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:7, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:20, a LCVR CDR2 sequence of SEQ ID NO:29, and a LCVR CDR3 sequence of SEQ ID NO:33, wherein the antibody or antigen-binding fragment thereof specifically binds to galectin-9 (Gal9).

[0293] Embodiment 63. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 62, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:68, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0294] Embodiment 64. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 62, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:69, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0295] Embodiment 65. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 62, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:70, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0296] Embodiment 66. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 62, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:76, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0297] Embodiment 67. An isolated monoclonal antibody or antigen-binding fragment thereof as described in embodiment 62, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO:77, and optionally further comprises the HFR1 sequence of SEQ ID NO:66, the HFR2 sequence of SEQ ID NO:67 and / or the HFR4 sequence of SEQ ID NO:71.

[0298] Embodiment 68. An isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of embodiments 62-67, wherein: the LCVR sequence of the antibody or antigen-binding fragment thereof further comprises an LFR1 sequence of SEQ ID NO:72, an LFR2 sequence of SEQ ID NO:73, an LFR3 sequence of SEQ ID NO:74 and / or an LFR4 sequence of SEQ ID NO:75.

[0299] Embodiment 69. The isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 62, wherein the HCVR sequence is SEQ ID NO:63 and / or the LCVR sequence is SEQ ID NO:65.

[0300] Embodiment 70. The isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 62 or 69, wherein the HCVR sequence is SEQ ID NO:63 and the LCVR sequence is SEQ ID NO:65.

[0301] Embodiment 71. An isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of embodiments 1-71, wherein the antibody is a humanized antibody.

[0302] Embodiment 72. An isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of embodiments 1-71, wherein the antigen-binding fragment thereof is Fab, Fab', F(ab')2, F d , single-chain Fv or scFv, disulfide-linked F v , V-NAR domain, IgNar, intracellular antibody, IgGACH2, mini antibody, F(ab')3, tetra antibody, tria antibody, diabody, domain antibody (dAb), DVD-Ig, Fcab, mAb2, (scFv)2, tandem scFv, TandAb, nanobody or scFv-Fc.

[0303] Embodiment 73. An isolated monoclonal antibody comprising the heavy chain sequence of SEQ ID NO:38 and the light chain sequence of SEQ ID NO:40.

[0304] Embodiment 74. An isolated monoclonal antibody comprising the heavy chain sequence of SEQ ID NO:42 and the light chain sequence of SEQ ID NO:44.

[0305] Embodiment 75. An isolated monoclonal antibody comprising the heavy chain sequence of SEQ ID NO:46 and the light chain sequence of SEQ ID NO:48.

[0306] Embodiment 76. An isolated monoclonal antibody comprising the heavy chain sequence of SEQ ID NO:50 and the light chain sequence of SEQ ID NO:52.

[0307] Embodiment 77. An isolated monoclonal antibody comprising the heavy chain sequence of SEQ ID NO:54 and the light chain sequence of SEQ ID NO:56.

[0308] Embodiment 78. An isolated monoclonal antibody comprising the heavy chain sequence of SEQ ID NO:58 and the light chain sequence of SEQ ID NO:60.

[0309] Embodiment 79. An isolated monoclonal antibody comprising the heavy chain sequence of SEQ ID NO:62 and the light chain sequence of SEQ ID NO:64.

[0310] Embodiment 80. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of embodiments 1-79, wherein the monoclonal antibody or antigen-binding fragment thereof has a K of less than about 5 nM, 2 nM, 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0,2 nM, 0,1 nM, 0.05 nM or 0.01 nM. d Binds human Gal9.

[0311] Embodiment 81. An isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of embodiments 1-80, which binds to Gal9 and inhibits the binding of Gal9 to a Gal9 receptor (e.g., TIM3 or CD44).

[0312] Embodiment 82. An isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of embodiments 1-80, which binds to Gal9 and does not inhibit the binding of Gal9 to a Gal9 receptor (e.g., TIM3 or CD44).

[0313] Embodiment 83. An isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of embodiments 1-82, which reverses T cell (such as CD4 + Gal9-induced Th1 apoptosis of T cells.

[0314] Embodiment 84. An isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of embodiments 1-83, which inhibits Gal9-induced Treg expansion.

[0315] Embodiment 85. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of embodiments 1-84, thereby treating the cancer in the subject.

[0316] Embodiment 86. A method for treating urothelial cancer, gastric cancer, gastroesophageal cancer, esophageal cancer, cervical cancer and / or head and neck squamous cell carcinoma in a subject in need thereof, the method comprising administering to the subject an effective amount of an isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of embodiments 1-84, thereby treating the subject's cancer.

[0317] Embodiment 87. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of embodiments 1-84 and an anti-PD-1 antibody, thereby treating the cancer in the subject.

[0318] Embodiment 88. A method for treating urothelial cancer, gastric cancer, gastroesophageal cancer, esophageal cancer, cervical cancer and / or head and neck squamous cell carcinoma in a subject in need thereof, the method comprising administering to the subject an effective amount of an isolated monoclonal antibody or antigen-binding fragment thereof, an anti-PD-1 antibody, and an anti-GITR antibody as described in any one of embodiments 1-84, thereby treating the subject's cancer.

[0319] Embodiment 89. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of embodiments 1-84 and an anti-PD-1 antibody, thereby treating the cancer in the subject.

[0320] Embodiment 90. A method for treating urothelial cancer, gastric cancer, gastroesophageal cancer, esophageal cancer, cervical cancer and / or head and neck squamous cell carcinoma in a subject in need thereof, the method comprising administering to the subject an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof, anti-PD-1 antibody and anti-GITR antibody as described in any one of embodiments 1-69, thereby treating the subject's cancer.

[0321] Embodiment 91. The method of embodiment 85, 87 or 89, wherein the cancer is a blood cancer or a solid tumor.

[0322] Embodiment 92. A method as described in embodiment 91, wherein the blood cancer is selected from the group consisting of: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia, chronic myeloid leukemia and hairy cell leukemia, AIDS-related lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, Hodgkin lymphoma, mycosis fungoides, non-Hodgkin lymphoma, primary central nervous system lymphoma, Sezary syndrome, cutaneous T-cell lymphoma, Waldenstrom's macroglobulinemia, diffuse large B-cell lymphoma (DLBCL) and multiple myeloma.

[0323] Embodiment 93. A method as described in embodiment 91, wherein the solid tumor is breast cancer, head and neck cancer, lung cancer, melanoma, uveal melanoma, colorectal cancer, renal cancer, urothelial carcinoma, ovarian cancer, endometrial cancer, liver cancer, pancreatic cancer, bile duct cancer, gastric cancer, gastroesophageal cancer, esophageal cancer, cervical cancer, head and neck squamous cell carcinoma or prostate cancer.

[0324] Embodiment 94. The method of any one of embodiments 85, 87, 89 or 91-93, further comprising administering to the patient a chemotherapeutic agent, an anti-angiogenic agent, a growth inhibitory agent, an immuno-oncology agent, a checkpoint inhibitor and / or an anti-tumor composition.

[0325] Embodiment 95. The method of embodiment 94, wherein the checkpoint inhibitor is anti-PD-1.

[0326] Embodiment 96. The method of embodiment 94, wherein the immuno-oncology agent is an anti-glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) antibody.

[0327] Embodiment 97. A polynucleotide encoding a heavy chain or light chain or an antigen binding portion thereof as described in any one of embodiments 1-84.

[0328] Embodiment 98. A vector comprising the polynucleotide of embodiment 97, wherein the vector is an expression vector selected from the group consisting of a mammalian expression vector, a yeast expression vector, an insect expression vector, and a bacterial expression vector.

[0329] Embodiment 99. A method of rescuing or promoting effector T cell proliferation, enhancing effector T cell activity, and / or identifying and treating cancer in a subject, the method comprising:

[0330] (i) determining the level of Gal9 in a sample from the subject, and

[0331] (ii) administering an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof of any one of embodiments 1-84 to a subject having a level of Gal9 that is higher than a reference level of Gal9,

[0332] Thereby rescuing or promoting effector T cell proliferation, enhancing effector T cell activity and / or identifying and treating the subject.

[0333] Embodiment 100. A method of rescuing or promoting effector T cell proliferation, enhancing effector T cell activity, and / or identifying and treating cancer in a subject, the method comprising:

[0334] (i) determining the level of Gal9 in a sample from the subject, and

[0335] (ii) administering an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof of any one of embodiments 1-84 and an anti-PD-1 antibody to a subject, wherein the subject has a Gal9 level that is higher than a reference level of Gal9,

[0336] Thereby rescuing or promoting effector T cell proliferation, enhancing effector T cell activity and / or identifying and treating the subject.

[0337] Embodiment 101. A method of rescuing or promoting effector T cell proliferation, enhancing effector T cell activity, and / or identifying and treating cancer in a subject, the method comprising:

[0338] (i) determining the level of Gal9 in a sample from the subject, and

[0339] (ii) administering an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of embodiments 1-84 and an anti-PD-1 antibody and an anti-GITR antibody to a subject, wherein the subject has a Gal9 level that is higher than a reference level of Gal9,

[0340] Thereby rescuing or promoting effector T cell proliferation, enhancing effector T cell activity and / or identifying and treating the subject.

[0341] Embodiment 102. The method of any one of embodiments 99-101, wherein the subject is diagnosed with cancer, is at risk of developing cancer, or has a recurrence of cancer.

[0342] Embodiment 103. A method as described in any of embodiments 99-102, wherein the reference level of Gal9 is the level of Gal9 measured in a sample from a healthy or control individual.

[0343] Embodiment 104. The method of any one of embodiments 99-103, wherein determining comprises comparing the level of Gal9 in the sample to the reference level.

[0344] Embodiment 105. The method of any one of embodiments 99-104, wherein the cancer is a blood cancer or a solid tumor.

[0345] Embodiment 106. The method of embodiment 105, wherein the blood cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia, chronic myeloid leukemia and hairy cell leukemia, AIDS-related lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, Hodgkin lymphoma, mycosis fungoides, non-Hodgkin lymphoma, primary central nervous system lymphoma, Sezary syndrome, cutaneous T-cell lymphoma, Waldenstrom's macroglobulinemia, diffuse large B-cell lymphoma (DLBCL) and multiple myeloma.

[0346] Embodiment 107. A method as described in embodiment 105, wherein the solid tumor is breast cancer, head and neck cancer, lung cancer, melanoma, uveal melanoma, colorectal cancer, renal cancer, urothelial carcinoma, ovarian cancer, endometrial cancer, liver cancer, pancreatic cancer, bile duct cancer, gastric cancer, gastroesophageal cancer, esophageal cancer, cervical cancer, head and neck squamous cell carcinoma or prostate cancer.

[0347] Embodiment 108. The method of any one of embodiments 99-107, wherein the sample is a blood sample, a plasma sample, a serum sample, a tumor biopsy sample, or a bone marrow (BM)-derived mononuclear cell (MNC) sample.

[0348] Embodiment 109. The method of any one of embodiments 99 or 102-106, further comprising administering to the patient a chemotherapeutic agent, an anti-angiogenic agent, a growth inhibitory agent, an immuno-oncology agent, a checkpoint inhibitor and / or an anti-tumor composition.

[0349] Embodiment 110. The method of embodiment 109, wherein the checkpoint inhibitor is anti-PD-1.

[0350] Embodiment 111. The method of claim 110, wherein the immuno-oncology agent is an anti-glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) antibody.

[0351] Example

[0352] Example 1

[0353] Affinity maturation of humanized anti-Gal9 antibody

[0354] To develop anti-Gal9 antibodies with improved binding affinity to human Gal9 compared to the original Ab A anti-Gal9 antibody, random mutations were performed in the following three CDRs: H1-H2-L2 or L1-L3-H3, and a variant library was generated.

[0355] The following two antibodies were identified as having the best binding affinity compared to Ab A anti-Gal9 antibody: anti-gal9 antibodies Ab 001 and Ab 002. The alignment of the sequences of the heavy and light chains are shown in Figure 1A and Figure 1B , where the CDRs are squared in the sequence.

[0356] The affinity binding of the antibodies was measured using surface plasmon resonance and solution equilibrium assays. As shown in Table 2, it was found that the anti-Gal9 antibodies Ab 001 and Ab 002 had greater binding affinity than the Ab A antibody, as determined by the K D Defined by the decrease in value.

[0357] Table 2: Binding data of affinity matured antibodies

[0358]

[0359] Separately, in order to develop an anti-Gal9 antibody with improved binding affinity to human Gal9 compared to the original Ab A anti-Gal9 antibody, a yeast display method was used.

[0360] The following five antibodies were identified as having the best binding affinity compared to Ab A anti-Gal9 antibody: anti-gal9 antibodies Ab 003, Ab 004, Ab 005, Ab 006 and Ab 007. The alignment of the sequences of the heavy and light chains are shown in Figure 1C and Figure 1D , where the CDRs are squared in the sequence.

[0361] The affinity binding of the antibodies was measured using surface plasmon resonance and solution equilibrium assays. As shown in Table 3, it was found that the anti-Gal9 antibodies Ab 003-007 had greater binding affinity than antibody Ab A, as determined by the K DSurprisingly, at least antibodies Ab 003-005 and Ab 007 also showed significantly improved affinity for mouse Gal9 relative to antibody Ab A.

[0362] Table 3: Binding data of affinity matured antibodies

[0363]

[0364] Example 2

[0365] Effect of anti-Gal9 antibody on Gal9-induced apoptosis

[0366] To characterize the affinity matured Ab 001 anti-Gal9 antibody compared to the parental Ab A anti-Gal9 antibody, a comparison of the effects of the antibodies on Gal9-induced apoptosis was performed.

[0367] like Figure 2B As shown in the figure, it was found that the affinity matured anti-Gal9 antibody was more potent in reducing CD4+ T cell apoptosis than the Ab A antibody (also shown in Figure 2A The calculated IC50 values ​​were 18.7 nM for Ab A and 1.9 nM for Ab 001.

[0368] T cell apoptosis assay will be used to evaluate the efficacy of Ab 002 antibody in inhibiting Gal9-induced apoptosis.

[0369] Additional characterization of the antibodies to be performed will include evaluation of cross-reactivity of the antibodies with other Gal9 family members, assessment of biochemical and biophysical properties (including, for example, aggregation data), deamination, oxidation assessment, proteolysis (particularly before and after heat stress).

[0370] Annexin V + and propidium iodide + (PI) Flow cytometric analysis of double positive cells to evaluate the inhibition of Gal9-induced CD4 + and / or CD8 + T cell apoptosis ability.

[0371] CD4 + or CD8 + T cells were activated with mitogenic stimuli anti-CD3 and anti-CD28 as well as Gal9. Activated T cells were co-cultured with anti-Gal9 antibody Ab 001 or isotype control. Figure 2C and Figure 2D As shown, inclusion of Ab 001 resulted in a decrease in CD4 + and CD8 + T cell apoptosis was significantly reduced.

[0372] Example 3

[0373] Evaluation of AML apoptosis ex vivo

[0374] The effects of anti-Gal9 affinity matured antibodies (Ab 001 and Ab 002) on apoptosis of leukemic stem cells (LSC, CD34+, CD38-) will be evaluated and compared to the effects induced by a non-affinity matured anti-Gal9 antibody (Ab A).

[0375] AML bone marrow samples will be collected and frozen until used in experiments. LSC cells will be sorted from the samples based on their CD34 and CD38 expression.

[0376] The expression level of Tim3 / Gal9 on the cell surface and the level of secreted / released gal9 will be assessed. The cells will be suspended and cultured for 96 h in the presence or absence of antibodies, and apoptosis will be assessed by flow cytometry. Internal controls will include other existing anti-Gal9 antibodies, positive controls (aGal9), and negative controls.

[0377] Example 4

[0378] Evaluation of the in vivo effects of affinity-matured anti-Gal9 antibodies on AML patient samples

[0379] To evaluate the efficacy of affinity-matured anti-Gal9 antibodies in vivo, NODscid gamma (NSG) immunocompromised mice will be transplanted using an in vivo systemic model of AML. FLT3, IDH1 / 2, and TP53 genes will be sequenced to identify mutants; cytogenetic, clinical, and immunophenotyping assessments will be performed.

[0380] The system transplant dynamics will be measured.

[0381] The engraftment period will last approximately 6-12 weeks and the AML engraftment will be assessed by flow cytometry to detect CD45 / CD33 cells. AML agents will then be tested. The endpoint of the experiment will be survival, at which time flow cytometric analysis of splenocytes, whole blood, bone marrow CD45 / CD33 will be performed, where animals are treated with single anti-Gal9 antibodies or a combination of anti-Gal9 antibodies and chemotherapy.

[0382] As an alternative to humanized AML models, an autologous AML platform will be used to enable testing of immuno-oncology therapeutics (as single agents or in combination) in an autologous co-culture system, while Champions' well-characterized AML patient model library can be utilized. Primary AML cells from patients will be co-cultured with matched T cells from the patient in the presence or absence of the antibody of interest (alone or in combination with another therapeutic agent). Cells will be grown in enriched growth medium, and multiple parameters will be evaluated, including phenotyping by flow cytometry, T cell activation, cytokine production by Luminex, and AML frequency.

[0383] As a proof-of-concept experiment, a solid tumor model using the MC38 mouse syngeneic model (colorectal cancer) will be used for further in vivo analysis. Monotherapy studies using anti-Gal9 antibodies or anti-PD-1 antibodies and dose-response experiments will be performed in MC38 animals. Combination studies of anti-Gal9 antibodies and anti-PD-1 will be performed to understand the in vivo mechanism of action.

[0384] Example 5

[0385] Evaluation of Ab 001's inhibitory effect on Gal9-induced TIM-3 dimerization

[0386] TIM-3 dimerization was assessed by Eurofins DiscoverX assay. The assay relies on enzyme fragment complementation, in which the enzyme β-galactosidase is divided into two fragments fused to the cytoplasmic tail of the receptor TIM-3, i.e., the enzyme acceptor (EA) and the enzyme donor (ED). When the ligand Gal9 binds to its receptor TIM-3, the receptor homodimerizes, bringing EA and ED together, which metabolize the assay substrate and produce quantifiable visible photons.

[0387] like Figure 3 As shown, the inclusion of anti-Gal9 antibodies Ab001 or Ab003 inhibited the ability of soluble Gal9 to form TIM-3 homodimers, as evidenced by quenching of emitted photons, compared to an isotype control.

[0388] Example 6

[0389] Efficacy of anti-Gal9 antibodies as combination therapy in a proof-of-concept mouse model

[0390] Anti-Gal9 antibodies were tested alone or in combination with checkpoint inhibitor antibodies to evaluate the efficacy of inhibiting tumor growth in the MC38 mouse syngeneic colon cancer model. The antibodies tested included anti-GITR, anti-PD-1, and anti-Gal9. Two anti-Gal9 antibodies, a commercially available anti-Gal9 antibody (anti-Gal9 Ab RG9-1) and one of the antibodies of the present invention (Ab 003), were used in separate experiments.

[0391] Although all monotherapies appeared to provide minimal tumor growth inhibition in this model compared to isotype controls, combination therapy with anti-Gal9 antibody and anti-GITR or anti-PD-1 resulted in modest tumor growth inhibition (49%-51% tumor growth inhibition for Ab 003 in double therapy). Triple therapy with anti-Gal9, anti-GITR, and anti-PD1 provided even greater tumor growth inhibition (67% tumor growth inhibition for Ab 003 in triple therapy).

[0392] Example 7

[0393] Clinical Overview of Phase 1a / 1b in Relapsed / Refractory Advanced Selected Solid Tumors

[0394] The Phase 1a / 1b clinical trial will evaluate the maximum tolerated dose (MTD) of the anti-Gal9 antibody of the present invention in patients with relapsed / refractory advanced solid tumors according to the BOIN design method.

[0395] Indications were selected based on Gal9 expression levels and were as follows: colorectal cancer, gastric cancer, gastroesophageal junction adenocarcinoma, esophageal cancer, urothelial carcinoma, cervical cancer, and head and neck squamous cell carcinoma. The dose escalation phase was a standard 3+3 Bayesian optimal interval design. After the recommended dose was determined, the dose expansion phase consisted of administering two dose levels of anti-Gal9, with or without pembrolizumab, until the minimum safe biologically effective dose was confirmed. A Phase II trial was planned after completion of the dose expansion phase.

[0396] Although the invention has been described with reference to the above embodiments, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the present invention is limited only by the following claims.

[0397] sequence:

[0398] add Underline :Signal sequence

[0399] CDR

[0400] >VH-CDR1

[0401] GYX1FX2X3YTIH (SEQ ID NO: 1)

[0402] X1 is T, E, P or A

[0403] X2 is T, G or S

[0404] X3 is E, S or D

[0405] >VH-CDR2

[0406] WFYPGSGSTX4YAQKFQG(SEQ ID NO:8)

[0407] X4 is E, W or V

[0408] >VH-CDR3

[0409] HGGYDGFDY (SEQ ID NO: 12)

[0410] >VL-CDR1

[0411] KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13)

[0412] X5 is Q or R

[0413] X6 is S or N

[0414] X7 is L, V or I

[0415] X8 is Y or W

[0416] X9 is S or P

[0417] X 10 is N, S, P or A

[0418] X 11 Is N or H

[0419] X 12 Is Q, N or Y

[0420] X 13 Is K or R

[0421] X 14 Is Y or H

[0422] >VL-CDR2

[0423] WX 15 SX 16 RX 17 X 18

[0424] X 15 A or G

[0425] X 16 is T, N, M or A

[0426] X 17 Is G or E

[0427] X 18is S, E, Y, P or T

[0428] >VL-CDR3

[0429] QQYYX 19 X 20 PFT(SEQ ID NO:30)

[0430] X 19 is S or F

[0431] X 20 is Y or F

[0432] >Ab A HC(SEQ ID NO:34)

[0433]

[0434] >Ab A VH(SEQ ID NO:35)

[0435]

[0436] >Ab A VH-CDR1(SEQ ID NO:2)

[0437] GYTFTEYTIH

[0438] >Ab A VH-CDR2(SEQ ID NO:9)

[0439] WFYPGSGSTEYAQKFQG

[0440] >Ab A VH-CDR3(SEQ ID NO:12)

[0441] HGGYDGFDY

[0442] >Ab A HFR1(SEQ ID NO:66)

[0443] QVQLVQSGAEVKKPGASVKVSCKAS

[0444] >Ab A HFR2(SEQ ID NO:67)

[0445] WVRQAPGQGLEWMG

[0446] >Ab A HFR3(SEQ ID NO:68)

[0447] RVTMTADTSISTAYMELSRLRSDDTAVYFCER>Ab A HFR4(SEQ ID NO:71)

[0448] <h2 style=";text-align:left;direction:ltr">WGQGTTVTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0449] <h2 style=";text-align:left;direction:ltr"> >Ab A LC(SEQ ID NO:36)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0450] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0451] <h2 style=";text-align:left;direction:ltr"> >Ab A VL(SEQ ID NO:37)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0452] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0453] <h2 style=";text-align:left;direction:ltr"> >Ab A VL-CDR1(SEQ ID NO:14)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0454] <h2 style=";text-align:left;direction:ltr"> KSSQSLLYSNNQKNYLA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0455] <h2 style=";text-align:left;direction:ltr"> >Ab A VL-CDR2(SEQ ID NO:22)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0456] <h2 style=";text-align:left;direction:ltr"> WASTRGS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0457] <h2 style=";text-align:left;direction:ltr"> >Ab A VL-CDR3(SEQ ID NO:31)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0458] <h2 style=";text-align:left;direction:ltr"> QQYYSYPFT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0459] <h2 style=";text-align:left;direction:ltr"> >Ab A LFR1(SEQ ID NO:72)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0460] <h2 style=";text-align:left;direction:ltr"> DIVMTQSPDSLAVSLGERATINC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0461] <h2 style=";text-align:left;direction:ltr"> >Ab A LFR2(SEQ ID NO:73)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0462] <h2 style=";text-align:left;direction:ltr"> WYQQKPGQPPKLLIY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0463] <h2 style=";text-align:left;direction:ltr"> >Ab A LFR3(SEQ ID NO:74)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0464] <h2 style=";text-align:left;direction:ltr"> GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC>Ab A LFR4(SEQ ID NO:75)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0465] <h2 style=";text-align:left;direction:ltr"> FGGGTKVEIK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0466] <h2 style=";text-align:left;direction:ltr"> >Ab 001 HC(SEQ ID NO:38)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0467] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0468] <h2 style=";text-align:left;direction:ltr"> >Ab 001 VH(SEQ ID NO:39)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0469] <h2 style=";text-align:left;direction:ltr">

[0470] >Ab 001 VH-CDR1(SEQ ID NO:3)

[0471] GYEFTEYTIH

[0472] >Ab 001 VH-CDR2(SEQ ID NO:10)

[0473] WFYPGSGSTWYAQKFQG

[0474] >Ab 001 VH-CDR3(SEQ ID NO:12)

[0475] HGGYDGFDY

[0476] >Ab 001 HFR1(SEQ ID NO:66)

[0477] QVQLVQSGAEVKKPGASVKVSCKAS

[0478] >Ab 001 HFR2(SEQ ID NO:67)

[0479] WVRQAPGQGLEWMG

[0480] >Ab 001 HFR3(SEQ ID NO:68)

[0481] RVTMTADTSISTAYMELSRLRSDDTAVYFCER>Ab 001 HFR4(SEQ ID NO:71)

[0482] WGQGTTVTVSS

[0483] >Ab 001 LC(SEQ ID NO:40)

[0484]

[0485] >Ab 001 VL(SEQ ID NO:41)

[0486]

[0487] >Ab 001 VL-CDR1(SEQ ID NO:15)

[0488] KSSQSLLYPNNQKNYLA

[0489] >Ab 001 VL-CDR2(SEQ ID NO:23)

[0490] <h2 style=";text-align:left;direction:ltr">WASTRGS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0491] <h2 style=";text-align:left;direction:ltr"> >Ab 001 VL-CDR3(SEQ ID NO:32)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0492] <h2 style=";text-align:left;direction:ltr"> QQYYSFPFT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0493] <h2 style=";text-align:left;direction:ltr"> >Ab 001 LFR1(SEQ ID NO:72)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0494] <h2 style=";text-align:left;direction:ltr"> DIVMTQSPDSLAVSLGERATINC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0495] <h2 style=";text-align:left;direction:ltr"> >Ab 001 LFR2(SEQ ID NO:73)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0496] <h2 style=";text-align:left;direction:ltr"> WYQQKPGQPPKLLIY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0497] <h2 style=";text-align:left;direction:ltr"> >Ab 001 LFR3(SEQ ID NO:74)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0498] <h2 style=";text-align:left;direction:ltr"> GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC>Ab 001 LFR4(SEQ ID NO:75)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0499] <h2 style=";text-align:left;direction:ltr"> FGGGTKVEIK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0500] <h2 style=";text-align:left;direction:ltr"> >Ab 002 HC(SEQ ID NO:42)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0501] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0502] <h2 style=";text-align:left;direction:ltr"> >Ab 002 VH(SEQ ID NO:43)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0503] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0504] <h2 style=";text-align:left;direction:ltr"> >Ab 002 VH-CDR1(SEQ ID NO:4)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0505] <h2 style=";text-align:left;direction:ltr"> GYTFGEYTIH<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0506] <h2 style=";text-align:left;direction:ltr"> >Ab 002 VH-CDR2(SEQ ID NO:11)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0507] <h2 style=";text-align:left;direction:ltr"> WFYPGSGSTVYAQKFQG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0508] <h2 style=";text-align:left;direction:ltr"> >Ab 002 VH-CDR3(SEQ ID NO:12)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0509] <h2 style=";text-align:left;direction:ltr"> HGGYDGFDY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0510] <h2 style=";text-align:left;direction:ltr"> >Ab 002 HFR1(SEQ ID NO:66)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0511] QVQLVQSGAEVKKPGASVKVSCKAS

[0512] >Ab 002 HFR2(SEQ ID NO:67)

[0513] WVRQAPGQGLEWMG

[0514] >Ab 002 HFR3(SEQ ID NO:68)

[0515] RVTMTADTSISTAYMELSRLRSDDTAVYFCER>Ab 002 HFR4(SEQ ID NO:71)

[0516] WGQGTTVTVSS

[0517] >Ab 002(E07)LC(SEQ ID NO:44)

[0518]

[0519] >Ab 002 VL(SEQ ID NO:45)

[0520]

[0521] >Ab 002 VL-CDR1(SEQ ID NO:14)

[0522] KSSQSLLYSNNQKNYLA

[0523] >Ab 002 VL-CDR2(SEQ ID NO:24)

[0524] WASTRGE

[0525] >Ab 002 VL-CDR3(SEQ ID NO:31)

[0526] QQYYSYPFT

[0527] >Ab 002 LFR1(SEQ ID NO:72)

[0528] DIVMTQSPDSLAVSLGERATINC

[0529] >Ab 002 LFR2(SEQ ID NO:73)

[0530] WYQQKPGQPPKLLIY

[0531] >Ab 002 LFR3(SEQ ID NO:74)

[0532] GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC>Ab 002 LFR4(SEQ ID NO:75)

[0533] FGGGTKVEIK

[0534] >Ab 003 HC(SEQ ID NO:46)

[0535]

[0536] >Ab 003 VH(SEQ ID NO:47)

[0537]

[0538] >Ab 003 VH-CDR1(SEQ ID NO:5)

[0539] GYPFSSYTIH

[0540] >Ab 003 VH-CDR2(SEQ ID NO:9)

[0541] WFYPGSGSTEYAQKFQG

[0542] >Ab 003 VH-CDR3(SEQ ID NO:12)

[0543] HGGYDGFDY

[0544] >Ab 003 HFR1(SEQ ID NO:66)

[0545] QVQLVQSGAEVKKPGASVKVSCKAS

[0546] >Ab 003 HFR2(SEQ ID NO:67)

[0547] WVRQAPGQGLEWMG

[0548] >Ab 003 HFR3(SEQ ID NO:69)

[0549] RVTMTADTSISTAYMELSRLRSDDTAVYFCEV>Ab 003 HFR4(SEQ ID NO:71)

[0550] WGQGTTVTVSS

[0551] >Ab 003 LC(SEQ ID NO:48)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0552] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0553] <h2 style=";text-align:left;direction:ltr"> >Ab 003 VL(SEQ ID NO:49)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0554] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0555] <h2 style=";text-align:left;direction:ltr"> >Ab 003 VL-CDR1(SEQ ID NO:16)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0556] <h2 style=";text-align:left;direction:ltr"> KSSQNVLWSSNNKNYLA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0557] <h2 style=";text-align:left;direction:ltr"> >Ab 003 VL-CDR2(SEQ ID NO:25)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0558] <h2 style=";text-align:left;direction:ltr"> WGSNRES<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0559] <h2 style=";text-align:left;direction:ltr"> >Ab 003 VL-CDR3(SEQ ID NO:33)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0560] <h2 style=";text-align:left;direction:ltr"> QQYYFYPFT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0561] <h2 style=";text-align:left;direction:ltr"> >Ab 003 LFR1(SEQ ID NO:72)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0562] <h2 style=";text-align:left;direction:ltr"> DIVMTQSPDSLAVSLGERATINC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0563] <h2 style=";text-align:left;direction:ltr"> >Ab 003 LFR2(SEQ ID NO:73)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0564] <h2 style=";text-align:left;direction:ltr"> WYQQKPGQPPKLLIY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0565] <h2 style=";text-align:left;direction:ltr"> >Ab 003 LFR3(SEQ ID NO:74)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0566] <h2 style=";text-align:left;direction:ltr"> GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC>Ab 003 LFR4(SEQ ID NO:75)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0567] <h2 style=";text-align:left;direction:ltr"> FGGGTKVEIK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0568] <h2 style=";text-align:left;direction:ltr"> >Ab 004 HC(SEQ ID NO:50)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0569] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0570] <h2 style=";text-align:left;direction:ltr"> ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0571] <h2 style=";text-align:left;direction:ltr"> KSLSLSPGK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0572] <h2 style=";text-align:left;direction:ltr">>Ab 004 VH(SEQ ID NO:51)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0573] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0574] <h2 style=";text-align:left;direction:ltr"> >Ab 004 VH-CDR1(SEQ ID NO:6)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0575] <h2 style=";text-align:left;direction:ltr"> GYAFTDYTIH<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0576] <h2 style=";text-align:left;direction:ltr"> >Ab 004 VH-CDR2(SEQ ID NO:9)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0577] <h2 style=";text-align:left;direction:ltr"> WFYPGSGSTEYAQKFQG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0578] <h2 style=";text-align:left;direction:ltr"> >Ab 004 VH-CDR3(SEQ ID NO:12)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0579] <h2 style=";text-align:left;direction:ltr"> HGGYDGFDY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0580] <h2 style=";text-align:left;direction:ltr"> >Ab 004 HFR1(SEQ ID NO:66)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0581] <h2 style=";text-align:left;direction:ltr"> QVQLVQSGAEVKKPGASVKVSCKAS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0582] <h2 style=";text-align:left;direction:ltr"> >Ab 004 HFR2(SEQ ID NO:67)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0583] <h2 style=";text-align:left;direction:ltr"> WVRQAPGQGLEWMG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0584] <h2 style=";text-align:left;direction:ltr"> >Ab 004 HFR3(SEQ ID NO:76)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0585] <h2 style=";text-align:left;direction:ltr"> RVTMTADTSISTAYMELSRLRSDDTAVYYCEV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0586] <h2 style=";text-align:left;direction:ltr"> >Ab 004 HFR4(SEQ ID NO:71)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0587] <h2 style=";text-align:left;direction:ltr"> WGQGTTVTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0588] <h2 style=";text-align:left;direction:ltr"> >Ab 004 LC(SEQ ID NO:52)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0589] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0590] <h2 style=";text-align:left;direction:ltr"> VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0591] <h2 style=";text-align:left;direction:ltr"> SFNRGEC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0592] <h2 style=";text-align:left;direction:ltr"> >Ab 004 VL(SEQ ID NO:53)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0593] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0594] <h2 style=";text-align:left;direction:ltr"> >Ab 004 VL-CDR1(SEQ ID NO:17)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0595] <h2 style=";text-align:left;direction:ltr"> KSSRNILYSPHNKNYLA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0596] <h2 style=";text-align:left;direction:ltr"> >Ab 004 VL-CDR2(SEQ ID NO:26)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0597] <h2 style=";text-align:left;direction:ltr"> WASMRGS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0598] <h2 style=";text-align:left;direction:ltr"> >Ab 004 VL-CDR3(SEQ ID NO:33)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0599] <h2 style=";text-align:left;direction:ltr"> QQYYFYPFT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0600] <h2 style=";text-align:left;direction:ltr"> >Ab 004 LFR1(SEQ ID NO:72)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0601] <h2 style=";text-align:left;direction:ltr"> DIVMTQSPDSLAVSLGERATINC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0602] <h2 style=";text-align:left;direction:ltr"> >Ab 004 LFR2(SEQ ID NO:73)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0603] <h2 style=";text-align:left;direction:ltr"> WYQQKPGQPPKLLIY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0604] <h2 style=";text-align:left;direction:ltr"> >Ab 004 LFR3(SEQ ID NO:74)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0605] <h2 style=";text-align:left;direction:ltr"> GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0606] <h2 style=";text-align:left;direction:ltr"> >Ab 004 LFR4(SEQ ID NO:75)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0607] <h2 style=";text-align:left;direction:ltr"> FGGGTKVEIK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0608] <h2 style=";text-align:left;direction:ltr"> >Ab 005 HC(SEQ ID NO:54)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0609] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0610] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0611] <h2 style=";text-align:left;direction:ltr"> >Ab 005 VH(SEQ ID NO:55)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0612] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0613] <h2 style=";text-align:left;direction:ltr"> >Ab 005 VH-CDR1(SEQ ID NO:7)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0614] <h2 style=";text-align:left;direction:ltr"> GYTFGDYTIH

[0615] >Ab 005 VH-CDR2(SEQ ID NO:9)

[0616] WFYPGSGSTEYAQKFQG

[0617] >Ab 005 VH-CDR3(SEQ ID NO:12)

[0618] HGGYDGFDY

[0619] >Ab 005 HFR1(SEQ ID NO:66)

[0620] QVQLVQSGAEVKKPGASVKVSCKAS

[0621] >Ab 005 HFR2(SEQ ID NO:67)

[0622] WVRQAPGQGLEWMG

[0623] >Ab 005 HFR3(SEQ ID NO:70)

[0624] RVTMTADTSISTAYMELSRLRSDDTAVYYCEI

[0625] >Ab 005 HFR4(SEQ ID NO:71)

[0626] WGQGTTVTVSS

[0627] >Ab 005 LC(SEQ ID NO:56)

[0628]

[0629] >Ab 005 VL(SEQ ID NO:57)

[0630]

[0631] >Ab 005 VL-CDR1(SEQ ID NO:18)

[0632] KSSQSVLYSANNRNYLA

[0633] >Ab 005 VL-CDR2(SEQ ID NO:27)

[0634] WGSAREY

[0635] >Ab 005 VL-CDR3(SEQ ID NO:33)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0636] <h2 style=";text-align:left;direction:ltr"> QQYYFYPFT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0637] <h2 style=";text-align:left;direction:ltr"> >Ab 005 LFR1(SEQ ID NO:72)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0638] <h2 style=";text-align:left;direction:ltr"> DIVMTQSPDSLAVSLGERATINC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0639] <h2 style=";text-align:left;direction:ltr"> >Ab 005 LFR2(SEQ ID NO:73)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0640] <h2 style=";text-align:left;direction:ltr"> WYQQKPGQPPKLLIY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0641] <h2 style=";text-align:left;direction:ltr"> >Ab 005 LFR3(SEQ ID NO:74)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0642] <h2 style=";text-align:left;direction:ltr"> GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC>Ab 005 LFR4(SEQ ID NO:75)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0643] <h2 style=";text-align:left;direction:ltr"> FGGGTKVEIK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0644] <h2 style=";text-align:left;direction:ltr"> >Ab 006 HC(SEQ ID NO:58)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0645] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0646] <h2 style=";text-align:left;direction:ltr"> >Ab 006 VH(SEQ ID NO:59)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0647] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0648] <h2 style=";text-align:left;direction:ltr"> >Ab 006 VH-CDR1(SEQ ID NO:7)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0649] <h2 style=";text-align:left;direction:ltr"> GYTFGDYTIH<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0650] <h2 style=";text-align:left;direction:ltr"> >Ab 006 VH-CDR2(SEQ ID NO:9)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0651] <h2 style=";text-align:left;direction:ltr"> WFYPGSGSTEYAQKFQG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0652] <h2 style=";text-align:left;direction:ltr"> >Ab 006 VH-CDR3(SEQ ID NO:12)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0653] <h2 style=";text-align:left;direction:ltr"> HGGYDGFDY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0654] <h2 style=";text-align:left;direction:ltr"> >Ab 006 HFR1(SEQ ID NO:66)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0655] <h2 style=";text-align:left;direction:ltr"> QVQLVQSGAEVKKPGASVKVSCKAS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0656] <h2 style=";text-align:left;direction:ltr">>Ab 006 HFR2(SEQ ID NO:67)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0657] <h2 style=";text-align:left;direction:ltr"> WVRQAPGQGLEWMG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0658] <h2 style=";text-align:left;direction:ltr"> >Ab 006 HFR3(SEQ ID NO:77)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0659] <h2 style=";text-align:left;direction:ltr"> RVTMTADTSISTAYMELSRLRSDDTAVYFCEI>Ab 006 HFR4(SEQ ID NO:71)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0660] <h2 style=";text-align:left;direction:ltr"> WGQGTTVTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0661] <h2 style=";text-align:left;direction:ltr"> >Ab 006 LC(SEQ ID NO:60)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0662] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0663] <h2 style=";text-align:left;direction:ltr"> >Ab 006 VL(SEQ ID NO:61)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0664] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0665] <h2 style=";text-align:left;direction:ltr"> >Ab 006 VL-CDR1(SEQ ID NO:19)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0666] <h2 style=";text-align:left;direction:ltr"> KSSQSLLYSSNYKNHLA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0667] <h2 style=";text-align:left;direction:ltr"> >Ab 006 VL-CDR2(SEQ ID NO:28)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0668] <h2 style=";text-align:left;direction:ltr"> WGSTREP<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0669] <h2 style=";text-align:left;direction:ltr"> >Ab 006 VL-CDR3(SEQ ID NO:33)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0670] <h2 style=";text-align:left;direction:ltr"> QQYYFYPFT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0671] <h2 style=";text-align:left;direction:ltr"> >Ab 006 LFR1(SEQ ID NO:72)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0672] <h2 style=";text-align:left;direction:ltr"> DIVMTQSPDSLAVSLGERATINC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0673] <h2 style=";text-align:left;direction:ltr"> >Ab 006 LFR2(SEQ ID NO:73)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0674] <h2 style=";text-align:left;direction:ltr"> WYQQKPGQPPKLLIY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0675] <h2 style=";text-align:left;direction:ltr"> >Ab 006 LFR3(SEQ ID NO:74)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0676] GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC>Ab 006 LFR4(SEQ ID NO:75)

[0677] FGGGTKVEIK

[0678] >Ab 007 HC(SEQ ID NO:62)

[0679]

[0680] >Ab 007 VH(SEQ ID NO:63)

[0681]

[0682] >Ab 007 VH-CDR1(SEQ ID NO:7)

[0683] GYTFGDYTIH

[0684] >Ab 007 VH-CDR2(SEQ ID NO:9)

[0685] WFYPGSGSTEYAQKFQG

[0686] >Ab 007 VH-CDR3(SEQ ID NO:12)HGGYDGFDY

[0687] >Ab 007 HFR1(SEQ ID NO:66)

[0688] QVQLVQSGAEVKKPGASVKVSCKAS

[0689] >Ab 007 HFR2(SEQ ID NO:67)

[0690] WVRQAPGQGLEWMG

[0691] >Ab 007 HFR3(SEQ ID NO:70)

[0692] RVTMTADTSISTAYMELSRLRSDDTAVYYCEI

[0693] >Ab 007 HFR4(SEQ ID NO:71)

[0694] WGQGTTVTVSS

[0695] >Ab 007 LC(SEQ ID NO:64)

[0696] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0697] <h2 style=";text-align:left;direction:ltr"> >Ab 007 VL(SEQ ID NO:65)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0698] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0699] <h2 style=";text-align:left;direction:ltr"> >Ab 007 VL-CDR1(SEQ ID NO:20)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0700] <h2 style=";text-align:left;direction:ltr"> KSSQNVLWSSNNRNYLA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0701] <h2 style=";text-align:left;direction:ltr"> >Ab 007 VL-CDR2(SEQ ID NO:29)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0702] <h2 style=";text-align:left;direction:ltr"> WGSTRET<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0703] <h2 style=";text-align:left;direction:ltr"> >Ab 007 VL-CDR3(SEQ ID NO:33)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0704] <h2 style=";text-align:left;direction:ltr"> QQYYFYPFT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0705] <h2 style=";text-align:left;direction:ltr"> >Ab 007 LFR1(SEQ ID NO:72)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0706] <h2 style=";text-align:left;direction:ltr"> DIVMTQSPDSLAVSLGERATINC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0707] <h2 style=";text-align:left;direction:ltr"> >Ab 007 LFR2(SEQ ID NO:73)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0708] <h2 style=";text-align:left;direction:ltr"> WYQQKPGQPPKLLIY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0709] <h2 style=";text-align:left;direction:ltr"> >Ab 007 LFR3(SEQ ID NO:74)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0710] <h2 style=";text-align:left;direction:ltr"> GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0711] <h2 style=";text-align:left;direction:ltr"> >Ab 007 LFR4(SEQ ID NO:75)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0712] <h2 style=";text-align:left;direction:ltr"> FGGGTKVEIK.

Claims

1. An isolated monoclonal antibody or an antigen-binding fragment thereof, the isolated monoclonal antibody or the antigen-binding fragment thereof comprising: (a) a heavy chain variable region (HCVR), the heavy chain variable region comprising (i) a HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), wherein X1 is selected from T, E, P or A; X2 is selected from T, G or S; and X3 is selected from E, S or D; (ii) a HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), wherein X4 is selected from E, W or V; and (iii) a HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) a light chain variable region (LCVR), the light chain variable region comprising (i) having the amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13) LCVR CDR1 sequence, wherein X5 is selected from Q or R; X6 is selected from S or N; X7 is selected from L, V or I; X8 is selected from Y or W; X9 is selected from S or P; X 10 Selected from N, S, P or A; X 11 Selected from N or H; X 12 Selected from Q, N or Y; X 13 Selected from K or R; and X 14 Selected from Y or H; (ii) having the amino acid sequence WX 15 SX 16 RX 17 X 18 LCVR CDR2 sequence, where X 15 Selected from A or G; X 16 Selected from T, N, M or A; X 17 Selected from G or E; and X 18 is selected from S, E, Y, P or T; and (iii) having the amino acid sequence QQYYX 19 X 20 The LCVR CDR3 sequence of PFT (SEQ ID NO: 30), wherein X 19 Selected from S or F; and X 20 Select from Y or F, wherein the antibody or antigen-binding fragment thereof specifically binds galectin-9 (Gal9), provided that the antibody does not comprise the HCVR CDR1 sequence GYTFTEYTIH (SEQ ID NO: 2), the HCVR CDR2 sequence WFYPGSGSTEYAQKFQG (SEQ ID NO: 9), the HCVR CDR3 sequence HGGYDGFDY (SEQ ID NO: 12), the LCVR CDR1 sequence KSSQSLLYSNNQKNYLA (SEQ ID NO: 14), the LCVR CDR2 sequence WASTRGS (SEQ ID NO: 22) and the LCVR CDR3 sequence QQYYSYPFT (SEQ ID NO: 31).

2. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the isolated monoclonal antibody or antigen-binding fragment thereof comprises: (a) HCVR comprising (i) a HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), wherein X1 is selected from T or E, and X2 is selected from T or G, and X3 is E; (ii) a HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), wherein X4 is selected from W or V; and (iii) a HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) a LCVR comprising (i) having the amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13), wherein X5 is Q, X6 is S, X7 is L, X8 is Y, X9 is selected from S or P, X 10 It is N, X 11 It is N, X 12 It's Q, X 13 is K; and X 14 It is Y; (ii) having the amino acid sequence WX 15 SX 16 RX 17 X 18 LCVR CDR2 sequence, where X 15 It's A, X 16 It's T, X 17 is G, and X 18 Selected from S or E; and (iii) having the amino acid sequence QQYYX 19 X 20 The LCVR CDR3 sequence of PFT (SEQ ID NO: 30), wherein X 19 It is S, and X 20 Select from Y or F, The antibody or antigen-binding fragment thereof specifically binds to Gal9.

3. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the isolated monoclonal antibody or antigen-binding fragment thereof comprises: (a) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO: 3, a HCVR CDR2 sequence of SEQ ID NO: 10, and a HCVR CDR3 sequence of SEQ ID NO: 12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO: 15, a LCVR CDR2 sequence of SEQ ID NO: 23, and a LCVR CDR3 sequence of SEQ ID NO: 32; or (b) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO: 4, a HCVR CDR2 sequence of SEQ ID NO: 11, and a HCVR CDR3 sequence of SEQ ID NO: 12; and a light chain variable region (LCVR) comprising a LCVR CDR1 sequence of SEQ ID NO: 14, a LCVR CDR2 sequence of SEQ ID NO: 24, and a LCVR CDR3 sequence of SEQ ID NO: 31, The antibody or antigen-binding fragment thereof specifically binds to Gal9.

4. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein: The HCVR sequence is SEQ ID NO:39; and / or, the LCVR sequence is SEQ ID NO:41, or The HCVR sequence is SEQ ID NO:43; and / or, the LCVR sequence is SEQ ID NO:

45.

5. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the antibody comprises: The HCVR sequence of SEQ ID NO:39 and the LCVR sequence of SEQ ID NO:41; or The HCVR sequence of SEQ ID NO:43 and the LCVR sequence of SEQ ID NO:

45.

6. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising: (a) HCVR comprising (i) a HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), wherein X1 is selected from T, P or A; X2 is selected from T, S or G; and X3 is selected from S or D; (ii) a HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), wherein X4 is E; and (iii) a HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) a LCVR comprising: (i) having the amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13) LCVR CDR1 sequence, wherein X5 is selected from Q or R; X6 is selected from S or N; X7 is selected from L, V or I; X8 is selected from Y or W; X9 is S, X 10 Selected from S, P or A; X 11 Selected from N or H; X 12 is N or Y; X 13 is K or R; and X 14 is Y or H; (ii) having the amino acid sequence WX 15 SX 16 RX 17 X 18 LCVR CDR2 sequence, where X 15 Selected from A or G; X 16 Selected from T, N, M or A; X 17 Selected from G or E; and X 18 is selected from S, Y, P or T; and (iii) having the amino acid sequence QQYYX 19 X 20 The LCVR CDR3 sequence of PFT (SEQ ID NO: 30), wherein X 19 is F, and X 20 It is Y. The antibody or antigen-binding fragment thereof specifically binds to Gal9.

7. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 6, wherein the isolated monoclonal antibody or antigen-binding fragment thereof comprises: (a) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:5, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a light chain variable region (LCVR) comprising a LCVR CDR1 sequence of SEQ ID NO:16, a LCVR CDR2 sequence of SEQ ID NO:25, and a LCVR CDR3 sequence of SEQ ID NO:33; or (b) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:6, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:17, a LCVR CDR2 sequence of SEQ ID NO:26, and a LCVR CDR3 sequence of SEQ ID NO:33; or (c) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:7, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:18, a LCVR CDR2 sequence of SEQ ID NO:27, and a LCVR CDR3 sequence of SEQ ID NO:33; or (d) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:7, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:19, a LCVR CDR2 sequence of SEQ ID NO:28, and a LCVR CDR3 sequence of SEQ ID NO:33; or (e) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:7, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO:20, a LCVR CDR2 sequence of SEQ ID NO:29, and a LCVR CDR3 sequence of SEQ ID NO:33, The antibody or antigen-binding fragment thereof specifically binds to Gal9.

8. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1, 6 or 7, wherein: The HCVR sequence is SEQ ID NO:47 and / or the LCVR sequence is SEQ ID NO:49, The HCVR sequence is SEQ ID NO:51 and / or the LCVR sequence is SEQ ID NO:53, The HCVR sequence is SEQ ID NO:55 and / or the LCVR sequence is SEQ ID NO:57, The HCVR sequence is SEQ ID NO:59 and / or the LCVR sequence is SEQ ID NO:61, or The HCVR sequence is SEQ ID NO:63 and / or the LCVR sequence is SEQ ID NO:

65.

9. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1, 6, 7 or 8, wherein the antibody comprises: The HCVR sequence of SEQ ID NO:47 and the LCVR sequence of SEQ ID NO:49; The HCVR sequence of SEQ ID NO:51 and the LCVR sequence of SEQ ID NO:53; The HCVR sequence of SEQ ID NO:55 and the LCVR sequence of SEQ ID NO:57; The HCVR sequence of SEQ ID NO:59 and the LCVR sequence of SEQ ID NO:61; or The HCVR sequence of SEQ ID NO:63 and the LCVR sequence of SEQ ID NO:

65.

10. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising: (a) HCVR comprising (i) a HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), wherein X1 is selected from E or P; X2 is selected from T or S; and X3 is selected from E or S; (ii) a HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), wherein X4 is selected from W or E; and (iii) a HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) a LCVR comprising: (i) having the amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13) LCVR CDR1 sequence, wherein X5 is Q, X6 is selected from S or N, X7 is selected from L or V, X8 is selected from Y or W, X9 is selected from P or S, X 10 Select from N or S, X 11 It is N, X 12 Select from Q or N, X 13 is K; and X 14 It is Y; (ii) having the amino acid sequence WX 15 SX 16 RX 17 X 18 LCVR CDR2 sequence, where X 15 Selected from A or G; X 16 Select from T or N; X 17 Select from G or E, and X 18 is S; and (iii) having the amino acid sequence QQYYX 19 X 20 The LCVR CDR3 sequence of PFT (SEQ ID NO: 30), wherein X 19 Selected from S or F; and X 20 Select from Y or F, The antibody or antigen-binding fragment thereof specifically binds to Gal9.

11. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 10, comprising: (a) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO: 3, a HCVR CDR2 sequence of SEQ ID NO: 10, and a HCVR CDR3 sequence of SEQ ID NO: 12; and a LCVR comprising a LCVR CDR1 sequence of SEQ ID NO: 15, a LCVR CDR2 sequence of SEQ ID NO: 23, and a LCVR CDR3 sequence of SEQ ID NO: 32; or (b) a HCVR comprising a HCVR CDR1 sequence of SEQ ID NO:5, a HCVR CDR2 sequence of SEQ ID NO:9, and a HCVR CDR3 sequence of SEQ ID NO:12; and a light chain variable region (LCVR) comprising a LCVR CDR1 sequence of SEQ ID NO:16, a LCVR CDR2 sequence of SEQ ID NO:25, and a LCVR CDR3 sequence of SEQ ID NO:33, The antibody or antigen-binding fragment thereof specifically binds to Gal9.

12. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein the antibody is a humanized antibody.

13. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the antigen-binding fragment thereof is Fab, Fab', F(ab')2, F d , single-chain Fv or scFv, disulfide-linked F v , V-NAR domain, IgNar, intracellular antibody, IgGACH2, mini antibody, F(ab')3, tetra antibody, tria antibody, diabody, domain antibody (dAb), DVD-Ig, Fcab, mAb2, (scFv)2, tandem scFv, TandAb, nanobody or scFv-Fc.

14. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-13, wherein the monoclonal antibody or antigen-binding fragment thereof has a K of less than about 5 nM, 2 nM, 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.05 nM or 0.01 nM. d Binds human Gal9.

15. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-14, which binds to Gal9 and inhibits the binding of Gal9 to a Gal9 receptor (e.g., TIM3 or CD44).

16. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-14, which binds to Gal9 and does not inhibit the binding of Gal9 to a Gal9 receptor (e.g., TIM3 or CD44).

17. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, wherein the isolated monoclonal antibody or antigen-binding fragment thereof reverses T cell (such as CD4 + Gal9-induced Th1 apoptosis of T cells.

18. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-17, which inhibits Gal9-induced Treg expansion.

19. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-18, thereby treating the cancer in the subject.

20. The method of claim 19, wherein the cancer is a hematological cancer or a solid tumor.

21. The method of claim 20, wherein the blood cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia, chronic myeloid leukemia and hairy cell leukemia, AIDS-related lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, Hodgkin lymphoma, mycosis fungoides, non-Hodgkin lymphoma, primary central nervous system lymphoma, Sezary syndrome, cutaneous T-cell lymphoma, Waldenstrom's macroglobulinemia, diffuse large B-cell lymphoma (DLBCL) and multiple myeloma.

22. The method of claim 20, wherein the solid tumor is breast cancer, head and neck cancer, lung cancer, melanoma, uveal melanoma, colorectal cancer, renal cancer, urothelial carcinoma, ovarian cancer, endometrial cancer, liver cancer, pancreatic cancer, bile duct cancer, gastric cancer, gastroesophageal cancer, esophageal cancer, cervical cancer, head and neck squamous cell carcinoma, or prostate cancer.

23. The method of any one of claims 19-22, further comprising administering to the patient a chemotherapeutic agent, an anti-angiogenic agent, a growth inhibitory agent, an immuno-oncology agent, a checkpoint inhibitor, and / or an anti-tumor composition.

24. The method of claim 23, wherein the checkpoint inhibitor is anti-PD-1.

25. The method of claim 23, wherein the immuno-oncology agent is an anti-glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) antibody.

26. A polynucleotide encoding the heavy chain or light chain or antigen binding portion thereof as described in any one of claims 1-13.

27. A vector comprising the polynucleotide of claim 26, wherein the vector is an expression vector selected from the group consisting of a mammalian expression vector, a yeast expression vector, an insect expression vector, and a bacterial expression vector.

28. A method for rescuing or promoting effector T cell proliferation, enhancing effector T cell activity and / or identifying and treating cancer in a subject, the method comprising: (i) determining the level of Gal9 in a sample from the subject, and (ii) administering an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 18 to a subject having a level of Gal9 that is higher than a reference level of Gal9, Thereby rescuing or promoting effector T cell proliferation, enhancing effector T cell activity and / or identifying and treating cancer in a subject.

29. The method of claim 28, wherein the subject is diagnosed with cancer, is at risk of developing cancer, or has a recurrence of cancer.

30. The method of claim 28 or 29, wherein the reference level of Gal9 is the level of Gal9 measured in a sample from a healthy or control individual.

31. The method of any one of claims 28-30, wherein determining comprises comparing the level of Gal9 in the sample to the reference level.

32. The method of any one of claims 28-31, wherein the cancer is a hematological cancer or a solid tumor.

33. The method of claim 32, wherein the blood cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia, chronic myeloid leukemia, and hairy cell leukemia, AIDS-related lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, Hodgkin lymphoma, mycosis fungoides, non-Hodgkin lymphoma, primary central nervous system lymphoma, Sezary syndrome, cutaneous T-cell lymphoma, Waldenstrom's macroglobulinemia, diffuse large B-cell lymphoma (DLBCL), and multiple myeloma.

34. The method of claim 32, wherein the solid tumor is breast cancer, head and neck cancer, lung cancer, melanoma, uveal melanoma, colorectal cancer, renal cancer, urothelial carcinoma, ovarian cancer, endometrial cancer, liver cancer, pancreatic cancer, bile duct cancer, gastric cancer, gastroesophageal cancer, esophageal cancer, cervical cancer, head and neck squamous cell carcinoma, or prostate cancer.

35. The method of any one of claims 28-34, wherein the sample is a blood sample, a plasma sample, a serum sample, a tumor biopsy sample, or a bone marrow (BM)-derived mononuclear cell (MNC) sample.

36. The method of any one of claims 28-35, further comprising administering to the patient a chemotherapeutic agent, an anti-angiogenic agent, a growth inhibitory agent, an immuno-oncology agent, a checkpoint inhibitor, and / or an anti-tumor composition.

37. The method of claim 36, wherein the checkpoint inhibitor is anti-PD-1.

38. The method of claim 36, wherein the immuno-oncology agent is an anti-glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) antibody.

Images