Modified anti-galectin-9 antibody and its use

Abstract

Disclosed herein are affinity matured anti-galectin-9 (Gal9) antibodies. The antibodies have an improved binding affinity for Gal9. Also disclosed herein are methods of using the affinity matured anti-Gal9 antibodies, including methods of treating cancer, methods of restoring or promoting the proliferation of effector T cells, methods of enhancing the activity of effector T cells, and / or methods of identifying and treating cancer in a subject, which include administration of the anti-Gal9 antibodies described herein. Also provided are polynucleotides encoding the heavy or light chains, or antigen-binding portions thereof, described herein, and vectors, particularly expression vectors, comprising the polynucleotides described herein.

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims the benefit of priority of U.S. Provisional Application Nos. 63 / 389,607 (filed on July 15, 2022) and 63 / 389,610 (filed on July 15, 2022) under 35 U.S.C. § 119(e). The disclosures of these prior applications are considered a part of the disclosure of this application and are hereby incorporated by reference in their entirety into the disclosure of this application.

[0002] [Incorporation of Sequence Listing] In this specification, the content of the accompanying sequence listing is incorporated by reference into this application. The xml file of the accompanying sequence listing, named 148640 - 004302_SL.xml, was created on July 13, 2023 and is 80,112 bytes in size.

[0003] [Summary of the Invention] The present invention generally relates to anti - galectin - 9 antibodies and more specifically to anti - galectin - 9 antibodies for use in the treatment of cancer.

Background Art

[0004] Galectin - 9 (i.e., Gal9) belongs to the galectin (i.e., S - type lectin) family of proteins. There are at least 15 types in vertebrates and 10 of them in humans. Despite being a soluble 34 - 39 kDa protein without a leader peptide, Gal9 is secreted via a non - classical mechanism. It preferentially interacts with β - galactoside residues of glycoproteins and glycolipids. In humans, Gal9 exists in three isoforms, namely long - type, medium - type, and short - type.

[0005] Gal9 belongs to the lectin family of β-galactoside-binding proteins, contains two different carbohydrate recognition domains (CRDs), which are linked by a flexible linker (tandem repeat type galectin). Gal9 can bind / ligate two ligands simultaneously and promote the association or organization of two or more monomeric receptors, thereby changing the rate of signal transduction and, in some cases, receptor endocytosis. Multiple 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 activities of several types of immune cells and creates an immunosuppressive environment in the tumor microenvironment (TME). Notably, Gal9 (i) induces apoptosis of CD4+ and CD8+ T cells, (ii) suppresses the generation of Th17 cells, (iii) induces the proliferation of inhibitory Tregs, (iv) promotes the proliferation of myeloid-derived suppressor cells, (v) promotes the M2-type tumor-associated macrophage (TAM) phenotype, and (vi) impairs the function of natural killer (NK) cells. Some of these effects of Gal9 have been confirmed in studies using Gal9 knockout mice.

[0006] Gal9 is one of the most studied ligands for T cell immunoglobulin and mucin domain-containing-3 (TIM-3), also known as hepatitis A virus cellular receptor 2 (HAVCR2), and is expressed in various hematological malignancies (e.g., chronic lymphocytic leukemia (CLL), myelodysplastic syndrome (MDS), Hodgkin and non-Hodgkin lymphoma, acute myeloid leukemia (AML)), and solid tumors (e.g., lung cancer, breast cancer, and hepatocellular carcinoma).

[0007] The interaction between Gal9 and TIM-3 / HAVCR2 has been found to attenuate T cell proliferation and effector function in the tumor microenvironment and chronic infection. Furthermore, Gal9 contributes to tumor cell transformation, cell cycle regulation, angiogenesis, and tumor formation by cell adhesion.

[0008] Gal9 is also directly expressed by regulatory T lymphocytes (i.e., Tregs), and its expression increases upon activation of Tregs. On the other hand, Gal9 is very weakly expressed by effector T lymphocytes (e.g., CD8 + cytotoxic T lymphocytes), and this expression disappears upon activation of effector T lymphocytes. Inhibition of Gal9 by anti-Gal9 antibodies has been found to inhibit the suppressive activity of Tregs.

[0009] Wu et al. (Immunity 41 (2): 270-282, 2014) reported that Gal9 is important in regulating the immune response. There are two subsets of Treg cells, namely "natural" Treg (nTreg) cells and "induced" Treg (iTreg) cells. Gal9 is highly expressed in iTreg cells and is essential for the generation and function of iTreg cells, but not in nTreg cells. Gal9 expression in iTreg cells is driven by the transcription factor Smad3, forming a feed-forward loop that further promotes Foxp3 expression. Gal9 enhances the stability and function of iTreg cells by directly binding to its receptor CD44. CD44 forms a complex with the transforming growth factor-β (TGF-β) receptor I (TGF-βRI) and activated Smad3. Gal9 signaling was further found to regulate the induction of iTreg cells mainly by acting through the CNS1 region of the Foxp3 locus. Exogenous Gal9, in addition to being an effector molecule of Treg cells, acts synergistically with TGF-β to enhance the differentiation and maintenance of iTreg cells.

[0010] Various types of T lymphocytes usually grow into, for example, "effector" cells, i.e., effector T lymphocytes, which perform specialized immune functions to defend the host organism. Thus, CD4 + T lymphocytes, i.e., helper T lymphocytes, secrete major cytokines that assist the humoral function of B lymphocytes (production of specific antibodies) and the cytotoxic activity of CD8 + T lymphocytes.

[0011] Tregs constitutively overexpress CD4 and CD25 molecules (and are thus also called "CD4 + CD25 + "), and constitutively overexpress the Foxp3 transcription factor. A small fraction of these CD4 + CD25 + T lymphocytes negatively regulate the immune response by recognizing various self-antigens via the T cell receptor (TCR). Thus, Tregs play a major role in the physiology of the immune system by protecting the body from the onset of autoimmune diseases.

[0012] Tregs exert immunosuppressive activity on effector T lymphocytes. Such activity, once activated, promotes tumor growth in pathological conditions such as tumors. Thus, the inhibitory activity of Tregs can be understood as an activity that reduces the anti-tumor immune response by inhibiting the function of effector T lymphocytes.

[0013] Gal9 is mainly associated with tumor immunosuppression resulting from its interaction with various immune receptors. For example, Gal9 inhibits the Th1 response and induces peripheral tolerance, as evidenced by the reduction of Th1 apoptosis upon Gal9 blockade, the increased susceptibility of Gal9 knockout mice to collagen-induced arthritis (CIA), and the prolonged survival of grafts and suppression of autoimmune diseases (AIDs) upon Gal9 administration. Gal9 also regulates the function of peripheral NK cells to promote maternal-fetal tolerance, promotes the proliferation of myeloid-derived suppressor cells (MDSCs), and synergizes with TGF-β to promote Treg proliferation.

[0014] High expression of Gal9 is evident in both the tumors and blood of patients with multiple solid tumors and hematological malignancies, and correlates with disease progression and poor prognosis. Recently, several reports have supported a therapeutic approach that combines anti-Gal9 antibodies with currently used immunotherapies, including immune checkpoint inhibition therapies.

[0015] Yang et al. (Nature Commun. 12, 832, 2021) showed the following: (i) PD-1 binds to the Gal9 C-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 Tim-3 and PD-1 / gal9 C-CRD complex contributes to the persistence of exhausted T cells by restricting Tim3-Gal9-driven apoptosis; and (iv) combination therapy with anti-PD-1 and anti-Gal9 antibodies can result in superior antitumor effects and overall outcomes (e.g., survival) compared to using each therapy individually. Furthermore, Yang et al. (supra) generated 16,291 immune cell transcriptomes from 48 tumor samples collected from 32 melanoma patients who received anti-PD-1 therapy and evaluated their association with Gal9 expression and treatment outcomes. Tumor-infiltrating lymphocytes (TILs) from anti-PD-1 non-responders expressed Gal9 at much higher levels compared to patients who responded to treatment.

[0016] Limagne et al. (Oncoimmunology 8: e1564505, 2019) evaluated a population of patients with metastatic non-small cell lung cancer (mNSCLC) and found that the accumulation of Tim3+ lymphoid cells and Gal9+ monocytic myeloid-derived suppressor cells (mMDSC) is associated with PD-1 blockade resistance.

[0017] Li et al. (Immunotherapy 15, 135, 2023) showed that the levels of Gal9 and PD-L1 are higher in pancreatic ductal adenocarcinoma (PDAC) tumors compared to normal tissues, and their levels are significantly associated with tumor infiltration into lymph nodes and TNM classification (tumor node metastasis staging). In a mouse KPC model (an established clinically relevant model of PDAC), significant tumor growth inhibition was observed in the combination therapy group of anti-PD-L1 and anti-Gal9 compared to the anti-PD-L1 or anti-Gal9 monotherapy groups, or the control group.

[0018] Collectively, these findings suggest that in certain human cancers, the combination therapy of anti-Gal9 and anti-PD-1 can be an effective treatment system.

[0019] The blood level of Gal9 is significantly higher in certain cancer patients compared to healthy controls. Therefore, there is an unmet need for anti-Gal9 antibodies for use in cancer treatment.

Summary of the Invention

[0020] Aspects of the invention described herein relate to antibodies against Gal9 and their use for the treatment of diseases related to the suppressive activity of regulatory T lymphocytes (Tregs).

[0021] In one aspect, the invention is an isolated monoclonal antibody or antigen-binding fragment thereof, comprising: (a) A heavy chain variable region (HCVR) comprising: (i) An HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), where 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) An HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), where X4 is selected from E, W, or V; and (iii) An HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) A light chain variable region (LCVR) comprising: (i) An LCVR CDR1 sequence having the amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13), where 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 10is selected from N, S, P, or A; X 11 is selected from N or H; X 12 is selected from Q, N, or Y; X 13 is selected from K or R; and X 14 is selected from Y or H; (ii) an LCVR CDR2 sequence having the amino acid sequence WX 15 SX 16 RX 17 X 18 where X 15 is selected from A or G; X 16 is selected from T, N, M, or A; X 17 is selected from G or E; and X 18 is selected from S, E, Y, P, or T; and (iii) an LCVR CDR3 sequence having the amino acid sequence QQYYX 19 X 20 PFT (SEQ ID NO: 30), where X 19 is selected from S or F; and X 20 is selected from Y or F; and includes provided that the antibody excludes an antibody comprising 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), providing an isolated monoclonal antibody or an antigen-binding fragment thereof that specifically binds to Gal9.

[0022] In one embodiment, the isolated monoclonal antibody or an antigen-binding fragment thereof (a) an HCVR comprising the following (i) An 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) An HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), wherein X4 is selected from W or V; and (iii) An HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) An LCVR comprising the following (i) An 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 10 is N; X 11 is N; X 12 is Q; X 13 is K; and X 14 is Y; (ii) An LCVR CDR2 sequence having the amino acid sequence WX 15 SX 16 RX 17 X 18 wherein X 15 is A; X 16 is T; X 17 is G; and X 18 is selected from S or E; and (iii) An LCVR CDR3 sequence having the amino acid sequence QQYYX 19 X 20 PFT (SEQ ID NO: 30), wherein X 19 is S; and X 20 is selected from Y or F; and comprising The antibody or antigen-binding fragment thereof specifically binds to Gal9.

[0023] In certain embodiments of the present invention, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, which (A) an HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 3, the HCVR CDR2 sequence of SEQ ID NO: 10, and the HCVR CDR3 sequence of SEQ ID NO: 12; and an LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 15, the LCVR CDR2 sequence of SEQ ID NO: 23, and the LCVR CDR3 sequence of SEQ ID NO: 32; or (B) an HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 4, the HCVR CDR2 sequence of SEQ ID NO: 11, and the HCVR CDR3 sequence of SEQ ID NO: 12; and a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 14, the LCVR CDR2 sequence of SEQ ID NO: 24, and the LCVR CDR3 sequence of SEQ ID NO: 31; and the antibody or antigen-binding fragment thereof specifically binds to Gal9, providing an isolated monoclonal antibody or antigen-binding fragment thereof.

[0024] In one embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof (a) an HCVR comprising (i) an HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), where 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) an HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), where X4 is E; and (iii) an HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) an LCVR comprising: (i) the amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14An LCVR CDR1 sequence having LA (SEQ ID NO: 13), 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 is selected from S, P, or A; X 11 is 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) An LCVR CDR2 sequence having the amino acid sequence WX 15 SX 16 RX 17 X 18 wherein X 15 is selected from A or G; X 16 is selected from T, N, M, or A; X 17 is selected from G or E; and X 18 is selected from S, Y, P, or T; and (iii) An LCVR CDR3 sequence having the amino acid sequence QQYYX 19 X 20 PFT (SEQ ID NO: 30), wherein X 19 is F; and X 20 is Y; and includes The antibody or antigen-binding fragment thereof specifically binds to Gal9.

[0025] In certain embodiments of the invention, the invention is an isolated monoclonal antibody or antigen-binding fragment thereof, (C) An HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 5, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 16, the LCVR CDR2 sequence of SEQ ID NO: 25, and the LCVR CDR3 sequence of SEQ ID NO: 33; (D) An (H)HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 6, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and an (L)LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 17, the LCVR CDR2 sequence of SEQ ID NO: 26, and the LCVR CDR3 sequence of SEQ ID NO: 33; (E) An (H)HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 7, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and an (L)LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 18, the LCVR CDR2 sequence of SEQ ID NO: 27, and the LCVR CDR3 sequence of SEQ ID NO: 33; (F) An (H)HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 7, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and an (L)LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 19, the LCVR CDR2 sequence of SEQ ID NO: 28, and the LCVR CDR3 sequence of SEQ ID NO: 33; or (G) An (H)HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 7, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and an (L)LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 20, the LCVR CDR2 sequence of SEQ ID NO: 29, and the LCVR CDR3 sequence of SEQ ID NO: 33; comprising providing an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to Gal9.

[0026] In another embodiment, the invention is an isolated monoclonal antibody or antigen-binding fragment thereof, (a) An (H)HCVR comprising (i) an 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) An HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), where X4 is selected from W or E; and (iii) An HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) A light chain variable region (LCVR) comprising: (i) An LCVR CDR1 sequence having the amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13), where 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 is selected from N or S; X 11 is N; X 12 is selected from Q or N; X 13 is K; and X 14 is Y; (ii) An LCVR CDR2 sequence having the amino acid sequence WX 15 SX 16 RX 17 X 18 where X 15 is selected from A or G; X 16 is selected from T or N; X 17 is selected from G or E; and X 18 is S; and (iii) An LCVR CDR3 sequence having the amino acid sequence QQYYX 19 X 20 PFT (SEQ ID NO: 30), where X 19 is selected from S or F; and X 20 is selected from Y or F; and comprising, The antibody or antigen-binding fragment thereof provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to Gal9.

[0027] In certain embodiments of the invention, the invention is an isolated monoclonal antibody or antigen-binding fragment thereof, comprising: (A) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 3, the HCVR CDR2 sequence of SEQ ID NO: 10, and the HCVR CDR3 sequence of SEQ ID NO: 12; and a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 15, the LCVR CDR2 sequence of SEQ ID NO: 23, and the LCVR CDR3 sequence of SEQ ID NO: 32; or (C) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 5, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 16, the LCVR CDR2 sequence of SEQ ID NO: 25, and the LCVR CDR3 sequence of SEQ ID NO: 33; and the antibody or antigen-binding fragment thereof provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to Gal9.

[0028] In one aspect, the HCVR sequence of the antibody of (A), (B), (C), (D), (E), (F), or (G) or an antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 68, and may further comprise 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 of (A), (B), (C), (D), (E), (F), or (G) or an antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 69, and may further comprise 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 of (A), (B), (C), (D), (E), (F), or (G) or an antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 70, and may further comprise 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 of (A), (B), (C), (D), (E), (F), or (G) or an antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 76, and may further comprise 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 of (A), (B), (C), (D), (E), (F), or (G) or an antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 77, and may further comprise 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 of (A), (B), (C), (D), (E), (F), or (G) or an antigen-binding fragment thereof may further comprise 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 HFR4 sequence of SEQ ID NO: 75.

[0029] 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: 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; or the HCVR sequence of SEQ ID NO: 47 and the LCVR sequence of SEQ ID NO: 49; or the HCVR sequence of SEQ ID NO: 51 and the LCVR sequence of SEQ ID NO: 53; or the HCVR sequence of SEQ ID NO: 55 and the LCVR sequence of SEQ ID NO: 57; or 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.

[0030] In one aspect, the antibody is a humanized antibody. In another aspect, the antigen-binding fragment thereof is Fab, Fab′, F(ab′)2, F d , single-chain Fv i.e., scFv, disulfide-bonded F v , V-NAR domain, IgNar, intrabody, IgGACH2, minibody, F(ab′)3, tetrabody, tribody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, tandem scFv, DART (registered trademark), TandAb, nanobody, or scFv-Fc. In other aspects, the monoclonal antibody or its antigen-binding fragment is K dis 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 and binds to human Gal9. In other embodiments, the antibody binds to Gal9 and inhibits Gal9 from binding to a Gal9 receptor (e.g., TIM3 or CD44). In another embodiment, the antibody binds to Gal9 and does not inhibit Gal9 from binding to a Gal9 receptor (e.g., TIM3 or CD44). In one embodiment, the antibody reverses Gal9-induced Th1 apoptosis in T cells (e.g., CD4 + T cells). In another embodiment, the antibody suppresses Gal9-induced Treg proliferation.

[0031] 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 cancer in the subject.

[0032] 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 lymphoma, mycosis fungoides, non-Hodgkin lymphoma, primary central nervous system lymphoma, Sézary syndrome, cutaneous T-cell lymphoma, Waldenström 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 cancer, ovarian cancer, endometrial cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, gastric cancer, gastroesophageal cancer, esophageal cancer, cervical cancer, head and neck squamous cell carcinoma, and prostate cancer. 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 an anti-PD-1. In other aspects, the immuno-oncology agent is an anti-glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) antibody.

[0033] In additional embodiments, the invention provides a polynucleotide encoding a heavy chain or a light chain, or an antigen-binding portion thereof, of any of the antibodies or antigen-binding fragments thereof described herein.

[0034] In one embodiment, the 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.

[0035] In another embodiment, the invention is a method of restoring or promoting the proliferation of effector T cells, enhancing the activity of effector T cells, and / or identifying and treating a subject, comprising: (i) determining the level of Gal9 in a sample from said subject, and (ii) administering to a subject having a Gal9 level higher than a reference level of Gal9, an effective amount of either the isolated monoclonal antibody or an antigen-binding fragment thereof described herein, thereby providing a method of restoring or promoting the proliferation of effector T cells, enhancing the activity of effector T cells, and / or identifying and treating a subject.

[0036] In one aspect, the subject is diagnosed with cancer, at risk of developing cancer, or has a cancer recurrence. In another aspect, the reference level of Gal9 is the level of Gal9 measured in a sample from a healthy or control individual. In some aspects, the determining step includes comparing the level of Gal9 in the sample to the reference level. 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: ALL, AML, CLL, CML, and hairy cell leukemia; AIDS-related lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, mycosis fungoides, non-Hodgkin lymphoma, primary central nervous system lymphoma, Sézary syndrome, cutaneous T-cell lymphoma, Waldenström macroglobulinemia, 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 cancer, ovarian cancer, endometrial cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, gastric cancer, gastroesophageal cancer, esophageal cancer, cervical cancer, head and neck squamous cell carcinoma, and prostate cancer. In one aspect, the sample is a blood sample, plasma sample, serum sample, tumor biopsy sample, or bone marrow (BM)-derived mononuclear cell (MNC) sample. In one aspect, the method further includes 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 an anti-PD-1 antibody. In other aspects, the immuno-oncology agent is an anti-GITR antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0037]

Figure 1A-D

Figure 2A-D

Figure 3

Mode for Carrying Out the Invention

[0038] Aspects of the invention described herein relate to antibodies against Gal9 and their use for the treatment of diseases associated with the suppressive activity of regulatory T lymphocytes (Tregs).

[0039] Before describing the compositions and methods, it is to be understood that the invention is not limited to the specific compositions, methods, and test conditions described, as such compositions, methods, and conditions may vary. Also, since the scope of the invention is limited only by the appended claims, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0040] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include the plural unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods and / or steps of the kind described herein, which will be apparent to those skilled in the art upon reading this disclosure.

[0041] All documents, patents, and patent applications mentioned herein are incorporated herein by reference to the same extent as if each individual document, patent, or patent application was specifically and individually incorporated by reference.

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, it being understood that modifications and variations are included within the spirit and scope of the present disclosure. Preferred methods and materials are described below.

[0043] Provided herein are affinity matured antibodies that specifically bind to Gal9. Affinity maturation is a process used to increase the affinity, avidity, and antigen-binding activity of antibodies (see, e.g., Teixeira et al., mAbs 14(1) e2115200, 2022).

[0044] Affinity maturation was performed against the antibody HFB9-2hz11 (Ab A) described in International Publication No. WO WO2021 / 139682. Seven antibodies with significantly improved Gal9 binding affinity were identified. These antibodies showed variability in the CDR regions and maintained and improved Gal9 binding compared to the parental antibody. Thus, in one aspect, the present invention is an isolated monoclonal antibody or an antigen-binding fragment thereof, comprising (a) A heavy chain variable region (HCVR) comprising (i) An HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), where 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) An HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), where X4 is selected from E, W, or V; and (iii) An HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) A light chain variable region (LCVR) comprising (i) An LCVR CDR1 sequence having the amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13), where 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 is selected from N, S, P, or A; X 11 is selected from N or H; X 12 is selected from Q, N, or Y; X 13 is selected from K or R; and X 14 is selected from Y or H; (ii) An amino acid sequence WX 15 SX 16 RX 17 X 18An LCVR CDR2 sequence having, where X 15 is selected from A or G; X 16 is selected from T, N, M, or A; X 17 is selected from G or E; and X 18 is selected from S, E, Y, P, or T; and (iii) An LCVR CDR3 sequence having the amino acid sequence QQYYX 19 X 20 PFT (SEQ ID NO: 30), where X 19 is selected from S or F; and X 20 is selected from Y or F; comprising, provided that the antibody excludes antibodies comprising 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), providing an isolated monoclonal antibody or an antigen-binding fragment thereof that specifically binds to Gal9.

[0045] In one embodiment, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises (a) An HCVR comprising (i) An HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), where X1 is selected from T or E; and X2 is selected from T or G; and X3 is E; (ii) An HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), where X4 is selected from W or V; and (iii) An HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) An LCVR comprising (i) An amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 having an LCVR CDR1 sequence of LA (SEQ ID NO: 13), where X5 is Q; X6 is S; X7 is L; X8 is Y; X9 is selected from S or P; X 10 is N; X 11 is N; X 12 is Q; X 13 is K; and X 14 is Y; (ii) An amino acid sequence WX 15 SX 16 RX 17 X 18 having an LCVR CDR2 sequence, where X 15 is A; X 16 is T; X 17 is G; and X 18 is selected from S or E; and (iii) An amino acid sequence QQYYX 19 X 20 having an LCVR CDR3 sequence of PFT (SEQ ID NO: 30), where X 19 is S; and X 20 is selected from Y or F; and including The antibody or its antigen-binding fragment specifically binds to Gal9.

[0046] A particular embodiment of the present invention is an isolated monoclonal antibody or its antigen-binding fragment, (A) An HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 3, the HCVR CDR2 sequence of SEQ ID NO: 10, and the HCVR CDR3 sequence of SEQ ID NO: 12; and an LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 15, the LCVR CDR2 sequence of SEQ ID NO: 23, and the LCVR CDR3 sequence of SEQ ID NO: 32; or (B) An HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 4, the HCVR CDR2 sequence of SEQ ID NO: 11, and the HCVR CDR3 sequence of SEQ ID NO: 12; and a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 14, the LCVR CDR2 sequence of SEQ ID NO: 24, and the LCVR CDR3 sequence of SEQ ID NO: 31; The antibody or antigen-binding fragment thereof comprises an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to Gal9.

[0047] In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof (a) An HCVR comprising (i) An HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), where 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) An HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), where X4 is E; and (iii) An HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) An LCVR comprising (i) An LCVR CDR1 sequence having the amino acid sequence KSSX5X6X7LX8X9XXNXLA (SEQ ID NO: 13), where 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 X 11 X 12 X 13 NX 14 LA, where X is selected from S, P, or A; X 10 is selected from N or H; X 11 is selected from N or Y; X 12 is selected from N or Y; X 13 is selected from K or R; and X 14 is selected from Y or H; (ii) An amino acid sequence WX15 SX 16 RX 17 X 18 An LCVR CDR2 sequence having X, where X 15 is selected from A or G; X 16 is selected from T, N, M, or A; X 17 is selected from G or E; and X 18 is selected from S, Y, P, or T; and (iii) an amino acid sequence QQYYX 19 X 20 An LCVR CDR3 sequence having PFT (SEQ ID NO: 30), where X 19 is F; and X 20 is Y; and The antibody or antigen-binding fragment thereof specifically binds to Gal9.

[0048] Certain embodiments of the invention are isolated monoclonal antibodies or antigen-binding fragments thereof, comprising (C) an HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 5, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 16, the LCVR CDR2 sequence of SEQ ID NO: 25, and the LCVR CDR3 sequence of SEQ ID NO: 33; (D) an HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 6, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and an LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 17, the LCVR CDR2 sequence of SEQ ID NO: 26, and the LCVR CDR3 sequence of SEQ ID NO: 33; (E) an HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 7, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and an LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 18, the LCVR CDR2 sequence of SEQ ID NO: 27, and the LCVR CDR3 sequence of SEQ ID NO: 33; (F) An (F)HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 7, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and an LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 19, the LCVR CDR2 sequence of SEQ ID NO: 28, and the LCVR CDR3 sequence of SEQ ID NO: 33; or (G) An (G)HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 7, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and an LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 20, the LCVR CDR2 sequence of SEQ ID NO: 29, and the LCVR CDR3 sequence of SEQ ID NO: 33; and the antibody or antigen-binding fragment thereof comprises an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to Gal9.

[0049] In another embodiment, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, (a) an HCVR comprising (i) an 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) an HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), wherein X4 is selected from W or E; and (iii) an HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) a light chain variable region (LCVR) comprising (i) the amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14An LCVR CDR1 sequence having LA (SEQ ID NO: 13), where 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 is selected from N or S; X 11 is N; X 12 is selected from Q or N; X 13 is K; and X 14 is Y; (ii) An LCVR CDR2 sequence having the amino acid sequence WX 15 SX 16 RX 17 X 18 where X 15 is selected from A or G; X 16 is selected from T or N; X 17 is selected from G or E; and X 18 is S; and (iii) An LCVR CDR3 sequence having the amino acid sequence QQYYX 19 X 20 having PFT (SEQ ID NO: 30), where X 19 is selected from S or F; and X 20 is selected from Y or F; and includes The antibody or antigen-binding fragment thereof provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to Gal9.

[0050] Certain embodiments of the invention are isolated monoclonal antibodies or antigen-binding fragments thereof, (A) An HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 3, the HCVR CDR2 sequence of SEQ ID NO: 10, and the HCVR CDR3 sequence of SEQ ID NO: 12; and an LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 15, the LCVR CDR2 sequence of SEQ ID NO: 23, and the LCVR CDR3 sequence of SEQ ID NO: 32; or (C) An HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 5, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 16, the LCVR CDR2 sequence of SEQ ID NO: 25, and the LCVR CDR3 sequence of SEQ ID NO: 33; The antibody or antigen-binding fragment thereof comprises an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to Gal9.

[0051] The terms "antibodies", "ab", "immunoglobulins", or "Ig" refer, in the broadest sense, to glycoproteins having similar structural features (i.e., molecules containing an antigen-binding site that binds immunospecifically 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). Antibodies exhibit binding specificity for a particular antigen, while immunoglobulins include both antibodies and other antibody-like molecules lacking antigen specificity. "Antibody" encompasses any polypeptide containing an antigen-binding site, regardless of its origin, the species from which it is derived, the method of production, and its properties. The term "antibody" may also be used broadly to refer to molecules containing the complementarity-determining regions (CDRs) 1, 2, and 3 of the heavy chain, and CDRs 1, 2, and 3 of the light chain, which are capable of binding to an antigen.

[0052] In some embodiments, the antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR). In some embodiments, the antibody comprises at least one heavy chain (HC) comprising a heavy chain variable region and at least a portion of a heavy chain constant region, and at least one light chain (LC) comprising a light chain variable region and at least a portion of a light chain constant region. In some embodiments, the antibody comprises two heavy chains (each heavy chain comprising a heavy chain variable region and at least a portion of a heavy chain constant region), and two light chains (each light chain comprising a light chain variable region and at least a portion of a light chain constant region).

[0053] "Native antibodies" and "intact immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains (L chains) and two identical heavy chains (H chains). Light chains from any vertebrate species can be classified into one of two distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be classified into different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which may be further subdivided into subclasses (isotypes). For example, IgG antibodies include, but are not limited to: IgG1 (comprising a γ1 constant region), IgG2 (comprising a γ2 constant region), IgG3 (comprising a γ3 constant region), and IgG4 (comprising a γ4 constant region) antibodies; IgA antibodies include, but are not limited to: IgA1 (comprising an α1 constant region) and IgA2 (comprising an α2 constant region) antibodies; and IgM antibodies include, but are not limited to: IgM1 (comprising a μ1 constant region) and IgM2 (comprising a μ2 constant region). The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional arrangement of different classes of immunoglobulins are well known.

[0054] A complete antibody may have one or more "effector functions", which refer to its biological activities resulting from the Fc region of the antibody (Fc region of the native sequence, or Fc region of an amino acid sequence variant, or other modified Fc region). Examples of antibody effector functions include: C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptor (BCR)); and cross-presentation of antigens by antigen-presenting cells or dendritic cells.

[0055] Each light chain is linked to the heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intra-chain disulfide bridges. Each heavy chain has a variable domain (V H ) at one end, followed by a plurality of constant domains. Each light chain has a variable domain (V L ) at one end and a constant domain at the other end; the constant domain of the light chain aligns with the first constant domain of the heavy chain, and the light chain variable domain aligns with the variable domain of the heavy chain. Specific amino acid residues are thought to form the interface between the light and heavy chain variable domains. Each variable region contains three segments called complementarity-determining regions (CDRs) or hypervariable regions, and the more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of the heavy and light chains each contain four FR regions, mainly adopt a β-sheet conformation, and are linked by three CDRs. These CDRs form loops and connections and, in some cases, form part of the β-sheet structure. The CDRs of each chain are held in close proximity by the FRs and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pages 647-669

[1991] ). The constant domains are not directly involved in the binding of the antibody to the antigen but exhibit various effector functions such as the involvement of the antibody in antibody-dependent cell cytotoxicity.

[0056] As used herein, the term "heavy chain variable region (HCVR)" refers to a region that includes at least CDR1 (CDR-H1), framework 2 (HFR2), CDR2 (CDR-H2), FR3 (HFR3), and CDR3 (CDR-H3) of the heavy chain. In some embodiments, the heavy chain variable region also includes at least a portion (e.g., the whole) of FR1 (HFR1) located on the N-terminal side of CDR-H1, and / or at least a portion (e.g., the whole) of FR4 (HFR4) located on the C-terminal side of CDR-H3.

[0057] As used herein, the term "heavy chain constant region" refers to a region that includes at least three heavy chain constant domains, namely CH1, CH2, and CH3. Non-limiting and exemplary heavy chain constant regions include γ, δ, and α. Non-limiting and exemplary heavy chain constant regions also include ε and μ. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody that includes a γ constant region is an IgG antibody, an antibody that includes a δ constant region is an IgD antibody, an antibody that includes an α constant region is an IgA antibody, an antibody that includes an ε constant region is an IgE antibody, and an antibody that includes a μ constant region is an IgM antibody.

[0058] As used herein, the term "light chain variable region (LCVR)" refers to a region that includes CDR1 (CDR-L1), framework (FR) 2 (LFR2), CDR2 (CDR-L2), FR3 (LFR3), and CDR3 (CDR-L3) of the light chain. In some embodiments, the light chain variable region also includes at least a portion (e.g., the whole) of FR1 (LFR1) located on the N-terminal side of CDR-L1, and / or at least a portion (e.g., the whole) of FR4 (LFR4) located on the C-terminal side of CDR-L3.

[0059] As used herein, the term "light chain constant region" refers to a region that includes the light chain constant domain, i.e., CL. Non-limiting and exemplary light chain constant regions include λ and κ.

[0060] As used herein, the term "light chain" refers to a polypeptide that includes at least a light chain variable region, with or without a leader sequence. In some embodiments, the light chain includes at least a portion of the light chain constant region. As used herein, the term "full-length light chain" refers to a polypeptide that includes a light chain variable region and a light chain constant region, with or without a leader sequence.

[0061] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies making up the population are identical, except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies have high specificity and are directed against a single antigenic site. Further, unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are free of contamination by other immunoglobulins. The modifier "monoclonal" indicates the property that the antibody is obtained from a substantially homogeneous population of antibodies and should not be construed to require production of the antibody by a particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or by recombinant DNA methods (e.g., see U.S. Patent No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), or isolated from yeast display and transgenic animals.

[0062] The terms "amino acid" and "naturally occurring amino acid" refer, in the broadest sense, to organic compounds containing both functional groups of an amino group and a carboxylic acid group, and from which proteins are formed, i.e., α-amino acids. Each α-amino acid contains a central carbon atom to which an amino group, a carboxylic acid, a hydrogen, and an "R" group are bonded. This "R" group varies among the amino acids and is the decisive difference that distinguishes each one. The 20 amino acids encoded by the eukaryotic genetic code and naturally incorporated into the polypeptide chain during protein construction are therefore referred to as "natural" amino acids. Other non-natural amino acids are also known, which are not found in proteins or are not naturally produced by standard cellular mechanisms (e.g., synthesis from intermediates produced in glycolysis) within eukaryotic cells. The 20 natural amino acids are classified according to 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 the special side chains of glycine, cysteine, and proline (containing side chains that are not easily classified by the above properties).

[0063] The 20 natural amino acids include the following: 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).

[0064] Of the 20 natural amino acids, 5 have side chains that can be charged. At pH = 7, 3 are positively charged: lysine (Lys, K), arginine (Arg, R), and histidine (His, H) (basic side chains); and 2 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. These do not ionize under normal conditions, with the notable exception of the catalytic serine in serine proteases. Nonpolar or hydrophobic amino acids include 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 a major driving force in the process of folding proteins into their functional three-dimensional structures. The side chains of these amino acids do not readily ionize and thus do not have a pKa. However, tyrosine is an exception, as the hydroxyl group of tyrosine deprotonates at high pH to form a negatively charged phenolate. For this reason, tyrosine can also be classified in the polar uncharged amino acid category, but due to its very low solubility in water, it fits well with the properties of hydrophobic amino acids.

[0065] Some side chains cannot be adequately described in terms of the categories of charge, polarity, or hydrophobicity. Glycine (Gly, G) can also be considered a polar amino acid because its small size results in solubility being mainly determined by the amino and carboxylic acid groups. However, due to having no side chain at all, glycine has a unique flexibility among amino acids and has diverse effects on protein folding. Cysteine (Cys, C) can also be classified into the polar amino acid category because it can readily form hydrogen bonds, but it is often found within protein structures that form covalent bonds with other cysteines called disulfide bonds. This bond affects protein folding and stability and is essential for antibody formation. Proline (Pro, P) has an alkyl side chain and can be considered hydrophobic, but because its side chain loops back and bonds to the α-amino group, it is particularly inflexible when incorporated into proteins. Similar to glycine, this gives it a unique influence on protein structure among amino acids. G, C, and P are generally classified as amino acids with "special case side chains".

[0066] The antibodies or antigen-binding fragments thereof described herein specifically bind to Gal9.

[0067] Galectins are defined by a shared consensus amino acid sequence that belongs to the animal lectin family and enables specific binding to β-galactoside-containing complex carbohydrates. The galectin family is ubiquitously expressed from lower organisms such as nematodes and sponges to higher mammalian species such as humans. The presence of these proteins across many species and the presence of highly conserved amino acid residues essential for ligand recognition in the carbohydrate recognition domain (CRD) suggest that galectins are involved in conserved important biological processes. The CRD of galectin usually consists of about 130 amino acid residues, with five- and six-stranded β-sheets tightly folded into a sandwich structure that recognizes the basic structure of N-acetyl lactosamine (LacNAc). To date, 15 members of the mammalian galectin family have been identified. Hirabayashi and Kasai proposed classifying the galectin subfamilies into prototype, chimeric, and tandem repeat types based on their domain composition. Prototype galectins (galectin-1, 2, 5, 7, 10, 11, 13, 14, and 15) contain a single CRD with a short N-terminal sequence, while tandem repeat galectins (galectin-4, 6, 8, 9, and 12) contain two non-identical CRDs linked by a short linker peptide sequence. The only chimeric galectin (galectin-3) has one CRD with an extended N-terminal that contains multiple repeats of a proline-tyrosine-glycine-rich motif. Gal9 is a tandem repeat galectin that was originally isolated from mouse embryonic kidney cells and later found to be widely distributed throughout rat and mouse tissues. In contrast, the expression of human Gal9 is limited to peripheral blood leukocytes and lymphoid tissues. The potent eosinophil chemoattractant ecalectin was originally cloned from a human T cell line but has been reported to be identical to human Gal9. Mammalian Gal9 has multiple isoforms, each with a linker of a different length.

[0068] Gal9 exhibits various biological functions, including cell aggregation, eosinophil chemotaxis, and apoptosis of mouse thymocytes, T cells, and human melanoma cells. Mouse Gal9 induces apoptosis of thymocytes in a lactose-inhibitable manner. The chemotactic activity of Gal9 depends on its sugar-binding activity and requires both CRDs. Therefore, the physiological function of Gal9 is thought to depend on its sugar recognition ability. Two physiological targets of mouse Gal9, Tim-3 and glucose transporter (GLUT)-2, have been reported. Tim-3 is specifically expressed on the surface of T helper type 1 (Th1) cells. Gal9 recognizes sugars (s) covalently bound to Tim-3; since the Gal9-Tim3 pathway induces cell death of Th1 cells, Gal9 is suggested to play an important role in the downregulation of Th1 responses.

[0069] However, Gal9 also interacts with GLUT-2 (a glucose transporter expressed on the surface of pancreatic β cells and essential for glucose-stimulated insulin secretion) on the extracellular surface. The recognition of GLUT-2 by Gal9 through the recognition of the sugar chain moiety is necessary for GLUT-2 to remain on the cell surface. The loss of sugar chain modification or the addition of sugar chains decreases the half-life of GLUT-2 on the cell surface, causes endocytosis of the receptor, and is accompanied by redistribution to endosomes and lysosomes.

[0070] The exact target sugar chain structure recognized by Gal9 in vivo is unidentified, but in vitro analysis has shown that human Gal9 has a high affinity for multiple oligosaccharides containing β-galactoside. 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 shows significant affinity for complex glycoconjugates such as Forssman pentasaccharide and polymeric N-acetyl lactosamine.

[0071] Since the biological activity of human Gal9 depends on the ligand specificity of each CRD and the multivalent binding brought about by the subsequent two CRDs, structural analysis of the specific sugar chain recognition properties of the NCRD of human Gal9 should provide insights into the physiological functions of Gal9.

[0072] In one aspect, the HCVR sequence of an antibody of (A), (B), (C), (D), (E), (F), or (G) or an antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 68, and may further comprise 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 an antibody of (A), (B), (C), (D), (E), (F), or (G) or an antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 69, and may further comprise 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 an antibody of (A), (B), (C), (D), (E), (F), or (G) or an antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 70, and may further comprise 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 an antibody of (A), (B), (C), (D), (E), (F), or (G) or an antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 76, and may further comprise 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 an antibody of (A), (B), (C), (D), (E), (F), or (G) or an antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 77, and may further comprise 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 an antibody of (A), (B), (C), (D), (E), (F), or (G) or an antigen-binding fragment thereof may further comprise 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 HFR4 sequence of SEQ ID NO: 75.

[0073] 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: 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; or the HCVR sequence of SEQ ID NO: 47 and the LCVR sequence of SEQ ID NO: 49; or the HCVR sequence of SEQ ID NO: 51 and the LCVR sequence of SEQ ID NO: 53; or the HCVR sequence of SEQ ID NO: 55 and the LCVR sequence of SEQ ID NO: 57; or 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.

[0074] In a specific embodiment, the present invention provides an isolated monoclonal antibody or an antigen-binding fragment thereof comprising: an HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 3, the HCVR CDR2 sequence of SEQ ID NO: 10, and the HCVR CDR3 sequence of SEQ ID NO: 12; and an LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 15, the LCVR CDR2 sequence of SEQ ID NO: 23, and the LCVR CDR3 sequence of SEQ ID NO: 32; wherein the antibody or its antigen-binding fragment specifically binds to Gal9. In one aspect, the HCVR sequence of the antibody or its antigen-binding fragment further comprises the HFR3 sequence of SEQ ID NO: 68, and may further comprise 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 embodiment, the HCVR sequence is SEQ ID NO: 39. In another aspect, the LCVR sequence of the antibody or its antigen-binding fragment may further comprise 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 HFR4 sequence of SEQ ID NO: 75. In one embodiment, the LCVR sequence is SEQ ID NO: 41. The antibody is specifically exemplified herein as having the heavy chain sequence of SEQ ID NO: 38 and the light chain sequence of SEQ ID NO: 40 (Ab 001).

[0075] In another specific embodiment, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 5, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 16, the LCVR CDR2 sequence of SEQ ID NO: 25, and the LCVR CDR3 sequence of SEQ ID NO: 33; wherein the antibody or antigen-binding fragment thereof specifically binds to Gal9. In one aspect, the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 69, and may further comprise 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 embodiment, the HCVR sequence is SEQ ID NO: 47. In another aspect, the LCVR sequence of the antibody or antigen-binding fragment thereof may further comprise 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 HFR4 sequence of SEQ ID NO: 75. In one embodiment, the LCVR sequence is SEQ ID NO: 49. The antibody is specifically exemplified herein as having the heavy chain sequence of SEQ ID NO: 46 and the light chain sequence of SEQ ID NO: 48 (Ab 003).

[0076] In one aspect, the antibody is a humanized antibody.

[0077] The "humanized" form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., the Fv, Fab, Fab′, F(ab′)2, or other antigen-binding subsequence of an antibody), which contains minimal sequences derived from non-human immunoglobulins. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which the residues of the recipient's complementarity-determining regions (CDRs) are replaced by the residues of the CDRs of a non-human species such as a mouse, rat, or rabbit (donor antibody) having the desired specificity, affinity, and binding capacity. In some cases, residues of the framework regions (FRs) of the human immunoglobulin are replaced by the corresponding non-human residues. Further, a humanized antibody may contain residues not found in the transferred CDRs or framework sequences of the recipient antibody. These modifications are made to further optimize and maximize the manufacturability, stability, and / or performance of the antibody. Generally, a humanized antibody contains substantially the entirety of at least one, usually two, variable domains, wherein all or substantially all of the CDRs correspond to those of the non-human immunoglobulin and all or substantially all of the FRs are of human immunoglobulin sequences. A humanized antibody also optimally contains at least a portion of the immunoglobulin constant region (Fc), usually of human immunoglobulin. For 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™ antibodies, the antigen-binding regions of which are derived from antibodies produced by immunizing macaques with the antigen of interest (see Weber, J et al., From rabbit antibody repertoires to rabbit monoclonal antibodies. Exp Mol Med 49, e305 (2017)).

[0078] 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, Biotechnology and Genetic Engineering Reviews, 29:2, 175-186, DOI: 10.1080 / 02648725.2013.801235). Preferably, the humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "imported" residues and are usually obtained from the "imported" variable domain. Humanization can essentially be performed according to the method of Winter et al. (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536

[1988] ) by substituting rodent CDRs or CDR sequences with the corresponding sequences of human antibodies. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), where the portion replaced by the corresponding sequence from a non-human species is substantially less compared to the complete human variable domain. In practice, humanized antibodies are usually human antibodies, where some CDR residues and sometimes some FR residues are replaced by residues derived from similar sites of rodent antibodies.

[0079] The selection of human variable domains (light and heavy chains) used in making humanized antibodies is very important for reducing antigenicity. According to the so-called "best-fit" method, the sequences of the variable domains of rodent antibodies are screened against the entire library of known human variable domain sequences. Then the human sequence closest to the rodent sequence is adopted as the human framework region (FR) of the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901

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

[1993] ).

[0080] It is even more important that the antibody be humanized while retaining high affinity for the antigen and other desirable biological properties. To achieve this goal, according to a preferred method, a humanized antibody is prepared by a process of analyzing the parent sequence and various theoretical humanized products using three-dimensional models of the parent sequence and the humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available for illustrating and displaying the predicted three-dimensional conformation of the selected candidate immunoglobulin sequences. Examination of these displays allows analysis of the likely role of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of the residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, by selecting and combining FR residues from the recipient and the grafted sequence, desired antibody properties such as improved affinity for the target antigen(s) can be achieved. Generally, CDR residues are most directly and significantly involved in affecting antigen binding.

[0081] Alternatively, transgenic animals (e.g., mice) can now be produced, which, upon immunization, can produce a complete repertoire of human antibodies without producing endogenous immunoglobulins. For example, in chimeric mice and germline mutant mice, it has been reported that homozygous deletion of the antibody heavy chain joining region (JH) gene completely inhibits endogenous antibody production. Introduction of a human germline immunoglobulin gene array into such germline mutant mice results in the production of human antibodies upon antigen stimulation. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993). Human antibodies may also be derived from antibody display libraries (e.g., 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).

[0082] Antibodies can be humanized by replacing the sequences of the Fv variable regions that are not directly involved in antigen binding with equivalent sequences derived from human Fv variable regions. General reviews on humanized chimeric antibodies are described in Morrison et al., (Science 229:1202-1207 (1985)) and Oi et al. (BioTechniques 4:214 (1986)). These methods include the steps of isolating, manipulating, and expressing a nucleic acid sequence encoding all or part of an immunoglobulin Fv variable region derived from at least one of the heavy or light chains. Sources of such nucleic acids are well known to those skilled in the art and can be obtained, for example, from antibody-producing hybridomas. Recombinant DNA encoding a humanized or chimeric antibody, or a fragment thereof, can then be cloned into an appropriate expression vector.

[0083] Alternatively, humanized antibodies can also be produced by CDR substitution (U.S. Patent 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)).

[0084] In another aspect, said antigen-binding fragment is Fab, Fab′, F(ab′)2, F d , single-chain Fv i.e., scFv, disulfide-bonded F v , V-NAR domain, IgNar, intrabodies, IgGACH2, minibodies, F(ab′)3, tetrabody, tribody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, tandem scFv, DART (registered trademark), TandAb, nanobody (see mdpi.com / 2073-4468 / 8 / 2 / 28 / pdf), or scFv-Fc,

[0085] Experimentally, antibodies can be cleaved by the proteolytic enzyme papain. This results in the cleavage of each heavy chain and the production of three separate antibody fragments. "Antibody fragments" include a part of a complete antibody, preferably the antigen-binding or variable region of a complete antibody. Examples of antibody fragments include Fab, Fab′, and F(ab′)2, Fc fragments or Fc fusion products, single-chain Fv (scFv), disulfide-bonded Fv (sdfv), and fragments containing VL or VH domains; diabodies, tribodies, etc. (Zapata et al. Protein Eng. 8(10):1057-1062

[1995] ). The term "antibody fragment" or "antigen binding portion" (of an antibody) includes, but is not limited to, fragments capable of binding to an antigen. Two units consisting of a light chain and a fragment of the heavy chain of approximately equal mass to the light chain are called Fab fragments (i.e., "antigen-binding" fragments). The third unit consists of two equal segments of the heavy chain and is called the Fc fragment. The Fc fragment is usually not involved in antigen-antibody binding but is important in subsequent processes involved in removing the antigen from the body.

[0086] The Fab fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. "Fab" refers to an antibody fragment with a molecular weight of approximately 50,000 daltons and having the activity to bind to an antigen. It includes approximately the first half of the heavy chain on the N-terminal side and the entire light chain, linked by a disulfide bridge. Fab can be specifically obtained by treating immunoglobulins with the protease papain. The Fab′ fragment differs from the Fab fragment in that several residues containing one or more cysteines derived from the antibody hinge region are added to the carboxy terminus of the heavy chain CH1 domain. Fab′-SH refers herein to a Fab′ in which the cysteine residue(s) of the constant domain has a free thiol group.

[0087] F(ab′)2 antibody fragments were originally produced as pairs of Fab′ fragments with cysteines in the hinge in between. Other chemical linkages of antibody fragments are also known. The term "F(ab′)2" refers to a fragment of approximately 100,000 daltons that has the activity to bind to an antigen. This fragment is slightly larger than two Fab fragments linked via disulfide bridges in the hinge region. These fragments are obtained by treating immunoglobulins with the protease pepsin. Fab fragments can be obtained by cleaving the disulfide bridges in the hinge region from the F(ab′)2 fragment.

[0088] The Fc region of an antibody is the tail region of the antibody and interacts with some proteins of cell surface receptors and the complement system. Due to this property, the antibody can activate the immune system. In the antibody isotypes IgG, IgA, and IgD, the Fc region is composed of two identical protein fragments derived from the second and third constant domains of the two heavy chains of the antibody; in IgM and IgE, the Fc region contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. The Fc region of IgG bears a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is essential for Fc receptor-mediated activity. The N-glycans added to this site are mainly core-fucosylated complex-type bi-antennary structures. In addition, a small amount of these N-glycans also bear bisecting GlcNAc and α-2,6-linked sialic acid residues.

[0089] Fc fusion proteins (also known as Fc chimeric fusion proteins, Fc-Ig, Ig-based chimeric fusion proteins, and Fc-tagged proteins) are genetically engineered molecules that combine the Fc domain of IgG with a peptide or protein of interest. Fc fusion proteins have become valuable reagents in in vivo and in vitro research. The binding partner fused to Fc can range from a single peptide, a ligand that is activated by binding to a cell surface receptor, a signaling molecule, the extracellular domain of a receptor that is activated by dimerization, or a bait protein used to identify binding partners in protein microarrays. One of the most important features of the Fc domain in vivo is its ability to significantly extend the plasma half-life of the protein of interest, which in the case of biotherapeutics leads to improved therapeutic efficacy; this is a factor that makes Fc fusion proteins attractive biotherapeutic agents.

[0090] 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 non-toxic to the recipient at the dosages and concentrations employed and can include the following: buffering agents (e.g., phosphoric, citric, and other organic acids); antioxidants (including ascorbic acid and methionine); preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides (e.g., XTEN) (see Haeckel A, et al., XTEN as Biological Alternative to PEGylation Allows Complete Expression of a Protease-Activatable Killin-Based Cytostatic. PLoS One. 2016 Jun 13;11(6):e0157193. doi: 10.1371 / journal.pone.0157193. PMID: 27295081; PMCID: PMC4905650); proteins (e.g., serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (e.g., sodium); metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants (e.g., TWEEN™, PLURONIC™, or polyethylene glycol (PEG)).

[0091] "Fv" is the smallest antibody fragment that contains the complete antigen recognition and binding site. The dimer of "scFv" corresponds to that in which two scFv molecules are linked by a peptide bond. This Fv chain is often the result of the expression of a fusion gene in which the genes encoding VH and VL are linked by a linker sequence encoding a peptide. A human scFv fragment may contain CDR regions that maintain an appropriate three-dimensional structure, preferably by means of recombinant technology. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain that are tightly non-covalently associated. In this structure, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only the three CDRs specific for the antigen) may have the ability to recognize and bind the antigen, but the affinity is lower than that of the entire binding site.

[0092] The "single-chain Fv" or "scFv" antibody fragment contains the VH and VL domains of the antibody, and these domains are present within a single polypeptide chain. Preferably, the Fv polypeptide further contains a polypeptide linker between the VH and VL domains, thereby enabling the sFv to form a structure desirable for antigen binding. For a review on sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0093] The "dsFv" fragment is a VH-VL heterodimer stabilized by a disulfide bridge; it can be bivalent (dsFv2). Bivalent sc(Fv)2 or fragments of multivalent antibodies can be formed by the spontaneous association of monovalent scFv or produced by linking scFv fragments with peptide-binding sequences.

[0094] The Fc fragment supports the biological properties of the antibody, specifically the ability to be recognized by immune effectors or the ability to activate complement. It consists of the constant fragment of the heavy chain beyond the hinge region.

[0095] "Diabodies" refer to small antibody fragments that have two antigen-binding sites. These fragments contain a variable heavy chain domain VH linked to a variable light chain domain VL within the same VH-VL polypeptide chain. By using a binding sequence that is too short for the two domains to pair within the same chain, pairing with two complementary domains on another chain is inevitable, thereby generating two antigen-binding sites.

[0096] For the production of antibody fragments, various techniques have been developed. Conventionally, these fragments were obtained by proteolysis of the complete antibody (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science, 229:81

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

[1992] ). As another approach, F(ab')2 fragments can be directly isolated from recombinant host cell cultures. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, the selected antibody is a single-chain Fv fragment (scFv). See WO93 / 16185.

[0097] In other embodiments, the monoclonal antibody or its antigen-binding fragment is K dbinds to human Gal9 with an affinity 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.

[0098] In some embodiments, the affinity matured monoclonal antibody or antigen-binding fragment thereof has an approximately 2- to 10-fold increased affinity for Gal9 as compared to the non-affinity matured Ab A. For example, the affinity matured monoclonal antibody or antigen-binding fragment thereof has an approximately 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater increased affinity for Gal9 as compared to the non-affinity matured Ab A.

[0099] The term "antigen-binding domain" refers to the portion of an antibody molecule that contains a region that specifically binds to or is complementary to part or all of an antigen. When the antigen is large, the antibody may bind only to a particular portion of the antigen. An "epitope" or "antigenic determinant" is the portion of an antigen molecule that is responsible for the interaction with the antigen-binding domain of an antibody. The antigen-binding domain can 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 can include the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH).

[0100] The "antigen" according to the present invention encompasses any substance that elicits an immune response. Specifically, the "antigen" is related to any substance, preferably a peptide or protein, that specifically reacts with an antibody or a T lymphocyte (T cell). According to the present invention, the term "antigen" includes any molecule containing at least one epitope. Preferably, the antigen in the context of the present invention is a molecule that induces an immune response (after being processed if necessary). According to the present invention, any suitable antigen that is a candidate for an immune response may be used, where 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 an antigen-presenting cell (including diseased cells, particularly cancer cells), and in the context of MHC molecules, this elicits an immune response against the antigen. The antigen is preferably a product corresponding to or derived from a natural antigen. Such natural antigens include tumor antigens.

[0101] The term "epitope" refers to an antigenic determinant in a molecule such as an antigen, i.e., a part or fragment of a molecule recognized by the immune system. The epitope of a protein such as a tumor antigen preferably includes a continuous or discontinuous portion of the said protein.

[0102] The terms "epitope", "antigen peptide", "antigen epitope", "immunogenic peptide", and "MHC binding peptide" may be used interchangeably herein, preferably related to an incomplete phenotype of an antigen, and preferably capable of eliciting an immune response against the antigen or against a cell that expresses or contains and preferably presents the antigen.

[0103] [[ID=,11]] The term "binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner. Various methods for measuring binding affinity or avidity are known in the art, any of which may be used for purposes of the present invention. Specific exemplary embodiments are described below.

[0104] As used herein, "specific binding" refers to the binding of an antibody to a predetermined antigen. Antibodies typically bind to antibodies at a concentration of about 10 -8 K below M D and has an affinity (dissociation constant K) that is at least 10-fold lower, preferably at least 100-fold lower, for the predetermined antigen compared to the binding affinity for a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). D Alternatively, the antibody binds at 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 and may bind with an affinity corresponding to at least 10-fold greater, preferably at least 100-fold greater, affinity (binding constant K) for a predetermined antigen compared to the binding affinity for a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). A The bond is expressed by

[0105] "k d ”(seconds -1 The term "K", as used herein, is intended to refer to the dissociation rate constant of a particular antibody-antigen interaction. This value is also referred to as the "off value." D " (M -1The term ) as used herein is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0106] In some embodiments, the Gal9 antibodies of the invention have an antibody activity against Gal9 (e.g., human Gal9) of ≦5 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 Dissociation constant (K d In certain embodiments, the Gal9 antibody has an affinity for Gal9 (e.g., human Gal9) of ≦5 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 Dissociation constant (K d )

[0107] "k a " (M -1 seconds -1 The term "K", as used herein, is intended to refer to the binding rate constant of a particular antibody-antigen interaction. A The term "" (M), as used herein, is intended to refer to the binding equilibrium constant of a particular antibody-antigen interaction.

[0108] The antibodies described herein may be characterized using a surface plasmon resonance (SPR) assay or using a solution equilibrium assay (SEA).

[0109] Surface plasmon resonance (SPR) is a physical phenomenon that occurs at the surface of a thin gold film on an SPR chip. In an SPR assay, the binding target to be analyzed (e.g., an antibody) is covalently bound to the surface with a dextran linker. When the analyte flows over the surface, the binding that occurs between the analyte and the surface can be directly measured as a change in the local refractive index. This generates binding and dissociation curves, as well as maximum binding amount data. SPR assays are generally more sensitive than plate-based assays and can detect a wider range of antibodies. This is mainly due to the absence of a washing step that typically removes most of the low-affinity target population. Also, due to the label-free nature of SPR, it eliminates the interference effect caused by cross-linking labels, simplifies the analysis, and significantly reduces false negatives. SPR assays can also enable in-line isotyping by adding anti-isotype antibodies after the binding step.

[0110] SPR assays or label-free interaction measurements can be applied to the determination of antibody specificity, the thermodynamic study of interactions, the elucidation of the mechanism of action, or the characterization of epitopes, or affinity screening.

[0111] The ability to reliably estimate affinity using unpurified samples is a prerequisite for efficient screening. By using a high-throughput affinity screening process to avoid time-consuming purification steps and enable the initial characterization of thousands of antibody candidates in a short time, the cost and duration of the entire project can be reduced. Essential requirements for such affinity screening tools are: (1) the reliable identification of affinity-improved candidates; (2) the estimation of the appropriate dissociation equilibrium constant (KD) for each clone; (3) compatibility with unpurified production matrices (e.g., bacterial extracts or cell culture supernatants); and (iv) high-throughput processing (e.g., by automation of all sample processing steps). State-of-the-art technologies such as surface plasmon resonance (SPR; e.g., Biacore) and biolayer interferometry (BLI; e.g., Octet) partially meet these criteria (4, 5), but it remains a challenge to discriminate affinities in the picomolar (pM) range with a reasonable throughput.

[0112] Solution equilibrium titration (SET) or solution equilibrium assay (SEA) uses highly sensitive electrochemiluminescence as a readout system. Since the binding partner is not labeled, the resulting KD represents a reasonable approximation of the actual affinity. In screening, diluted bacterial lysates or cell culture supernatants are equilibrated with four different concentrations of the soluble target molecule, followed by quantification of unbound antibody on 384-well Meso Scale Discovery (MSD) plates coated with each antigen. To determine the KD value from the obtained 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.

[0113] In one aspect, the antibody binds to Gal9 and inhibits Gal9 from binding to a Gal9 receptor (e.g., TIM3 or CD44). In another aspect, the antibody binds to Gal9 and does not inhibit Gal9 from binding to a Gal9 receptor (e.g., TIM3 or CD44). In some embodiments, a Gal9 antibody having any of the properties described herein inhibits Gal9 signaling by at least 25%, 50%, 75%, 80%, 90%, or 100%.

[0114] In another aspect, the antibody inhibits Gal9-induced Th1 apoptosis in T cells (e.g., CD4 + T cells).

[0115] In one aspect, the antibody suppresses Gal9-induced Treg proliferation.

[0116] In another embodiment, the invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of any of the isolated monoclonal antibodies or antigen-binding fragments described herein, thereby treating cancer in the subject.

[0117] As used herein, the term "subject" refers to any individual or patient in whom the method of interest is performed. Generally, the subject is a human, but as will be understood 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, livestock (including cows, horses, goats, sheep, pigs, chickens, etc.), and primates (including monkeys, chimpanzees, orangutans, and gorillas) are included in the definition of a subject.

[0118] The term "treatment" is used interchangeably herein with the term "therapeutic method" and refers to both of the following: 1) a therapeutic treatment or means for curing, retarding the progression of, alleviating the symptoms of, and / or halting the progression of a diagnosed pathological condition or disorder, and 2) prophylactic / preventive means. Those in need of treatment can include not only individuals who already have a particular medical disease, but also those who may ultimately develop a disorder (i.e., those in need of preventive means). "Treatment" includes in mammals including humans, any administration or application of a therapeutic agent to a disease (also referred to herein as a "disorder" or "condition"), and also includes inhibition of a disease or the progression of the disease, inhibition or retardation of the disease or its progression, halting of its progression, partial or complete alleviation of the disease, partial or complete alleviation of one or more symptoms of the disease, or restoration or repair of a lost, missing, or defective function; or stimulation of an inefficient process. The term "treatment" also includes reduction in the severity of any phenotypic characteristic, and / or reduction in the incidence, degree, or likelihood of that characteristic.

[0119] The terms "therapeutically effective amount", "effective dose", "therapeutically effective dose", "effective amount", etc. refer to an amount of a compound of interest that causes a biological or medical response in a tissue, system, animal, or human that is sought by a researcher, veterinarian, physician, or other clinician. Generally, said response is either an improvement in the 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, the age, sex, weight of the individual, and the ability of the antibody and antigen-binding fragments thereof to cause a desired response in the individual. The therapeutically effective amount encompasses an amount where any toxic or adverse effects of the Gal9 antibody and antigen-binding fragments thereof are outweighed by the therapeutically beneficial effects.

[0120] The terms "administration of" and / or "administering" should be understood to mean providing 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. Thus, the route of administration includes, but is not limited to: intradermal, subcutaneous, intravenous, intramuscular, intravitreal, intraperitoneal, intraarterial, intrathecal, intraarticular, intraorbital, intracardiac, intradermal (intradermal), transdermal, intratracheal, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal, oral, sublingual buccal, rectal, vaginal, nasal-ocular administration, and infusion, inhalation, and nebulization. As used herein, the phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral and topical administration. In various embodiments, the Gal9 antibody is administered subcutaneously or intravenously.

[0121] The antibodies and antigen-binding fragments thereof described in this specification can be prepared as pharmaceutical compositions comprising the antibody or its antigen-binding fragment 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 common in the art, which is used together with a therapeutic agent to constitute a "pharmaceutical composition" and is administered to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosages and concentrations used and is compatible with the other components of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation used. For example, when the therapeutic agent is administered orally, the carrier may be a gel capsule. When the therapeutic agent is administered subcutaneously, the carrier ideally does not irritate the skin and does not cause an injection site reaction.

[0122] Pharmaceutical compositions can be administered in various unit dosage forms depending on the method of administration. Suitable unit dosage forms include, but are not limited to: powders, tablets, pills, capsules, troches, suppositories, patches, nasal sprays, injections, implantable sustained-release formulations, lipid complexes, etc.

[0123] The composition in question can be formulated as a preparation in solid, semi-solid, liquid, or gaseous form; this includes, but is not limited to: tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols.

[0124] In various embodiments, a composition comprising a Gal9 antibody and antigen-binding fragments thereof is provided as a formulation with a wide range 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. 2021 Jul;110(7):2590-2608.e56. doi: 10.1016 / j.xphs.2021.03.017. Epub 2021 Mar 28. PMID: 33789155). A variety of pharmaceutically acceptable carriers are available, including vehicles, adjuvants, and diluents. Additionally, a variety of pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, tonicity adjusters, stabilizers, wetting agents, etc., are also available. Non-limiting examples of carriers include saline solutions, buffered saline solutions, dextrose, water, glycerol, ethanol, and combinations thereof.

[0125] In various embodiments, a composition comprising a Gal9 antibody and antigen-binding fragments thereof can be formulated for injection, including subcutaneous administration, in an aqueous or non-aqueous solvent (e.g., vegetable oil or other oils, synthetic fatty acid glycerides, higher fatty acid esters, or propylene glycol); and, optionally, with conventional additives (e.g., solubilizers, isotonicity agents, suspending agents, emulsifying agents, stabilizers, and preservatives) by dissolving, suspending, or emulsifying.

[0126] In various embodiments, the composition can be formulated for inhalation using, for example, a pressurizable acceptable propellant (e.g., dichlorodifluoromethane, propane, nitrogen, etc.).

[0127] The composition can also be formulated in various embodiments as sustained-release microcapsules using, for example, biodegradable or non-biodegradable polymers. Non-limiting examples of biodegradable formulations include those containing poly(lactic-co-glycolic acid) (PLGA) polymers. Non-limiting examples of non-biodegradable formulations include those containing polyglycerol fatty acid esters. Some methods for making such formulations are described, for example, in EP 1125584 A1.

[0128] Also provided is a pharmaceutical dosage pack comprising one or more containers, each containing one or more doses of a Gal9 antibody and its antigen-binding fragment. In some embodiments, unit doses are provided, which contain a predetermined amount of the composition comprising the Gal9 antibody and its antigen-binding fragment, with or without one or more additional agents. In some embodiments, such unit doses are supplied in single-use prefilled syringes for injection. In various embodiments, the composition contained in the unit dose may include salts solutions, sucrose, etc.; buffering agents such as phosphoric acid, etc.; and / or may be formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition can be provided as a lyophilized powder that can be reconstituted by the addition of a suitable liquid (e.g., sterile water). In some embodiments, the composition includes 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 includes heparin and / or proteoglycan.

[0129] The pharmaceutical composition is administered in an amount effective for the treatment or prevention of a particular indication. A therapeutically effective amount typically depends on the body weight of the subject being treated, its physical or health condition, the extent of the condition being treated, or the age of the subject being treated.

[0130] In some embodiments, the Gal9 antibody and antigen-binding fragments thereof can be administered in an amount in the range of about 50 μg / kg body weight to about 50 mg / kg body weight per administration. In some embodiments, the Gal9 antibody and antigen-binding fragments thereof can be administered in an amount in the range of about 100 μg / kg body weight to about 50 mg / kg body weight per administration. In some embodiments, the Gal9 antibody and antigen-binding fragments thereof can be administered in an amount in the range of about 100 μg / kg body weight to about 20 mg / kg body weight per administration. In some embodiments, the Gal9 antibody and antigen-binding fragments thereof can be administered in an amount in the range of about 0.5 mg / kg body weight to about 20 mg / kg body weight per administration.

[0131] In some embodiments, the Gal9 antibody and antigen-binding fragments thereof can be administered in an amount in the range of about 10 mg to about 1000 mg per administration. In some embodiments, the Gal9 antibody and antigen-binding fragments thereof can be administered in an amount in the range of about 20 mg to about 500 mg per administration. In some embodiments, the Gal9 antibody and antigen-binding fragments thereof can be administered in an amount in the range of about 20 mg to about 300 mg per administration. In some embodiments, the Gal9 antibody and antigen-binding fragments thereof can be administered in an amount in the range of about 20 mg to about 200 mg per administration.

[0132] Compositions of Gal9 antibodies and antigen-binding fragments thereof can be administered to a subject as needed. In some embodiments, an effective dose of a Gal9 antibody and antigen-binding fragments thereof is administered to a subject one or more times. In various embodiments, an effective dose of a Gal9 antibody and antigen-binding fragments thereof is administered to a subject once a month, less than once a month (e.g., every two months, every three months, or every six months, etc.). In other embodiments, an effective dose of a Gal9 antibody and antigen-binding fragments thereof is administered more than once a month (e.g., every two weeks, once a week, twice a week, three times a week, once a day, or multiple times a day, etc.). An effective dose of a Gal9 antibody and antigen-binding fragments thereof is administered to a subject at least once. In some embodiments, an effective dose of a Gal9 antibody and antigen-binding fragments thereof can be administered multiple times over a period of at least one month, at least six months, or at least one year, etc. In some embodiments, the Gal9 antibody and antigen-binding fragments thereof are administered to a subject as needed to alleviate one or more symptoms of the condition.

[0133] The antibodies described herein can be used for the treatment of cancer.

[0134] Cancer is a group of diseases associated with abnormal cell growth and the potential to invade or metastasize to other parts of the body. In 2015, approximately 9.05 million people had cancer, approximately 14.1 million new cases occurred annually, and approximately 8.8 million people died (15.7% of deaths). The most common types of cancer in men are lung cancer, prostate cancer, colorectal cancer, and gastric cancer. The most common types in women are breast cancer, colorectal cancer, lung cancer, and cervical cancer.

[0135] The term "cancer" refers to a group of diseases characterized by abnormal and uncontrolled cell growth, which starts at a certain site (primary site) and has the potential to invade and metastasize to other sites (metastatic sites, metastases), distinguishing cancer (malignant tumor) from benign tumor. Virtually all organs can be affected, resulting in more than 100 types of cancers that humans can develop. Cancer can be caused by many factors, including: genetic predisposition, viral infection, exposure to ionizing radiation, exposure to environmental pollutants, smoking and / or alcohol consumption, obesity, dietary disorders, lack of exercise, or any combination thereof.

[0136] As used herein, "neoplasm" or "tumor" (including its grammatical variations) means a new abnormal growth of tissue, which can be either benign or cancerous. In related aspects, a neoplasm indicates a neoplastic disease or disorder and includes, but is not limited to, various cancers. For example, such cancers can include: prostate cancer, pancreatic cancer, biliary tract 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.

[0137] In one aspect, the cancer is a hematological cancer or a solid tumor.

[0138] Cancers that occur in hematopoietic tissues (e.g., bone marrow) or cells of the immune system are referred to as hematological cancers or blood cancers. Hematological cancers affect the production and function of blood cells and are classified into three main types: leukemia, lymphoma, and multiple myeloma.

[0139] As used herein, "leukemia" refers to a blood cancer caused by the rapid production of abnormal white blood cells. Examples of leukemia include: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia, chronic myeloid leukemia, and hairy cell leukemia.

[0140] As used herein, "lymphoma" refers to a type of blood cancer that affects the lymphatic system. Examples of lymphomas include, but are not limited to: AIDS-related lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, mycosis fungoides, non-Hodgkin lymphoma, primary central nervous system lymphoma, Sézary syndrome, cutaneous T-cell lymphoma, and Waldenström macroglobulinemia.

[0141] As used herein, "myeloma" refers to a cancer of plasma cells. Examples of myelomas include, but are not limited to: chronic myeloproliferative neoplasms, Langerhans cell histiocytosis, multiple myeloma, plasma cell neoplasms, myelodysplastic syndromes, and myelodysplastic / myeloproliferative neoplasms.

[0142] In some embodiments, the Gal9 antibodies and antigen-binding fragments thereof of the present invention may be used alone or in combination with any other suitable compound known to be capable of treating the disease or indication.

[0143] In some aspects, administration can be combined with one or more additional therapeutic agents. Phrases such as "combination therapy," "combined with," etc. refer to the simultaneous use of multiple drugs or treatments to enhance efficacy. The compositions of the present invention may be used in combination with, for example, other drugs or treatments used in the treatment of cancer. Specifically, administration of the compositions of the present invention to a subject can be combined with any anti-cancer therapy. Such therapy can be administered before, simultaneously with, or after administration of the compositions of the present invention.

[0144] In certain embodiments, the Gal9 antibody is administered to a subject (e.g., a subject having cancer) simultaneously or sequentially with another treatment. For example, the Gal9 antibody can be administered in combination with one or more of the following: radiation therapy, surgery, or chemotherapy (e.g., targeted chemotherapy or immunotherapy). The start time points of the administration of the two agents can be, for example, at intervals of 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days, 7 days, or more than one week. Alternatively, the administration of the second agent can be started, 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 more than one week after the administration of the first agent.

[0145] In some embodiments, 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, Sézary syndrome, cutaneous T-cell lymphoma, Waldenström macroglobulinemia, diffuse large B-cell lymphoma (DLBCL), and multiple myeloma.

[0146] The FAB classification (French-American-British classification) of acute myeloid leukemia (AML) classifies AML into different disease stages. The FAB subtypes are shown in Table 1.

[0147] JPEG2025524650000002.jpg74166

[0148] Examples of solid cancers include, but are not limited to: carcinomas, sarcomas, squamous cell carcinomas, small cell lung cancers, pituitary carcinomas, esophageal cancers, astrocytomas, soft tissue sarcomas, non-small cell lung cancers, lung adenocarcinomas, lung squamous cell carcinomas, peritoneal cancers, hepatocellular carcinomas, gastrointestinal cancers, pancreatic cancers, glioblastomas, cervical cancers, ovarian cancers, liver cancers, bladder cancers, liver tumors, breast cancers, colon cancers, colorectal cancers, endometrial cancers or corpus cancers, salivary gland cancers, kidney cancers, renal cancers, liver cancers, prostate cancers, vulvar cancers, thyroid cancers, hepatic carcinomas, brain cancers, endometrial cancers, testicular cancers, cholangiocarcinomas, gallbladder cancers, gastric cancers, melanomas, gastroesophageal cancers, esophageal cancers, cervical cancers, and various head and neck cancers.

[0149] In other embodiments, the solid tumor is selected from the group consisting of: breast cancer, head and neck cancer, lung cancer, melanoma, uveal melanoma, colorectal cancer, renal carcinoma, urothelial cancer, ovarian cancer, endometrial cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, gastric cancer, gastroesophageal cancer, esophageal cancer, cervical cancer, head and neck squamous cell carcinoma, or prostate cancer.

[0150] In one embodiment, 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.

[0151] A "chemotherapeutic agent" is a compound that may be useful in the treatment of cancer. Examples of chemotherapeutic agents include, but are not limited to, the following: alkylating agents (e.g., thiotepa and cyclophosphamide); alkyl sulfonates (e.g., busulfan, improsulfan, and piposulfan); aziridines (e.g., benzodopa, carboquone, meturedopa, and uredopa); ethyleneimines and methylamelamines (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine); acetogenins (particularly, bratasin and bratasinone); camptothecin (including the synthetic analog topotecan); bryostatin; calistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs KW-2189 and CB1-TM1); eribulin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards (e.g., chlorambucil, chloronaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard); nitrosoureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine); antibiotics (e.g., enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin γ2 (gammall) and calicheamicin ω2 (omegall) (see, e.g., Agnew, Chem lntl. Ed. Engl, 33: 183-186 (1994)); dynemicin (including dynemicin A); bisphosphonates (e.g., clodronic acid); esperamicin;and neocarzinostatin chromophore and related chromoprotein-engineered antibiotic chromophores); aclacinomysins; actinomycin; authramycin; azaserine; bleomycin; cactinomycin; carabicin; calminomycin; cardinophilin; chromomycinis; dactinomycin; daunorubicin; detorubicin; 6-diazo-5-oxo-L-norleucine; doxorubicin (including morpholino doxorubicin, cyanomorpholino doxorubicin, 2-pyrrolino doxorubicin, and deoxydoxorubicin); epirubicin; esorubicin; idarubicin; marcellomycin; mitomycin (e.g., mitomycin C); mycophenolic acid; nogalamycin; olivomycin; peplomycin; potfiromycin; puromycin; quelamycin; rhodrubicin; streptonigrin; streptozocin; tubercidin; ubenimex; dinostatin; zorubicin; antimetabolites (e.g., methotrexate and 5-fluorouracil (5-FU)); folic acid analogs (e.g., denopterin, methotrexate, pteropterin, trimetrexate); purine analogs (e.g., fludarabine, 6-mercaptopurine, thiampurine, thioguanine); pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine); androgens (e.g., calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone); antiadrenal agents (e.g., aminoglutethimide, mitotane, trilostane); folic acid supplements (e.g., frolinic acid); aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demeclocycline; diacont; elfomithine; elliptinium acetate;Epothilone; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Maytansinoids (e.g., Maytansine and Ansamitocins); Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pentostatin; Phenamet; Pirarubicin; Losoxantrone; Podophyllinic acid; 2-Ethylhydrazide; Procarbazine; Polysaccharide complex (JHS Natural Products, Eugene, Oregon); Razoxane; Rizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triazicone; 2,2′,2″-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, Verracurin A, Loridin A, and Anguizine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside (“Ara-C”); Cyclophosphamide; Thiotepa; Taxoids (e.g., Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, New Jersey), Paclitaxel in Cremophor-free albumin-modified nanoparticle formulation (American Pharmaceutical Partners, Schaumberg, Illinois), nab-Paclitaxel (Celgene), and Docetaxel (Rhone-Poulenc Rorer, Antony, France)); Chlorambucil; Gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogs (e.g., Cisplatin, Oxaliplatin, and Carboplatin); Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Xeloda; Ibandronic acid; Irinotecan (Camptosar, CPT-11) (including combination regimens of irinotecan with 5-FU and leucovorin); Topoisomerase inhibitor RFS2000;Difluoromethylornithine (DFMO); retinoids (e.g., retinoic acid); capecitabine; combretastatin; leucovorin (LV); oxaliplatin (including the oxaliplatin treatment regimen (FOLFOX)); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib), and VEGF-A that inhibit cell proliferation; and any pharmaceutically acceptable salt, acid, or derivative of any of the foregoing.

[0152] Non-limiting examples of additional chemotherapeutic agents include the following: antihormonal agents that act to modulate or inhibit the hormonal action on cancer (e.g., antiestrogen agents and selective estrogen receptor modulators (SERMs), such as tamoxifen (including tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene); aromatase inhibitors that inhibit the enzyme aromatase that regulates estrogen production in the adrenal gland (e.g., 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, and anastrozole, etc.); and antiandrogen agents (e.g., flutamide, nilutamide, bicalutamide, leuprolide, and goserelin); as well as troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit gene expression in signal transduction pathways involved in abnormal cell proliferation (e.g., PKC-α, Raf, and H-Ras, etc.); ribozymes (e.g., VEGF expression inhibitors (e.g., ribozymes), and HER2 expression inhibitors); vaccines (e.g., gene therapy vaccines, such as vaccine, vaccine, and vaccine); rIL-2; topoisomerase 1 inhibitors; rmRH; and any pharmaceutically acceptable salt, acid, or derivative of any of the foregoing.

[0153] The term "anti-angiogenesis agent" or "angiogenesis inhibitor" refers to a low molecular weight substance, polynucleotide (e.g., including inhibitory RNA (RNAi or siRNA)), polypeptide, isolated protein, recombinant protein, antibody, or a conjugate or fusion protein thereof that directly or indirectly inhibits angiogenesis, vasculogenesis, or undesirable vascular permeability. It should be understood that anti-angiogenesis agents include agents that bind to angiogenesis factors or their receptors and block their angiogenic activity. For example, an anti-angiogenesis agent can be an antibody or other antagonist to an angiogenesis agent, such as an antibody to VEGF-A (e.g., bevacizumab) or an antibody to a VEGF-A receptor (e.g., the KDR receptor or the Flt-1 receptor), an anti-PDGFR inhibitor (e.g., imatinib mesylate), a small molecule that blocks VEGF receptor signaling (e.g., PTK787 / ZK2284, SU6668, / SUI 1248 (sunitinib malate), AMG706, or those described, for example, in international patent application WO 2004 / 113304). Anti-angiogenesis agents also include natural angiogenesis inhibitors (e.g., 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 listing anti-angiogenesis therapies in malignant melanoma); Ferrara & Alitalo (1999) Nature Medicine 5 (12): 1359-1364; Tonini et al. (2003) Oncogene 22: 6549-6556 (e.g., Table 2 listing known angiogenesis factors); and Sato (2003) Int. J. Clin. Oncol. 8: 200-206 (e.g., Table 1 listing anti-angiogenesis agents used in clinical trials).

[0154] As used herein, the term "growth inhibitory agent" refers to a compound or composition that inhibits the growth of cells (e.g., cells expressing VEGF) in vitro or in vivo. Thus, a growth inhibitory agent can significantly reduce the proportion of cells in the S phase (e.g., cells expressing VEGF). Examples of growth inhibitory agents include, but are not limited to, agents that block the progression of the cell cycle (at stages other than the S phase) (e.g., agents that induce G1 arrest and M arrest). Classical M-phase blockers include the following: vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors (e.g., doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin). Agents that arrest the G1 phase also affect S-phase arrest, and examples thereof include DNA alkylating agents (e.g., tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C). Further information is described, for example, on page 13 of Chapter 1, "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. in Mendelsohn and Israel, eds., The Molecular Basis of Cancer (W.B. Saunders, Philadelphia, 1995). Taxanes (paclitaxel and docetaxel) are both anticancer agents derived from yew. Docetaxel (Rhone-Poulenc Rorer) is derived from the European yew and is a semisynthetic analog of paclitaxel (Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize the microtubules by preventing depolymerization, thereby inhibiting cell mitosis.

[0155] The term "anti-neoplastic composition" refers to a composition useful in treating cancer, which contains at least one effective therapeutic agent. Examples of therapeutic agents include, but are not limited to, for example: chemotherapeutic agents, growth inhibitors, cytotoxic agents, agents used in radiotherapy, anti-angiogenic agents, cancer immunotherapeutic agents (also referred to as immuno-oncology agents), apoptosis 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 (e.g., anti-PD-1 antibody BMS-936558), PD-L1 inhibitors (e.g., anti-PD-L1 antibody MPDL3280A), PD-L2 inhibitors (e.g., anti-PD-L2 antibody), VISTA inhibitors (e.g., anti-VISTA antibody), cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets: ErbB2, ErbB3, ErbB4, PDGFR-β, BlyS, APRIL, BCMA, PD-1, PD-L1, PD-L2, CTLA4, VISTA, or VEGF receptor(s), TRAIL / Apo2, and other bioactive agents and organic chemical agents, etc. Combinations thereof are also included in the present invention.

[0156] "Checkpoint inhibitor therapy" is a current form of cancer treatment that utilizes immune checkpoints that affect the function of the immune system. Immune checkpoints can be stimulatory or inhibitory. Tumors can utilize these checkpoints to protect themselves from attack by the immune system. Checkpoint therapy can block inhibitory checkpoints to restore the function of the immune system. Checkpoint proteins include the following: 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 antigen 4 (CTLA-4), A2AR (adenosine A2A receptor), B7-H3 (i.e., CD276), B7-H4 (i.e., VTCN1), BTLA (B and T lymphocyte attenuator, i.e., 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 3), and VISTA (V domain Ig suppressor of T cell activation).

[0157] 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 the downregulation of the immune system and the promotion of self-tolerance by suppressing T cell inflammatory activity. PD-1 is an immune checkpoint that defends against autoimmunity through the following dual mechanisms: promoting apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes while suppressing apoptosis of regulatory T cells (anti-inflammatory, inhibitory T cells).

[0158] PD-1 has two ligands, PD-L1 and PD-L2, which belong to the B7 family. The PD-L1 protein is upregulated in macrophages and dendritic cells (DCs) in response to LPS and GM-CSF treatment, and in T cells and B cells by TCR and B cell receptor signaling. On the other hand, in resting mice, PD-L1 mRNA can be detected in the heart, lung, thymus, spleen, and kidney. PD-L1 is expressed by IFN-γ treatment in almost all mouse tumor cell lines, including PA1 myeloma, P815 mastocytoma, and B16 melanoma. The expression of PD-L2 is more restricted and is mainly expressed in DCs and a small number of tumor cell lines.

[0159] CTLA4 or CTLA-4 (cytotoxic T lymphocyte antigen 4), also known as CD152 (cluster of differentiation 152), is a protein receptor that functions as an immune checkpoint and downregulates the immune response. CTLA4 is constitutively expressed in regulatory T cells but is only upregulated in normal T cells after activation, which is a particularly prominent phenomenon in cancer. CTLA4 belongs to the immunoglobulin superfamily, is expressed by activated T cells, and transmits inhibitory signals to T cells. CTLA4 has homology with the T cell costimulatory protein CD28, and both molecules bind to CD80 and CD86 (also called B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA-4 binds to CD80 and CD86 with higher affinity and avidity than CD28, and thus can outcompete CD28 for binding to its ligands. While CTLA4 transmits inhibitory signals to T cells, CD28 transmits stimulatory signals. CTLA4 is also found in regulatory T cells and contributes to its inhibitory function. The expression of CTLA-4 increases by T cell activation through the T cell receptor and CD28.

[0160] There are multiple checkpoint inhibitors currently being 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 ipilimumab (Yervoy). There are also multiple other checkpoint inhibitors in development, including anti-B7-H3 antibody (MGA271), anti-KIR antibody (lirilumab), and anti-LAG3 antibody (BMS-986016).

[0161] As used herein, "immune-oncology agent" refers to an anti-cancer agent that specifically targets cancer by targeting the immune system; this generally refers to therapeutic antibodies. In some embodiments, the immune-oncology agent is an anti-glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) antibody.

[0162] Tumor necrosis factor receptor superfamily 18 (TNFRSF18), also known as glucocorticoid-induced TNFR-related protein (GITR) or CD357, and also called activation-induced TNFR family receptor (AITR), is encoded by the TNFRSF18 gene on chromosome 1.

[0163] GITR belongs to the TNFR superfamily and has high homology with other members of TNFRSF such as CD137, OX40, or CD27 in the cytoplasmic domain characterized by cysteine pseudorepeats. GITR is constitutively expressed in CD25+CD4+ regulatory T cells and its expression is upregulated after activation in all T cell subsets. GITR is also expressed in mouse neutrophils and NK cells. GITR interacts with a ligand (GITRL) expressed in antigen-presenting cells (APCs) and endothelial cells.

[0164] 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 cause autoimmune diseases. Different responses to GITR signaling depend on GITR expression in different immune cell types. How GITR signaling is regulated in different cells remains unclear. GITR agonist 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 agonist antibodies is enhanced by combination with anti-PD-1 or anti-CTLA-4 therapy.

[0165] In additional embodiments, the invention provides a polynucleotide encoding a heavy chain or a light chain, or an antigen-binding portion thereof, of any of the antibodies or antigen-binding fragments thereof described herein.

[0166] The invention also provides a nucleic acid molecule comprising a polynucleotide encoding one or more chains of an antibody (e.g., an anti-Gal9 antibody) described herein. In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding a heavy chain or a light chain of an antibody described herein. In some embodiments, the nucleic acid molecule comprises both a polynucleotide encoding a heavy chain and a polynucleotide encoding a light chain of an antibody described herein. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide encoding a heavy chain, and a second nucleic acid molecule comprises a second polynucleotide encoding a light chain.

[0167] In some such embodiments, the heavy and light chains are expressed from one nucleic acid molecule or as two separate polypeptides from two separate nucleic acid molecules. In some embodiments, for example, when the antibody is a scFv, a single polynucleotide encodes a single polypeptide in which both the heavy and light chains are linked.

[0168] In some embodiments, the polynucleotide encoding the heavy or light chain of the antibody described herein includes a nucleotide sequence encoding a leader sequence that, after translation, is located at the N-terminus of the heavy or light chain. As described above, the leader sequence may be the native leader sequence of the heavy or light chain or another exogenous leader sequence.

[0169] As used herein, the terms "nucleic acid" or "oligonucleotide" refer to polynucleotides 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, and as linear or covalently closed circular molecules. Nucleic acids can be isolated. The term "isolated nucleic acid" means that the nucleic acid has been (i) amplified in vitro, for example via polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified by separation, for example by cleavage and gel electrophoresis, (iv) synthesized, for example by chemical synthesis, or (vi) extracted from a sample. Nucleic acids can be used for introduction into cells (i.e., transfection of cells) in the form of RNA, specifically prepared by in vitro transcription from a DNA template. The RNA can be further modified by stabilizing sequences, capping, and polyadenylation prior to application.

[0170] The term "sample" can include whole blood, plasma, serum, buffy coat, body fluids, lymphocytes, tissue, amniotic fluid, cultured cells, etc. The term "sample" can also refer to urine samples, saliva samples, cell-free DNA, and specimens from the skin, mucosa, or other body regions or surfaces being tested by swab. In various embodiments, the means used to collect the sample can contain a preservative. The preservative can include preservatives such as hydrochloric acid, boric acid, acetic acid, toluene, or thymol.

[0171] Nucleic acids can be extracted from a sample by any method known in the art, including using an organic solvent such as a mixture of phenol and chloroform, followed by precipitation with ethanol. An example of another method for extracting cell-free nucleic acids includes, for example, using polylysine-coated silica particles. Alternatively, cell-free DNA can be extracted using a commercially available kit such as the QIAamp® DNA Mini Kit (Qiagen, Germantown, Maryland).

[0172] The extracted nucleic acid is amplified by any of the alternative methods for amplification well-known in the art, including, for example, the following: Luminex's Xmap® technology (which can simultaneously analyze up to 500 bioassays through reading biological tests on the surface of micro-polystyrene beads); multiplex PCR (which can amplify multiple DNA sequences simultaneously); multiplex ligation-dependent probe amplification (MLPA) (which amplifies multiple targets using a pair of primers); quantitative PCR (qPCR) (which measures and quantifies amplification in real time); ligation chain reaction (LCR) (which amplifies using primers that cover the entire sequence, thereby preventing amplification of sequences with mutations); rolling circle amplification (RCA) (which ligates both ends of a sequence by a ligase and then amplifies the circular DNA); helicase-dependent amplification (HDA) (which utilizes a helicase for separation of double-stranded DNA); loop-mediated isothermal amplification (LAMP) (which uses a DNA polymerase with high strand displacement activity); nucleic acid sequence-based amplification (designed specifically for RNA targets); strand displacement amplification (SDA) (which utilizes a strand-displacing DNA polymerase and initiates replication at a nick generated by a strand-limiting restriction endonuclease or nicking enzyme at a site included in the primer); and multiple displacement amplification (MDA) (which is based on the use of a high-processivity strand-displacing DNA polymerase derived from bacteriophage O29). The amplification methods used herein have already been used and tested and are well-known in the art.

[0173] As used herein, "amplified DNA" or "PCR product" refers to an amplified DNA fragment of a determined size. Various techniques for detecting PCR products are available and are well known in the art. Detection methods for PCR products include, but are not limited to, the following: gel electrophoresis (using agarose or polyacrylamide gels, with ethidium bromide staining (DNA intercalating dye), labeled probes (radioactive or non-radioactive labels, Southern blotting), labeled deoxyribonucleotides (for direct incorporation of radioactive or non-radioactive labels), or silver staining (for direct visualization of amplified PCR products)); restriction endonuclease digestion (utilizing agarose or polyacrylamide gels, or high performance liquid chromatography (HPLC)); dot blot (hybridizing amplified DNA with a specific labeled probe (radioactive or non-radioactive label)); high pressure liquid chromatography (using ultraviolet detection); electrochemiluminescence (a combination of voltage-induced chemical reaction / photon detection); and direct sequencing (using radioactive or fluorescently labeled deoxyribonucleotides to determine the exact nucleotide sequence of the DNA fragment of interest); oligoligation assay (OLA), PCR, qPCR, DNA sequencing, fluorescence, gel electrophoresis, magnetic beads, allele-specific primer extension (ASPE), and / or direct hybridization.

[0174] Generally, nucleic acids can be extracted, isolated, amplified, or analyzed by various techniques such as those described below: Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, Woodbury, NY 2,028 pages (2012); or U.S. Patent No. 7,957,913; U.S. Patent No. 7,776,616; U.S. Patent No. 5,234,809; U.S. Published Patent Application No. 2010 / 0285578; and U.S. Published Patent Application No. 2002 / 0190663.

[0175] Examples of nucleic acid analysis include, but are not limited to, sequencing and DNA-protein interactions. Sequencing can be performed by any method known in the art. DNA sequencing techniques include the following: classical dideoxy sequencing reaction (Sanger method) (using labeled terminators or primers and gel separation on a slab or capillary), and next-generation sequencing methods such as sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, Illumina / Solexa sequencing, allele-specific hybridization to a library of labeled oligonucleotide probes, sequencing by synthesis using allele-specific hybridization to a library of labeled clones (subsequent ligation is performed), real-time monitoring of incorporation of labeled nucleotides during the polymerization step, polony sequencing, and SOLiD sequencing. Isolated molecules can be sequenced by sequential or single-round extension reactions using polymerase or ligase, and by single-round or sequential differential hybridization using a library of probes.

[0176] DNA-protein interactions can be analyzed by chromatin immunoprecipitation sequencing (also known as ChIP-seq). ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. This can be used to accurately map the global binding sites for any target protein. Conventionally, ChIP-on-chip has been the most common technique used for the study of these protein-DNA interactions. The ChIP method is well known and described in the art.

[0177] In one embodiment, the present invention provides a vector comprising the polynucleotide described herein, wherein the vector is an expression vector selected from the group consisting of mammalian expression vectors, yeast expression vectors, insect expression vectors, and bacterial expression vectors.

[0178] There is provided a vector comprising a polynucleotide encoding the heavy chain and / or light chain of the antibody described herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, the vector comprises a first polynucleotide sequence encoding 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, the heavy chain and the light chain are expressed as part of a single polypeptide, for example, when the antibody is a scFv.

[0179] In some embodiments, the first vector comprises a polynucleotide encoding the heavy chain and the second vector comprises a polynucleotide encoding the light chain. In some embodiments, the first vector and the second vector are transfected into a host cell in similar amounts (e.g., similar molar amounts or similar mass amounts). In some embodiments, the first vector and the second vector are transfected into a host cell at a molar ratio or mass ratio between 5:1 and 1:5. In some embodiments, a mass ratio between 1:1 and 1:5 is used for the vector encoding the heavy chain and the vector encoding the light chain. In some embodiments, a mass ratio of 1:2 is used for the vector encoding the heavy chain and the vector encoding the light chain.

[0180] In some embodiments, a vector optimized for the expression of a polypeptide in CHO cells or CHO-derived cells, or in NSO cells, is selected. Examples of vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20: 880-889 (2004). In some embodiments, a vector is selected for the in vivo expression of a Gal9 antibody and antigen-binding fragments thereof in an animal (including a human). In some such embodiments, the expression of the polypeptide(s) is under the control of a promoter(s) that functions tissue-specifically. For example, liver-specific promoters are described, for example, in PCT Publication No. WO 2006 / 076288.

[0181] The terms "vector", "expression vector", or "plasmid DNA" refer herein to a recombinant nucleic acid construct that has been engineered by human intervention. The recombinant nucleic acid construct contains two or more nucleotide sequences, and the products thereof may be linked in a form not found naturally within a cell. Specifically, two or more nucleotide sequences may be operably linked, for example, a gene encoding a protein of interest, one or more protein tags, functional domains, and the like.

[0182] Vectors suitable for use in the preparation of proteins and / or protein conjugates include those selected from the following: baculovirus, phage, plasmid, phagemid, cosmid, fosmid, bacterial artificial chromosome, viral DNA, P1-based artificial chromosome, yeast plasmid, and yeast artificial chromosome. For example, viral DNA vectors can be selected from the following: vaccinia virus, adenovirus, fowl pox virus, pseudorabies virus, and derivatives of SV40. One type of vector is the genome-integrating vector, i.e., the "integrated vector", which has the potential to integrate into the chromosomal DNA of the host cell. Another type of vector is the episomal vector, which is, for example, a nucleic acid capable of extrachromosomal replication. A vector capable of expressing a gene in an operably linked state is referred to herein as an "expression vector". Viral vectors include the following: adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, vaccinia virus, measles virus, herpes virus, and bovine papillomavirus vector (see Kay et al., Proc. Natl. Acad. Sci. USA 94:12744-12746 (1997) for a review of viral and non-viral vectors). Viral vectors are modified such that the original tropism and pathogenicity of the virus are altered or removed. The genome of the virus can also be modified to enhance its infectivity and to be adapted for packaging nucleic acids encoding the polypeptide of interest.

[0183] The nucleic acid construct of the present invention can be introduced into a cell, and the cell can be modified so that a chimeric protein can be expressed in the cell. Various methods suitable for introducing nucleic acids into cells are known in the art and include viral and non-viral mediated techniques. Examples of typical non-viral mediated techniques include, but are not limited to: electroporation, calcium phosphate-mediated introduction, nucleofection, sonoporation, heat shock, magnetofection, liposome-mediated introduction, microinjection, microprojectile-mediated introduction (nanoparticles), cationic polymer-mediated introduction (DEAE-dextran, polyethyleneimine, polyethylene glycol (PEG), etc.), or cell fusion. Other transfection methods include, for example, the following proprietary transfection reagents: Lipofectamine™, Dojindo Hilymax™, Fugene™, jetPEI™, Effectene™, and DreamFect™.

[0184] The nucleic acid construct of the present invention can be introduced into a host cell, and the cell can be modified so that a chimeric protein can be expressed in the cell. Various host cells suitable for the expression of chimeric proteins are known in the art. Examples of typical cells used for transfection include, but are not limited to: bacterial cells, eukaryotic cells, yeast cells, insect cells, or plant cells. For example, Escherichia coli (E. coli), Bacillus, Streptomyces, Pichia pastoris, Salmonella typhimurium, Drosophila S2, Spodoptera SJ9, CHO, COS (e.g., COS-7), 3T3-F442A, HeLa, HUVEC, HUAEC, NIH 3T3, Jurkat, 293, 293H, or 293F.

[0185] 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 (e.g., yeast), plant cells, insect cells, and mammalian cells. Such expression can be carried out, for example, according to procedures known in the art. Examples of eukaryotic cells that can be used for the expression of 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); PER.C6® cells (Crucell); and NSO cells. In some embodiments, the heavy and / or light chains of the antibodies described herein can be expressed in yeast. See, for example, U.S. Patent Publication No. US 2006 / 0270045 A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform the desired post-translational modifications on the heavy and / or light chains of the Gal9 antibody. For example, in some embodiments, CHO cells produce polypeptides with a higher level of sialylation compared to the same polypeptide produced in 293 cells.

[0186] The introduction of one or more nucleic acids into the desired host cell can be accomplished by any method including, but not limited to: calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, etc. Non-limiting examples of methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press (2001). The nucleic acids can be transfected transiently or stably into the desired host cell according to any suitable method.

[0187] In some embodiments, one or more polypeptides can be produced in vivo in an animal modified or transfected with one or more nucleic acid molecules encoding the polypeptide, according to any suitable method.

[0188] The polynucleotide can be delivered to cells (e.g., a plurality of different cells or cell types, including target cells or cell types and / or non-target cell types) by a vector (e.g., an expression vector). Examples of vectors include, but are not limited to: (a) non-viral vectors (e.g., 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 (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, and AAV vectors). Viral vectors have a number of advantages for nucleic acid delivery, including high infectivity and / or tropism for specific target cells or tissues. In some cases, viral vectors can be used for delivery of the polynucleotides described herein.

[0189] In another embodiment, the invention is a method of restoring or promoting the proliferation of effector T cells, enhancing the activity of effector T cells, and / or identifying and treating a subject, comprising: (i) determining the level of Gal9 in a sample from the subject, and (ii) administering to the subject having a Gal9 level higher than a reference level of Gal9 an effective amount of either the isolated monoclonal antibody or an antigen-binding fragment thereof described herein, thereby providing a method of restoring or promoting the proliferation of effector T cells, enhancing the activity of effector T cells, and / or identifying and treating a subject.

[0190] The term "disease associated with the suppressive activity of regulatory T lymphocytes" means any disease (other than autoimmune diseases) in which the suppressive activity of regulatory T lymphocytes plays a role, particularly by promoting the development or persistence of the disease. Specifically, it has been shown that the suppressive activity of regulatory T lymphocytes promotes the development of tumors. Therefore, the present invention more specifically targets cancers in which the suppressive activity of T lymphocytes plays a role.

[0191] In pathological situations, Tregs cause inappropriate immunosuppression, which can, for example, promote tumor growth. Tregs are associated with reducing the anti-tumor immune response and promoting the development of many cancer types, particularly by inappropriately inhibiting the activity of effector T lymphocytes.

[0192] During activation, Gal9 is directly expressed by Tregs, while it is expressed very weakly or not at all by effector T lymphocytes. Therefore, targeting Gal9, for example, by using a Gal9-specific antibody, can specifically inhibit the suppressive activity of regulatory T lymphocytes without the risk of causing depletion of effector T lymphocytes. The antibodies according to the present invention are directed against Gal9 and inhibit the suppressive activity of regulatory T lymphocytes, and thus can be used for the treatment of diseases or conditions associated with the suppressive activity of regulatory T lymphocytes, specifically for the treatment of cancer.

[0193] Cancers that can be treated by the method / use of the present invention include those in which regulatory T lymphocytes exert their suppressive activity, for example, cancers in which a relatively large amount of regulatory T lymphocytes are present in the tumor tissue or in the circulation. The proliferation of regulatory T lymphocytes (which can be measured by the frequency of Tregs) generally correlates with an increase in Treg activation. The frequency of regulatory T lymphocytes can be evaluated by any method known in the art, for example, by flow cytometry (FACS) analysis of intratumoral lymphocytes or circulating lymphocytes, or by immunohistological staining of tumor tissue.

[0194] In one aspect, the subject is diagnosed with cancer, at risk of developing cancer, or has cancer recurrence.

[0195] In another aspect, the reference level of Gal9 is the level of Gal9 measured in a sample from a healthy or control individual.

[0196] As used herein, "reference sample", "reference cell", or "reference tissue" refers to a sample, cell, or tissue obtained from a source known or believed not to be affected by the disease or condition to be identified using the methods or compositions of the present invention. In one embodiment, the reference sample, reference cell, or reference tissue is obtained from a healthy body part of the same subject or patient for whom the disease or condition is to be identified using the compositions or methods of the present invention. In one embodiment, the reference sample, reference cell, or reference tissue is obtained from a healthy body part of at least one individual who is not the subject or patient for whom the disease or condition is to be identified using the compositions or methods of the present invention. In some embodiments, the reference sample, reference cell, or reference tissue was previously collected from a patient before the onset of the disease or condition, or at an early stage of the disease or condition.

[0197] In some aspects, the determining step includes comparing the level of Gal9 in the sample to the reference level.

[0198] 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 lymphoma, Hodgkin lymphoma, mycosis fungoides, non-Hodgkin lymphoma, primary central nervous system lymphoma, Sézary syndrome, cutaneous T-cell lymphoma, Waldenström 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 cancer, ovarian cancer, endometrial cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, gastric cancer, gastroesophageal cancer, esophageal cancer, cervical cancer, head and neck squamous cell carcinoma, or prostate cancer.

[0199] 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.

[0200] 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 an anti-PD-1. In other aspects, the immuno-oncology agent is an anti-glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) antibody.

[0201] [Additional Embodiments] Embodiment 1. An isolated monoclonal antibody or antigen-binding fragment thereof comprising: (a) an HCVR comprising (i) an HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), where 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) an HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), where X4 is selected from E, W, or V; and (iii) an HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) LCVR, which includes: (i) Amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LCVR CDR1 sequence having LA (SEQ ID NO: 13), 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; 10 is selected from N, S, P, or A; X 11 is selected from N or H; X 12 is selected from Q, N, or Y; X 13 is selected from K or R; and X 14 is selected from Y or H; (ii) amino acid sequence WX 15 SX 16 RX 17 X 18 LCVR CDR2 sequence, wherein X 15 is selected from A or G; X 16 is selected from T, N, M, or A; X 17 is selected from G or E; and X 18 is selected from S, E, Y, P, or T; and (iii) amino acid sequence QQYYX 19 X 20 LCVR CDR3 sequence having PFT (SEQ ID NO: 30), where X 19 is selected from S or F; and X 20 is selected from Y or F; the antibody or antigen-binding fragment thereof specifically binds to galectin-9 (Gal9); Provided, however, that the antibody excludes antibodies comprising 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). An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0202] Embodiment 2. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 1, wherein: (a) An HCVR comprising the following (i) An HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), where X1 is selected from T or E; and X2 is selected from T or G; and X3 is E; (ii) An HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), where X4 is selected from W or V; and (iii) An HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) An LCVR comprising the following (i) An LCVR CDR1 sequence having the amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13), where X5 is Q; X6 is S; X7 is L; X8 is Y; X9 is selected from S or P; X 10 is N; X 11 is N; X 12 is Q; X 13 is K; and X 14 is Y; (ii) An amino acid sequence WX 15 SX 16 RX17 X 18 having an LCVR CDR2 sequence, where X 15 is A; X 16 is T; X 17 is G; and X 18 is selected from S or E; and (iii) an amino acid sequence QQYYX 19 X 20 having an LCVR CDR3 sequence of PFT (SEQ ID NO: 30), where X 19 is S; and X 20 is selected from Y or F; and the antibody or antigen-binding fragment thereof specifically binds to Gal9, an isolated monoclonal antibody or antigen-binding fragment thereof.

[0203] Embodiment 3. An isolated monoclonal antibody or antigen-binding fragment thereof according to Embodiment 1 or 2, wherein: (A) an HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 3, the HCVR CDR2 sequence of SEQ ID NO: 10, and the HCVR CDR3 sequence of SEQ ID NO: 12; and an LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 15, the LCVR CDR2 sequence of SEQ ID NO: 23, and the LCVR CDR3 sequence of SEQ ID NO: 32; or (B) an HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 4, the HCVR CDR2 sequence of SEQ ID NO: 11, and the HCVR CDR3 sequence of SEQ ID NO: 12; and a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 14, the LCVR CDR2 sequence of SEQ ID NO: 24, and the LCVR CDR3 sequence of SEQ ID NO: 31; and the antibody or antigen-binding fragment thereof specifically binds to Gal9, an isolated monoclonal antibody or antigen-binding fragment thereof.

[0204] Embodiment 4. An isolated monoclonal antibody or antigen-binding fragment thereof according to Embodiment 1, wherein: (a) an HCVR comprising the following (i) An HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), where 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) An HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), where X4 is E; and (iii) An HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) An LCVR comprising: (i) An LCVR CDR1 sequence having the amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13), where 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 is selected from S, P, or A; X 11 is 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) An LCVR CDR2 sequence having the amino acid sequence WX 15 SX 16 RX 17 X 18 where X 15 is selected from A or G; X 16 is selected from T, N, M, or A; X 17 is selected from G or E; and X 18 is selected from S, Y, P, or T; and (iii) An LCVR CDR3 sequence having the amino acid sequence QQYYX 19 X 20 PFT (SEQ ID NO: 30), where X 19 is F; and X 20which comprises "is Y"; the antibody or antigen-binding fragment thereof specifically binds to Gal9, an isolated monoclonal antibody or antigen-binding fragment thereof.

[0205] Embodiment 5. An isolated monoclonal antibody or antigen-binding fragment thereof according to Embodiment 1 or 4, wherein: (C) a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 5, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 16, the LCVR CDR2 sequence of SEQ ID NO: 25, and the LCVR CDR3 sequence of SEQ ID NO: 33; or (D) a HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 6, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and a LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 17, the LCVR CDR2 sequence of SEQ ID NO: 26, and the LCVR CDR3 sequence of SEQ ID NO: 33; or (E) a HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 7, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and a LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 18, the LCVR CDR2 sequence of SEQ ID NO: 27, and the LCVR CDR3 sequence of SEQ ID NO: 33; or (F) a HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 7, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and a LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 19, the LCVR CDR2 sequence of SEQ ID NO: 28, and the LCVR CDR3 sequence of SEQ ID NO: 33; or (G)HCVRs that contain the HCVR CDR1 sequence of SEQ ID NO: 7, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and LCVRs that contain the LCVR CDR1 sequence of SEQ ID NO: 20, the LCVR CDR2 sequence of SEQ ID NO: 29, and the LCVR CDR3 sequence of SEQ ID NO: 33; including, the antibody or antigen-binding fragment thereof specifically binds to Gal9, an isolated monoclonal antibody or antigen-binding fragment thereof.

[0206] Embodiment 6. The isolated monoclonal antibody or antigen-binding fragment thereof according to Embodiment 1, wherein: (a) HCVR containing the following (i) an HCVR CDR1 sequence having the amino acid sequence GYX1FX2X3YTIH (SEQ ID NO: 1), where X1 is selected from E or P; X2 is selected from T or S; and X3 is selected from E or S; (ii) an HCVR CDR2 sequence having the amino acid sequence WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8), where X4 is selected from W or E; and (iii) an HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) a light chain variable region (LCVR) containing the following: (i) an LCVR CDR1 sequence having the amino acid sequence KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13), where 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 is selected from N or S; X 11 is N; X 12 is selected from Q or N; X 13 is K; and X 14 is Y; (ii) the amino acid sequence WX15 SX 16 RX 17 X 18 An LCVR CDR2 sequence having X, where X 15 is selected from A or G; X 16 is selected from T or N; X 17 is selected from G or E; and X 18 is S; and (iii) an amino acid sequence QQYYX 19 X 20 An LCVR CDR3 sequence having PFT (SEQ ID NO: 30), where X 19 is selected from S or F; and X 20 is selected from Y or F; and includes The antibody or antigen-binding fragment thereof specifically binds to Gal9, An isolated monoclonal antibody or antigen-binding fragment thereof.

[0207] Embodiment 7. An isolated monoclonal antibody or antigen-binding fragment thereof according to Embodiment 1 or 6, wherein: (A) An HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 3, the HCVR CDR2 sequence of SEQ ID NO: 10, and the HCVR CDR3 sequence of SEQ ID NO: 12; and an LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 15, the LCVR CDR2 sequence of SEQ ID NO: 23, and the LCVR CDR3 sequence of SEQ ID NO: 32; or (C) An HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 5, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 16, the LCVR CDR2 sequence of SEQ ID NO: 25, and the LCVR CDR3 sequence of SEQ ID NO: 33; and includes The antibody or antigen-binding fragment thereof specifically binds to Gal9, An isolated monoclonal antibody or antigen-binding fragment thereof.

[0208] Embodiment 8. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising: an HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 3, the HCVR CDR2 sequence of SEQ ID NO: 10, and the HCVR CDR3 sequence of SEQ ID NO: 12; and an LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 15, the LCVR CDR2 sequence of SEQ ID NO: 23, and the LCVR CDR3 sequence of SEQ ID NO: 32, wherein said antibody or antigen-binding fragment thereof specifically binds to Gal9, an isolated monoclonal antibody or antigen-binding fragment thereof.

[0209] Embodiment 9. The isolated monoclonal antibody or antigen-binding fragment thereof according to Embodiment 8, wherein: the HCVR sequence of said antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 68, and may further comprise 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, an isolated monoclonal antibody or antigen-binding fragment thereof.

[0210] Embodiment 10. The isolated monoclonal antibody or antigen-binding fragment thereof according to Embodiment 8, wherein: the HCVR sequence of said antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 69, and may further comprise 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, an isolated monoclonal antibody or antigen-binding fragment thereof.

[0211] Embodiment 11. The isolated monoclonal antibody or antigen-binding fragment thereof according to Embodiment 8, wherein: the HCVR sequence of said antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 70, and may further comprise 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, An isolated monoclonal antibody or antigen-binding fragment thereof.

[0212] Embodiment 12. An isolated monoclonal antibody or antigen-binding fragment thereof according to 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; An isolated monoclonal antibody or antigen-binding fragment thereof.

[0213] Embodiment 13. An isolated monoclonal antibody or antigen-binding fragment thereof according to embodiment 8, wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises an HFR3 sequence of SEQ ID NO: 77, and optionally further comprises an HFR1 sequence of SEQ ID NO: 66, an HFR2 sequence of SEQ ID NO: 67, and / or an HFR4 sequence of SEQ ID NO: 71; An isolated monoclonal antibody or antigen-binding fragment thereof.

[0214] Embodiment 14. An isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 8 to 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 HFR4 sequence of SEQ ID NO: 75; An isolated monoclonal antibody or antigen-binding fragment thereof.

[0215] Embodiment 15. An isolated monoclonal antibody or antigen-binding fragment thereof according to embodiment 8, wherein: said HCVR sequence is SEQ ID NO: 39, and / or said LCVR sequence is SEQ ID NO: 41; An isolated monoclonal antibody or antigen-binding fragment thereof.

[0216] Embodiment 16. An isolated monoclonal antibody or antigen-binding fragment thereof according to Embodiment 8 or 5, wherein: wherein: the HCVR sequence is SEQ ID NO: 39, and the LCVR sequence is SEQ ID NO: 41; An isolated monoclonal antibody or antigen-binding fragment thereof.

[0217] Embodiment 17. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region (HCVR) comprising the HCVR CDR1 sequence of SEQ ID NO: 4, the HCVR CDR2 sequence of SEQ ID NO: 11, and the HCVR CDR3 sequence of SEQ ID NO: 12; and a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 14, the LCVR CDR2 sequence of SEQ ID NO: 24, and the LCVR CDR3 sequence of SEQ ID NO: 31, wherein the antibody or antigen-binding fragment thereof specifically binds to galectin-9 (Gal9). An isolated monoclonal antibody or antigen-binding fragment thereof.

[0218] Embodiment 18. An isolated monoclonal antibody or antigen-binding fragment thereof according to 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 may further comprise 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. An isolated monoclonal antibody or antigen-binding fragment thereof.

[0219] Embodiment 19. An isolated monoclonal antibody or antigen-binding fragment thereof according to 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 may further comprise 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. An isolated monoclonal antibody or antigen-binding fragment thereof.

[0220] Embodiment 20. An isolated monoclonal antibody or antigen-binding fragment thereof as described in Embodiment 17, wherein here: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 70, and may further comprise 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, Isolated monoclonal antibody or antigen-binding fragment thereof.

[0221] Embodiment 21. An isolated monoclonal antibody or antigen-binding fragment thereof as described in Embodiment 17, wherein here: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 76, and may further comprise 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, Isolated monoclonal antibody or antigen-binding fragment thereof.

[0222] Embodiment 22. An isolated monoclonal antibody or antigen-binding fragment thereof as described in Embodiment 17, wherein here: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 77, and may further comprise 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, Isolated monoclonal antibody or antigen-binding fragment thereof.

[0223] Embodiment 23. An isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of Embodiments 17 to 22, wherein here: the LCVR sequence of the antibody or antigen-binding fragment thereof 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 HFR4 sequence of SEQ ID NO: 75, Isolated monoclonal antibody or antigen-binding fragment thereof. [[ID=,31]]

[0224] Embodiment 24. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 17, wherein the HCVR sequence is SEQ ID NO: 43, and / or the LCVR sequence is SEQ ID NO: 45, an isolated monoclonal antibody or an antigen-binding fragment thereof.

[0225] Embodiment 25. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 17 or 24, wherein the HCVR sequence is SEQ ID NO: 43 and the LCVR sequence is SEQ ID NO: 45, an isolated monoclonal antibody or an antigen-binding fragment thereof.

[0226] Embodiment 26. An isolated monoclonal antibody or an antigen-binding fragment thereof, comprising: a HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 5, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and a light chain variable region (LCVR) comprising the LCVR CDR1 sequence of SEQ ID NO: 16, the LCVR CDR2 sequence of SEQ ID NO: 25, and the LCVR CDR3 sequence of SEQ ID NO: 33, wherein the antibody or its antigen-binding fragment specifically binds to Gal9, an isolated monoclonal antibody or an antigen-binding fragment thereof.

[0227] Embodiment 27. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 26, wherein the HCVR sequence of the antibody or its antigen-binding fragment further comprises the HFR3 sequence of SEQ ID NO: 68, and may further comprise 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, an isolated monoclonal antibody or an antigen-binding fragment thereof.

[0228] Embodiment 28. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 26, wherein wherein: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 69, and may further comprise 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. An isolated monoclonal antibody or antigen-binding fragment thereof.

[0229] Embodiment 29. An isolated monoclonal antibody or antigen-binding fragment thereof according to 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 may further comprise 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. An isolated monoclonal antibody or antigen-binding fragment thereof.

[0230] Embodiment 30. An isolated monoclonal antibody or antigen-binding fragment thereof according to 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 may further comprise 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. An isolated monoclonal antibody or antigen-binding fragment thereof.

[0231] Embodiment 31. An isolated monoclonal antibody or antigen-binding fragment thereof according to 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 may further comprise 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. An isolated monoclonal antibody or antigen-binding fragment thereof.

[0232] Embodiment 32. An isolated monoclonal antibody or antigen-binding fragment thereof according to any one of Embodiments 26 to 31, wherein the LCVR sequence of the antibody or antigen-binding fragment thereof 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 HFR4 sequence of SEQ ID NO: 75 An isolated monoclonal antibody or antigen-binding fragment thereof.

[0233] Embodiment 33. An isolated monoclonal antibody or antigen-binding fragment thereof according to Embodiment 26, wherein the HCVR sequence is SEQ ID NO: 47 and / or the LCVR sequence is SEQ ID NO: 49 An isolated monoclonal antibody or antigen-binding fragment thereof.

[0234] Embodiment 34. An isolated monoclonal antibody or antigen-binding fragment thereof according to Embodiment 26 or 33, wherein the HCVR sequence is SEQ ID NO: 47 and the LCVR sequence is SEQ ID NO: 49 An isolated monoclonal antibody or antigen-binding fragment thereof.

[0235] Embodiment 35. An isolated monoclonal antibody or antigen-binding fragment thereof, comprising: an HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 7, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and an LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 18, the LCVR CDR2 sequence of SEQ ID NO: 27, and the LCVR CDR3 sequence of SEQ ID NO: 33, wherein the antibody or antigen-binding fragment thereof specifically binds to galectin-9 (Gal9). An isolated monoclonal antibody or antigen-binding fragment thereof.

[0236] Embodiment 36. An isolated monoclonal antibody or antigen-binding fragment thereof according to Embodiment 35, wherein Here: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 68, and may further comprise 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. An isolated monoclonal antibody or antigen-binding fragment thereof.

[0237] Embodiment 37. An isolated monoclonal antibody or antigen-binding fragment thereof according to Embodiment 35, Here: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 69, and may further comprise 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. An isolated monoclonal antibody or antigen-binding fragment thereof.

[0238] Embodiment 38. An isolated monoclonal antibody or antigen-binding fragment thereof according to Embodiment 35, Here: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 70, and may further comprise 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. An isolated monoclonal antibody or antigen-binding fragment thereof.

[0239] Embodiment 39. An isolated monoclonal antibody or antigen-binding fragment thereof according to Embodiment 35, Here: the HCVR sequence of the antibody or antigen-binding fragment thereof further comprises the HFR3 sequence of SEQ ID NO: 76, and may further comprise 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. An isolated monoclonal antibody or antigen-binding fragment thereof.

[0240] Embodiment 40. An isolated monoclonal antibody or antigen-binding fragment thereof according to Embodiment 35, Here: the HCVR sequence of the antibody or its antigen-binding fragment further includes the HFR3 sequence of SEQ ID NO: 77, and may further include 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. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0241] Embodiment 41. An isolated monoclonal antibody or an antigen-binding fragment thereof according to any one of Embodiments 35 to 40, Here: the LCVR sequence of the antibody or its antigen-binding fragment further includes 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 HFR4 sequence of SEQ ID NO: 75. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0242] Embodiment 42. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 35, Here, the HCVR sequence is SEQ ID NO: 51, and / or the LCVR sequence is SEQ ID NO: 53. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0243] Embodiment 43. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 35 or 42, Here, the HCVR sequence is SEQ ID NO: 51, and the LCVR sequence is SEQ ID NO: 53. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0244] Embodiment 44. An isolated monoclonal antibody or an antigen-binding fragment thereof, comprising: An HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 7, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and an LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 18, the LCVR CDR2 sequence of SEQ ID NO: 27, and the LCVR CDR3 sequence of SEQ ID NO: 33. The antibody or antigen-binding fragment thereof specifically binds to galectin-9 (Gal9), an isolated monoclonal antibody or antigen-binding fragment thereof.

[0245] Embodiment 45. An isolated monoclonal antibody or antigen-binding fragment thereof according to 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 may further comprise 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, an isolated monoclonal antibody or antigen-binding fragment thereof.

[0246] Embodiment 46. An isolated monoclonal antibody or antigen-binding fragment thereof according to 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 may further comprise 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, an isolated monoclonal antibody or antigen-binding fragment thereof.

[0247] Embodiment 47. An isolated monoclonal antibody or antigen-binding fragment thereof according to 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 may further comprise 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, an isolated monoclonal antibody or antigen-binding fragment thereof.

[0248] Embodiment 48. An isolated monoclonal antibody or antigen-binding fragment thereof according to Embodiment 44, Here: the HCVR sequence of the antibody or its antigen-binding fragment further comprises the HFR3 sequence of SEQ ID NO: 76, and may further comprise 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. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0249] Embodiment 49. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 44, wherein Here: the HCVR sequence of the antibody or its antigen-binding fragment further comprises the HFR3 sequence of SEQ ID NO: 77, and may further comprise 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. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0250] Embodiment 50. An isolated monoclonal antibody or an antigen-binding fragment thereof according to any one of Embodiments 44 to 49, wherein Here: the LCVR sequence of the antibody or its antigen-binding fragment 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 HFR4 sequence of SEQ ID NO: 75. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0251] Embodiment 51. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 44, wherein the HCVR sequence is SEQ ID NO: 55, and / or the LCVR sequence is SEQ ID NO: 57. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0252] Embodiment 52. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 44 or 51, wherein the HCVR sequence is SEQ ID NO: 55 and the LCVR sequence is SEQ ID NO: 57. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0253] Embodiment 53. An isolated monoclonal antibody or an antigen-binding fragment thereof, wherein: it is an HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 7, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and an LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 19, the LCVR CDR2 sequence of SEQ ID NO: 28, and the LCVR CDR3 sequence of SEQ ID NO: 33, the antibody or its antigen-binding fragment specifically binds to galectin-9 (Gal9), An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0254] Embodiment 54. The isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 53, wherein: here: the HCVR sequence of the antibody or its antigen-binding fragment further comprises the HFR3 sequence of SEQ ID NO: 68, and may further comprise 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, An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0255] Embodiment 55. The isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 53, wherein: here: the HCVR sequence of the antibody or its antigen-binding fragment further comprises the HFR3 sequence of SEQ ID NO: 69, and may further comprise 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, An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0256] Embodiment 56. The isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 53, wherein: Here: the HCVR sequence of the antibody or its antigen-binding fragment further comprises the HFR3 sequence of SEQ ID NO: 70, and may further comprise 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. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0257] Embodiment 57. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 53, Here: the HCVR sequence of the antibody or its antigen-binding fragment further comprises the HFR3 sequence of SEQ ID NO: 76, and may further comprise 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. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0258] Embodiment 58. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 53, Here: the HCVR sequence of the antibody or its antigen-binding fragment further comprises the HFR3 sequence of SEQ ID NO: 77, and may further comprise 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. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0259] Embodiment 59. An isolated monoclonal antibody or an antigen-binding fragment thereof according to any one of Embodiments 53 to 58, Here: the LCVR sequence of the antibody or its antigen-binding fragment 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 HFR4 sequence of SEQ ID NO: 75. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0260] Embodiment 60. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 53, Here, the HCVR sequence is SEQ ID NO: 59, and / or the LCVR sequence is SEQ ID NO: 61. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0261] Embodiment 61. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 53 or 60, where the HCVR sequence is SEQ ID NO: 59 and the LCVR sequence is SEQ ID NO: 61. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0262] Embodiment 62. An isolated monoclonal antibody or an antigen-binding fragment thereof, comprising: an HCVR comprising the HCVR CDR1 sequence of SEQ ID NO: 7, the HCVR CDR2 sequence of SEQ ID NO: 9, and the HCVR CDR3 sequence of SEQ ID NO: 12; and an LCVR comprising the LCVR CDR1 sequence of SEQ ID NO: 20, the LCVR CDR2 sequence of SEQ ID NO: 29, and the LCVR CDR3 sequence of SEQ ID NO: 33, wherein the antibody or its antigen-binding fragment specifically binds to galectin-9 (Gal9). An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0263] Embodiment 63. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 62, wherein: the HCVR sequence of the antibody or its antigen-binding fragment further comprises the HFR3 sequence of SEQ ID NO: 68, and may further comprise 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. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0264] Embodiment 64. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 62, Here: the HCVR sequence of the antibody or its antigen-binding fragment further comprises the HFR3 sequence of SEQ ID NO: 69, and may further comprise 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. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0265] Embodiment 65. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 62, Here: the HCVR sequence of the antibody or its antigen-binding fragment further comprises the HFR3 sequence of SEQ ID NO: 70, and may further comprise 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. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0266] Embodiment 66. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 62, Here: the HCVR sequence of the antibody or its antigen-binding fragment further comprises the HFR3 sequence of SEQ ID NO: 76, and may further comprise 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. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0267] Embodiment 67. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment 62, Here: the HCVR sequence of the antibody or its antigen-binding fragment further comprises the HFR3 sequence of SEQ ID NO: 77, and may further comprise 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. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0268] Embodiment 68. An isolated monoclonal antibody or an antigen-binding fragment thereof according to any one of Embodiments 62 to 67, Here: the LCVR sequence of the antibody or its antigen-binding fragment 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 HFR4 sequence of SEQ ID NO: 75. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0269] Embodiment No. 69. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment No. 62, wherein the HCVR sequence is SEQ ID NO: 63 and / or the LCVR sequence is SEQ ID NO: 65. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0270] Embodiment No. 70. An isolated monoclonal antibody or an antigen-binding fragment thereof according to Embodiment No. 62 or 69, wherein the HCVR sequence is SEQ ID NO: 63 and the LCVR sequence is SEQ ID NO: 65. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0271] Embodiment No. 71. An isolated monoclonal antibody or an antigen-binding fragment thereof according to any one of Embodiments No. 1 to 71, wherein the antibody is a humanized antibody. An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0272] Embodiment No. 72. An isolated monoclonal antibody or an antigen-binding fragment thereof according to any one of Embodiments No. 1 to 71, wherein the antigen-binding fragment is Fab, Fab′, F(ab′)2, F d , single-chain Fv, i.e., scFv, disulfide-bonded F v, a V-NAR domain, IgNar, an intrabody, IgGACH2, a minibody, F(ab′)3, a tetrabody, a tribody, a diabody, a domain antibody (dAb), DVD-Ig, Fcab, mAb2, (scFv)2, a tandem scFv, DART (registered trademark), TandAb, a nanobody, or an scFv-Fc, An isolated monoclonal antibody or an antigen-binding fragment thereof.

[0273] 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.

[0274] 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.

[0275] 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.

[0276] 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.

[0277] 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.

[0278] 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.

[0279] 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.

[0280] Embodiment 80. An isolated monoclonal antibody or an antigen-binding fragment thereof according to any one of Embodiments 1 to 79, wherein the monoclonal antibody or its antigen-binding fragment is K dless 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 and binds to human Gal9, an isolated monoclonal antibody or an antigen-binding fragment thereof.

[0281] Embodiment 81. An isolated monoclonal antibody or an antigen-binding fragment thereof according to any one of Embodiments 1 to 80, which binds to Gal9 and inhibits the binding of Gal9 to a Gal9 receptor (e.g., TIM3 or CD44), an isolated monoclonal antibody or an antigen-binding fragment thereof.

[0282] Embodiment 82. An isolated monoclonal antibody or an antigen-binding fragment thereof according to any one of Embodiments 1 to 80, which binds to Gal9 and does not inhibit the binding of Gal9 to a Gal9 receptor (e.g., TIM3 or CD44), an isolated monoclonal antibody or an antigen-binding fragment thereof.

[0283] Embodiment 83. An isolated monoclonal antibody or an antigen-binding fragment thereof according to any one of Embodiments 1 to 82, which arrests Gal9-induced Th1 apoptosis in T cells (e.g., CD4 + T cells), an isolated monoclonal antibody or an antigen-binding fragment thereof.

[0284] Embodiment 84. An isolated monoclonal antibody or an antigen-binding fragment thereof according to any one of Embodiments 1 to 83, which suppresses Gal9-induced Treg proliferation, an isolated monoclonal antibody or an antigen-binding fragment thereof.

[0285] Embodiment 85. A method of treating cancer in a subject in need thereof, The method includes administering to the subject an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 84, thereby treating cancer in the subject. Method.

[0286] 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 includes administering to the subject an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 84, thereby treating cancer in the subject. Method.

[0287] Embodiment 87. A method for treating cancer in a subject in need thereof, The method includes administering to the subject an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 84, and an anti-PD-1 antibody, thereby treating cancer in the subject. Method.

[0288] 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 includes administering to the subject an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 84, an anti-PD-1 antibody, and an anti-GITR antibody, thereby treating cancer in the subject. Method.

[0289] Embodiment 89. A method for treating cancer in a subject in need thereof, The method includes administering to the subject an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 84, and an anti-PD-1 antibody, thereby treating cancer in the subject. Method.

[0290] 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, wherein the method comprises administering to the subject an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 69, an anti-PD-1 antibody, and an anti-GITR antibody, thereby treating cancer in the subject. Method.

[0291] Embodiment 91. The method according to Embodiment 85, 87, or 89, wherein the cancer is a hematological cancer or a solid tumor. Method.

[0292] Embodiment 92. The method according to Embodiment 91, wherein the hematological 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, Sézary syndrome, cutaneous T-cell lymphoma, Waldenström macroglobulinemia, diffuse large B-cell lymphoma (DLBCL), and multiple myeloma. Method.

[0293] Embodiment 93. The method according to Embodiment 91, wherein the solid tumor is breast cancer, head and neck cancer, lung cancer, melanoma, uveal melanoma, colorectal cancer, renal cancer, urothelial cancer, ovarian cancer, endometrial cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, gastric cancer, gastroesophageal cancer, esophageal cancer, cervical cancer, head and neck squamous cell carcinoma, or prostate cancer. Method.

[0294] Embodiment 94. The method according to any one of Embodiments 85, 87, 89, or 91 to 93, 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. Method.

[0295] Embodiment 95. The method according to embodiment 94, wherein the checkpoint inhibitor is an anti-PD-1. Method.

[0296] Embodiment 96. The method according to embodiment 94, wherein the immuno-oncology agent is an anti-glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) antibody. Method.

[0297] Embodiment 97. A polynucleotide encoding a heavy chain or a light chain, or an antigen-binding portion thereof, according to any one of embodiments 1 to 84.

[0298] Embodiment 98. A vector comprising the polynucleotide according to 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. Vector.

[0299] Embodiment 99. A method of restoring or promoting the proliferation of effector T cells, enhancing the activity of effector T cells, and / or identifying and treating cancer in a subject, comprising: (i) determining the level of Gal9 in a sample from the subject, and (ii) administering to a subject having a Gal9 level higher than a reference level of Gal9 an effective amount of the isolated monoclonal antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 84, thereby restoring or promoting the proliferation of effector T cells, enhancing the activity of effector T cells, and / or identifying and treating the subject. Method

[0300] Embodiment 100. A method of restoring or promoting the proliferation of effector T cells, enhancing the activity of effector T cells, and / or identifying and treating cancer in a subject, comprising: (i) determining the level of Gal9 in a sample from the subject, and (ii) administering to a subject having a Gal9 level higher than a reference level of Gal9, an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 84, and an anti-PD-1 antibody, thereby restoring or promoting the proliferation of effector T cells, enhancing the activity of effector T cells, and / or identifying and treating the subject. Method

[0301] Embodiment 101. A method of restoring or promoting the proliferation of effector T cells, enhancing the activity of effector T cells, and / or identifying and treating cancer in a subject, comprising: (i) determining the level of Gal9 in a sample from the subject, and (ii) administering to a subject having a Gal9 level higher than a reference level of Gal9, an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 84, and an anti-PD-1 antibody and an anti-GITR antibody, thereby restoring or promoting the proliferation of effector T cells, enhancing the activity of effector T cells, and / or identifying and treating the subject. Method

[0302] Embodiment 102. The method according to any one of Embodiments 99 to 101, wherein the subject is diagnosed with cancer, at risk of developing cancer, or has cancer recurrence. Method

[0303] Embodiment 103. The method according to any one of Embodiments 99 to 102, wherein the reference level of Gal9 is the level of Gal9 measured in a sample from a healthy or control individual, method.

[0304] Embodiment 104. The method according to any one of Embodiments 99 to 103, wherein the step of determining includes a step of comparing the level of Gal9 in the sample with the reference level, method.

[0305] Embodiment 105. The method according to any one of Embodiments 99 to 104, wherein the cancer is a blood cancer or a solid tumor, method.

[0306] Embodiment 106. The method according to 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, Sézary syndrome, cutaneous T-cell lymphoma, Waldenström macroglobulinemia, diffuse large B-cell lymphoma (DLBCL), and multiple myeloma, method.

[0307] Embodiment 107. The method according to Embodiment 105, wherein the solid tumor is breast cancer, head and neck cancer, lung cancer, melanoma, uveal melanoma, colorectal cancer, renal cancer, urothelial cancer, ovarian cancer, endometrial cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, gastric cancer, gastroesophageal cancer, esophageal cancer, cervical cancer, head and neck squamous cell carcinoma, or prostate cancer, method.

[0308] Embodiment 108. The method according to any one of Embodiments 99 to 107, The sample is a blood sample, a plasma sample, a serum sample, a tumor biopsy sample, or a mononuclear cell (MNC) sample derived from bone marrow (BM). Method.

[0309] Embodiment 109. A method according to any one of Embodiment 99 or Embodiments 102 to 106, wherein 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. Method.

[0310] Embodiment 110. A method according to Embodiment 109, wherein the checkpoint inhibitor is an anti-PD-1. Method.

[0311] Embodiment 111. A method according to claim 110, wherein the immuno-oncology agent is an anti-glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) antibody. Method.

Example

[0312] [Example 1: Affinity Maturation of Humanized Anti-Gal9 Antibody] To develop anti-Gal9 antibodies with improved binding affinity for human Gal9 compared to the original Ab A anti-Gal9 antibody, random mutations were introduced in three CDRs: H1-H2-L2 or L1-L3-H3, and a library of variants was generated.

[0313] Two antibodies, anti-Gal9 antibody Ab 001 and Ab 002, with the most excellent binding affinity compared to the Ab A anti-Gal9 antibody were identified. The sequence alignments of the heavy and light chains are shown in FIGS. 1A and 1B, respectively, where the CDRs are boxed in the sequences.

[0314] The affinity binding of the antibodies was measured using surface plasmon resonance and solution equilibrium assays. As shown in Table 2, anti-Gal9 antibodies Ab 001 and Ab 002 were found to have a higher binding affinity than Ab A antibody, as indicated by the decrease in K D values.

[0315] JPEG2025524650000003.jpg53166

[0316] Separately, the yeast display method was used to develop anti-Gal9 antibodies with improved binding affinity for human Gal9 compared to the original Ab A anti-Gal9 antibody.

[0317] Five antibodies with the best binding affinity compared to Ab A anti-Gal9 antibody were identified: anti-Gal9 antibodies Ab 003, Ab 004, Ab 005, Ab 006, and Ab 007. The sequence alignments of the heavy and light chains are shown in FIGS. 1C and 1D, respectively, where the CDRs are boxed in the sequences.

[0318] The affinity binding of the antibodies was measured using surface plasmon resonance and solution equilibrium assays. As shown in Table 3, anti-Gal9 antibodies Ab 003-007 were found to have a higher binding affinity than antibody Ab A, as indicated by the decrease in K D values. Surprisingly, at least antibodies Ab 003-005 and Ab 007 were also shown to substantially improve their affinity for mouse Gal9 compared to antibody Ab A.

[0319] JPEG2025524650000004.jpg65166

[0320] [Example 2: Effect of anti-Gal9 antibodies on Gal9-induced apoptosis] 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 each antibody on Gal9 - induced apoptosis was performed.

[0321] As shown in Figure 2B, the affinity - matured anti - Gal9 antibody was found to more potently inhibit apoptosis of CD4+ T cells compared to 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.

[0322] The T - cell apoptosis assay was used to evaluate the efficacy of the Ab 002 antibody in inhibiting Gal9 - induced apoptosis.

[0323] Further characterization of the antibody includes the following: evaluation of cross - reactivity of the antibody to other Gal9 family members, evaluation of biochemical and biophysical properties (e.g., including aggregation data), evaluation of deamination, oxidation, proteolysis (especially before and after heat stress).

[0324] The ability of the anti - Gal9 antibody Ab 001 to inhibit Gal9 - induced CD4 + and / or CD8 + T - cell apoptosis was evaluated by flow - cytometry analysis of annexin V + and propidium iodide + (PI) double - positive cells.

[0325] CD4 + or CD8 + T - cells were activated with Gal9 together with anti - CD3 and anti - CD28, which are mitogenic stimuli. The activated T - cells were co - cultured with the anti - Gal9 antibody Ab 001 or an isotype control. As shown in Figures 2C and 2D, inclusion of Ab 001 led to a significant decrease in apoptosis of both CD4 + and CD8 + T - cells compared to the isotype control.

[0326] [Example 3: Evaluation of AML Apoptosis Ex Vivo] The effects of anti-Gal9 affinity matured antibodies (Ab 001 and Ab 002) were evaluated in the apoptosis of leukemia stem cells (LSC, CD34+, CD38-), and compared with the effects induced by a non-affinity matured anti-Gal9 antibody (Ab A).

[0327] AML bone marrow samples were collected and cryopreserved until use in the experiment. LSC cells were sorted from the samples based on their CD34 and CD38 expression.

[0328] The expression levels of Tim3 / Gal9 were evaluated on the cell surface, and the same was true for the levels of secreted / releases Gal9. Cells were cultured in suspension for 96 hours in the presence or absence of the antibody, and apoptosis was evaluated by flow cytometry. The internal controls included other existing anti-Gal9 antibodies, a positive control (aGal9), and a negative control.

[0329] [Example 4: Evaluation of the In Vivo Effects of Affinity Matured Anti-Gal9 Antibodies on AML Patient Samples] To evaluate the in vivo efficacy of the affinity matured anti-Gal9 antibody, transplantation was performed in NOD scid gamma (NSG) immunodeficient mice using an in vivo systemic model of AML. The FLT3, IDH1 / 2, and TP53 genes were sequenced to identify mutations; cytogenetic, clinical, and immunophenotypic evaluations were performed.

[0330] Systemic engraftment kinetics were measured.

[0331] The transplantation period lasted approximately 6 - 12 weeks, after which AML engraftment was evaluated by flow cytometry detecting CD45 / CD33 cells. Thereafter, AML therapeutic agents were tested. The endpoint of this experiment was survival, at which time flow cytometry analysis of splenocytes, whole blood, and bone marrow CD45 / CD33 was performed, using animals treated with the anti-Gal9 antibody alone or in combination with chemotherapy.

[0332] As an alternative to the humanized AML model, the use of an autologous AML platform enables the testing of immuno-oncology therapeutic agents (either as monotherapies or in combination) in an autologous co-culture system, which utilizes Champions' well-characterized AML patient model bank. Primary AML cells from patients are co-cultured with autologous T cells in the presence or absence of the antibody of interest (either alone or in combination with another therapeutic agent). The cells are cultured in enriched growth medium, and various parameters are evaluated, including phenotypic analysis by flow cytometry, T cell activation, cytokine production by Luminex, and AML frequency.

[0333] As a proof-of-concept experiment, a solid tumor model using the MC38 mouse syngeneic model (colorectal cancer) is used for further in vivo analysis. Monotherapy tests using anti-Gal9 antibody or anti-PD-1 antibody are conducted in MC38 animals, and dose-response tests are also performed. The combination test of anti-Gal9 antibody and anti-PD-1 is conducted to understand the mechanism of action in vivo.

[0334] [Example 5: Evaluation of the Gal9-induced TIM-3 dimerization inhibitory effect of Ab 001] The dimerization of TIM-3 was evaluated by the Eurofins DiscoverX assay. This assay relies on enzyme fragment complementation, where the enzyme β-galactosidase is split into two fragments, an enzyme acceptor (EA) and an enzyme donor (ED), and fused to the cytoplasmic tail of the receptor TIM-3. When the ligand Gal9 binds to its receptor TIM-3, the receptor homodimerizes, and the aggregation of EA and ED metabolizes the assay substrate, generating quantifiable photons.

[0335] As shown in Figure 3, the inclusion of anti-Gal9 antibodies Ab001 or Ab003 inhibited the ability of soluble Gal9 to form TIM-3 homodimers, as indicated by the quenching of emitted photons, compared to the isotype control.

[0336] [Example 6: Efficacy of anti-Gal9 antibody as combination therapy in a proof-of-concept mouse model] The anti-Gal9 antibody was tested alone or in combination with checkpoint inhibitor antibodies, and its efficacy in inhibiting tumor growth in the MC38 syngeneic colon cancer model was evaluated. The antibodies tested included anti-GITR, anti-PD-1, and anti-Gal9. Two anti-Gal9 antibodies were used in separate experiments, a commercially available anti-Gal9 antibody (anti-Gal9 Ab RG9-1) and one of the antibodies of the present invention (Ab 003).

[0337] All monotherapies appeared to result in minimal tumor growth inhibition compared to isotype controls in this model, while combination therapies of anti-Gal9 antibody with anti-GITR or anti-PD-1 resulted in moderate tumor growth inhibition (49 - 51% tumor growth inhibition with the dual therapy of Ab 003). Triple therapy with anti-Gal9, anti-GITR, and anti-PD-1 resulted in higher tumor growth inhibition (67% tumor growth inhibition with the triple therapy of Ab 003).

[0338] [Example 7: Clinical outline of Phase 1a / 1b in recurrent / refractory advanced solid tumors] In the Phase 1a / 1b clinical trial, the maximum tolerated dose (MTD) of the anti-Gal9 antibody of the present invention is evaluated in patients with recurrent / refractory advanced solid tumors according to the BOIN design method.

[0339] The indications are selected based on Gal9 expression levels and are as follows: colorectal cancer, gastric cancer, gastroesophageal junction adenocarcinoma, esophageal cancer, urothelial cancer, cervical cancer, and head and neck squamous cell carcinoma. The dose escalation phase is a standard 3+3 Bayesian optimal interval design. Once the recommended dose is established, in the dose expansion phase, two dose levels of anti-Gal9 are administered with or without pembrolizumab until the minimum safe biological effective dose is confirmed. A Phase II trial is planned after completion of the dose expansion phase.

[0340] The present invention has been described with reference to the above embodiments, but it will be understood that modifications and variations are included within the spirit and scope of the present invention. Therefore, the present invention is limited only by the following claims.

[0341] [Sequence Listing:] JPEG2025524650000005.jpg13166

[0342] >VH-CDR1 GYX1FX2X3YTIH (SEQ ID NO: 1) X1 is T, E, P, or A X2 is T, G, or S X3 is E, S, or D

[0343] >VH-CDR2 WFYPGSGSTX4YAQKFQG (SEQ ID NO: 8) X4 is E, W, or V

[0344] >VH-CDR3 HGGYDGFDY (SEQ ID NO: 12)

[0345] >VL-CDR1 KSSX5X6X7LX8X9X 10 X 11 X 12 X 13 NX 14 LA (SEQ ID NO: 13) X5 is Q or R X6 is S or N X7 is L, V, or I X8 is Y or W X9 is S or P X 10 is N, S, P, or A X 11 is N or H X 12 is Q, N, or Y X 13 is K or R X 14 is Y or H

[0346] >VL-CDR2 WX 15 SX 16 RX 17 X 18 X 15 is A or G X 16 is T, N, M, or A X 17 is G or E X 18 is S, E, Y, P, or T

[0347] >VL-CDR3 QQYYX 19 X 20 PFT (SEQ ID NO: 30) X 19 is S or F X 20 is Y or F

[0348] >Ab A HC (SEQ ID NO: 34) JPEG2025524650000006.jpg62166

[0349] >Ab A VH (SEQ ID NO: 35) JPEG2025524650000007.jpg21166

[0350] >Ab A VH-CDR1 (SEQ ID NO: 2) GYTFTEYTIH

[0351] >Ab A VH-CDR2 (SEQ ID NO: 9) WFYPGSGSTEYAQKFQG

[0352] >Ab A VH-CDR3 (SEQ ID NO: 12) HGGYDGFDY

[0353] >Ab A HFR1 (SEQ ID NO: 66) QVQLVQSGAEVKKPGASVKVSCKAS

[0354] >Ab A HFR2 (Accession No. 67) WVRQAPGQGLEWMG

[0355] >Ab A HFR3 (Accession No. 68) RVTMTADTSISTAYMELSRLRSDDTAVYFCER

[0356] >Ab A HFR4 (Accession No. 71) WGQGTTVTVSS

[0357] >Ab A LC (Accession No. 36) JPEG2025524650000008.jpg34166

[0358] >Ab A VL (Accession No. 37) JPEG2025524650000009.jpg14166 I

[0359] >Ab A VL-CDR1 (Accession No. 14) KSSQSLLYSNNQKNYLA

[0360] >Ab A VL-CDR2 (Accession No. 22) WASTRGS

[0361] >Ab A VL-CDR3 (Accession No. 31) QQYYSYPFT

[0362] >Ab A LFR1 (Accession No. 72) DIVMTQSPDSLAVSLGERATINC

[0363] >Ab A LFR2 (Accession No. 73) WYQQKPGQPPKLLIY

[0364] >Ab A LFR3 (Accession No. 74) GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC

[0365] >Ab A LFR4 (Accession No. 75) FGGGTKVEIK

[0366] >Ab 001 HC (Accession No. 38) JPEG2025524650000010.jpg62166

[0367] >Ab 001 VH (Accession No. 39) JPEG2025524650000011.jpg21166

[0368] >Ab 001 VH-CDR1 (Accession No. 3) GYEFTEYTIH

[0369] >Ab 001 VH-CDR2 (Accession No. 10) WFYPGSGSTWYAQKFQG

[0370] >Ab 001 VH-CDR3 (Accession No. 12) HGGYDGFDY

[0371] >Ab 001 HFR1 (Accession No. 66) QVQLVQSGAEVKKPGASVKVSCKAS

[0372] >Ab 001 HFR2 (Accession No. 67) WVRQAPGQGLEWMG

[0373] >Ab 001 HFR3 (Accession No. 68) RVTMTADTSISTAYMELSRLRSDDTAVYFCER

[0374] >Ab 001 HFR4 (Accession No. 71) WGQGTTVTVSS

[0375] >Ab 001 LC (Accession No. 40) JPEG2025524650000012.jpg34166

[0376] >Ab 001 VL (Accession No. 41) JPEG2025524650000013.jpg14166

[0377] >Ab 001 VL-CDR1 (Accession No. 15) KSSQSLLYPNNQKNYLA

[0378] >Ab 001 VL-CDR2 (Accession No. 23) WASTRGS

[0379] >Ab 001 VL-CDR3 (Accession No. 32) QQYYSFPFT

[0380] >Ab 001 LFR1 (Accession No. 72) DIVMTQSPDSLAVSLGERATINC

[0381] >Ab 001 LFR2 (Accession No. 73) WYQQKPGQPPKLLIY

[0382] >Ab 001 LFR3 (Accession No. 74) GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC

[0383] >Ab 001 LFR4 (Accession No. 75) FGGGTKVEIK

[0384] >Ab 002 HC (Accession No. 42) JPEG2025524650000014.jpg62166

[0385] >Ab 002 VH (Accession No. 43) JPEG2025524650000015.jpg21166

[0386] >Ab 002 VH-CDR1 (Accession No. 4) GYTFGEYTIH

[0387] >Ab 002 VH-CDR2 (SEQ ID NO: 11) WFYPGSGSTVYAQKFQG

[0388] >Ab 002 VH-CDR3 (SEQ ID NO: 12) HGGYDGFDY

[0389] >Ab 002 HFR1 (SEQ ID NO: 66) QVQLVQSGAEVKKPGASVKVSCKAS

[0390] >Ab 002 HFR2 (SEQ ID NO: 67) WVRQAPGQGLEWMG

[0391] >Ab 002 HFR3 (SEQ ID NO: 68) RVTMTADTSISTAYMELSRLRSDDTAVYFCER

[0392] >Ab 002 HFR4 (SEQ ID NO: 71) WGQGTTVTVSS

[0393] >Ab 002 (E07) LC (SEQ ID NO: 44) JPEG2025524650000016.jpg34166

[0394] >Ab 002 VL (SEQ ID NO: 45) JPEG2025524650000017.jpg14166

[0395] >Ab 002 VL-CDR1 (SEQ ID NO: 14) KSSQSLLYSNNQKNYLA

[0396] >Ab 002 VL-CDR2 (SEQ ID NO: 24) WASTRGE

[0397] >Ab 002 VL-CDR3 (SEQ ID NO: 31) QQYYSYPFT

[0398] >Ab 002 LFR1 (Accession No. 72) DIVMTQSPDSLAVSLGERATINC

[0399] >Ab 002 LFR2 (Accession No. 73) WYQQKPGQPPKLLIY

[0400] >Ab 002 LFR3 (Accession No. 74) GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC

[0401] >Ab 002 LFR4 (Accession No. 75) FGGGTKVEIK

[0402] >Ab 003 HC (Accession No. 46) JPEG2025524650000018.jpg63166

[0403] >Ab 003 VH (Accession No. 47) JPEG2025524650000019.jpg20166

[0404] >Ab 003 VH-CDR1 (Accession No. 5) GYPFSSYTIH

[0405] 」 >Ab 003 VH-CDR2 (Accession No. 9) WFYPGSGSTEYAQKFQG

[0406] >Ab 003 VH-CDR3 (Accession No. 12) HGGYDGFDY

[0407] >Ab 003 HFR1 (Accession No. 66) QVQLVQSGAEVKKPGASVKVSCKAS

[0408] >Ab 003 HFR2 (Accession No. 67) WVRQAPGQGLEWMG

[0409] >Ab 003 HFR3 (Accession No. 69) RVTMTADTSISTAYMELSRLRSDDTAVYFCEV

[0410] >Ab 003 HFR4 (Accession No. 71) WGQGTTVTVSS

[0411] >Ab 003 LC (Accession No. 48) JPEG2025524650000020.jpg34166

[0412] >Ab 003 VL (Accession No. 49) JPEG2025524650000021.jpg21166

[0413] >Ab 003 VL-CDR1 (Accession No. 16) KSSQNVLWSSNNKNYLA

[0414] >Ab 003 VL-CDR2 (Accession No. 25) WGSNRES

[0415] >Ab 003 VL-CDR3 (Accession No. 33) QQYYFYPFT

[0416] >Ab 003 LFR1 (Accession No. 72) DIVMTQSPDSLAVSLGERATINC

[0417] >Ab 003 LFR2 (Accession No. 73) WYQQKPGQPPKLLIY

[0418] >Ab 003 LFR3 (Accession No. 74) GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC

[0419] >Ab 003 LFR4 (Accession No. 75) FGGGTKVEIK

[0420] >Ab 004 HC (Accession No. 50) JPEG2025524650000022.jpg62166

[0421] >Ab 004 VH (Accession No. 51) JPEG2025524650000023.jpg21166

[0422] >Ab 004 VH-CDR1 (Accession No. 6) GYAFTDYTIH

[0423] >Ab 004 VH-CDR2 (Accession No. 9) WFYPGSGSTEYAQKFQG

[0424] >Ab 004 VH-CDR3 (Accession No. 12) HGGYDGFDY

[0425] >Ab 004 HFR1 (Accession No. 66) QVQLVQSGAEVKKPGASVKVSCKAS

[0426] >Ab 004 HFR2 (Accession No. 67) WVRQAPGQGLEWMG

[0427] >Ab 004 HFR3 (Accession No. 76) RVTMTADTSISTAYMELSRLRSDDTAVYYCEV

[0428] >Ab 004 HFR4 (Accession No. 71) WGQGTTVTVSS

[0429] >Ab 004 LC (Accession No. 52) JPEG2025524650000024.jpg34166

[0430] >Ab 004 VL (Accession No. 53) JPEG2025524650000025.jpg14166

[0431] >Ab 004 VL-CDR1 (Accession No. 17) KSSRNILYSPHNKNYLA

[0432] >Ab 004 VL-CDR2 (Accession No. 26) WASMRGS

[0433] >Ab 004 VL-CDR3 (Accession No. 33) QQYYFYPFT

[0434] >Ab 004 LFR1 (Accession No. 72) DIVMTQSPDSLAVSLGERATINC

[0435] >Ab 004 LFR2 (Accession No. 73) WYQQKPGQPPKLLIY

[0436] >Ab 004 LFR3 (Accession No. 74) GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC

[0437] >Ab 004 LFR4 (Accession No. 75) FGGGTKVEIK

[0438] >Ab 005 HC (Accession No. 54) JPEG2025524650000026.jpg62166

[0439] >Ab 005 VH (Accession No. 55) JPEG2025524650000027.jpg20166

[0440] >Ab 005 VH-CDR1 (Accession No. 7) GYTFGDYTIH

[0441] >Ab 005 VH-CDR2 (SEQ ID NO: 9) WFYPGSGSTEYAQKFQG

[0442] >Ab 005 VH-CDR3 (SEQ ID NO: 12) HGGYDGFDY

[0443] >Ab 005 HFR1 (SEQ ID NO: 66) QVQLVQSGAEVKKPGASVKVSCKAS

[0444] >Ab 005 HFR2 (SEQ ID NO: 67) WVRQAPGQGLEWMG

[0445] >Ab 005 HFR3 (SEQ ID NO: 70) RVTMTADTSISTAYMELSRLRSDDTAVYYCEI

[0446] >Ab 005 HFR4 (SEQ ID NO: 71) WGQGTTVTVSS

[0447] >Ab 005 LC (SEQ ID NO: 56) JPEG2025524650000028.jpg35166

[0448] >Ab 005 VL (SEQ ID NO: 57) JPEG2025524650000029.jpg14166

[0449] >Ab 005 VL-CDR1 (SEQ ID NO: 18) KSSQSVLYSANNRNYLA

[0450] >Ab 005 VL-CDR2 (SEQ ID NO: 27) WGSAREY

[0451] >Ab 005 VL-CDR3 (SEQ ID NO: 33) QQYYFYPFT

[0452] >Ab 005 LFR1 (Accession No. 72) DIVMTQSPDSLAVSLGERATINC

[0453] >Ab 005 LFR2 (Accession No. 73) WYQQKPGQPPKLLIY

[0454] >Ab 005 LFR3 (Accession No. 74) GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC

[0455] >Ab 005 LFR4 (Accession No. 75) FGGGTKVEIK

[0456] >Ab 006 HC (Accession No. 58) JPEG2025524650000030.jpg62166

[0457] >Ab 006 VH (Accession No. 59) JPEG2025524650000031.jpg21166

[0458] >Ab 006 VH-CDR1 (Accession No. 7) GYTFGDYTIH

[0459] >Ab 006 VH-CDR2 (Accession No. 9) WFYPGSGSTEYAQKFQG

[0460] >Ab 006 VH-CDR3 (Accession No. 12) HGGYDGFDY

[0461] >Ab 006 HFR1 (Accession No. 66) QVQLVQSGAEVKKPGASVKVSCKAS

[0462] >Ab 006 HFR2 (Accession No. 67) WVRQAPGQGLEWMG

[0463] >Ab 006 HFR3 (Accession No. 77) RVTMTADTSISTAYMELSRLRSDDTAVYFCEI

[0464] >Ab 006 HFR4 (Accession No. 71) WGQGTTVTVSS

[0465] >Ab 006 LC (Accession No. 60) JPEG2025524650000032.jpg35166

[0466] >Ab 006 VL (Accession No. 61) JPEG2025524650000033.jpg15166

[0467] >Ab 006 VL-CDR1 (Accession No. 19) KSSQSLLYSSNYKNHLA

[0468] >Ab 006 VL-CDR2 (Accession No. 28) WGSTREP

[0469] >Ab 006 VL-CDR3 (Accession No. 33) QQYYFYPFT

[0470] >Ab 006 LFR1 (Accession No. 72) DIVMTQSPDSLAVSLGERATINC

[0471] >Ab 006 LFR2 (Accession No. 73) WYQQKPGQPPKLLIY

[0472] >Ab 006 LFR3 (Accession No. 74) GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC

[0473] >Ab 006 LFR4 (Accession No. 75) FGGGTKVEIK

[0474] >Ab 007 HC (Sequence number 62) JPEG2025524650000034.jpg61166

[0475] >Ab 007 VH (Sequence number 63) JPEG2025524650000035.jpg20166

[0476] >Ab 007 VH-CDR1 (Sequence number 7) GYTFGDYTIH

[0477] >Ab 007 VH-CDR2 (Sequence number 9) WFYPGSGSTEYAQKFQG

[0478] >Ab 007 VH-CDR3 (Sequence number 12) HGGYDGFDY

[0479] >Ab 007 HFR1 (Sequence number 66) QVQLVQSGAEVKKPGASVKVSCKAS

[0480] >Ab 007 HFR2 (Sequence number 67) WVRQAPGQGLEWMG

[0481] >Ab 007 HFR3 (Sequence number 70) RVTMTADTSISTAYMELSRLRSDDTAVYYCEI

[0482] >Ab 007 HFR4 (Sequence number 71) WGQGTTVTVSS

[0483] >Ab 007 LC (Sequence number 64) JPEG2025524650000036.jpg35166

[0484] >Ab 007 VL (Sequence number 65) JPEG2025524650000037.jpg13166

[0485] >Ab 007 VL-CDR1 (SEQ ID NO: 20) KSSQNVLWSSNNRNYLA

[0486] >Ab 007 VL-CDR2 (SEQ ID NO: 29) WGSTRET

[0487] >Ab 007 VL-CDR3 (SEQ ID NO: 33) QQYYFYPFT

[0488] >Ab 007 LFR1 (SEQ ID NO: 72) DIVMTQSPDSLAVSLGERATINC

[0489] >Ab 007 LFR2 (SEQ ID NO: 73) WYQQKPGQPPKLLIY

[0490] >Ab 007 LFR3 (SEQ ID NO: 74) GVPDRFSGSGSGTDFLTISSLQAEDVAVYYC

[0491] >Ab 007 LFR4 (SEQ ID NO: 75) FGGGTKVEIK

Claims

1. 1. An isolated monoclonal antibody or antigen-binding fragment thereof: (a) a heavy chain variable region (HCVR) comprising: (i) amino acid sequence GYX １ FX ２ X ３ HCVR CDR1 sequence having YTIH (SEQ ID NO: 1), Here, X １ is selected from T, E, P, or A; X ２ is selected from T, G, or S; and X ３ is selected from E, S, or D; (ii) amino acid sequence WFYPGSGSTX ４ HCVR CDR2 sequence having the sequence: YAQKFQG (SEQ ID NO: 8); Here, X ４ is selected from E, W, or V; and (iii) an HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) a light chain variable region (LCVR) comprising: (i) amino acid sequence KSSX ５ X ６ X ７ LX ８ X ９ X １０ X １１ X １２ X １３ NX １４ LCVR CDR1 sequence having: LA (SEQ ID NO: 13); Here, X ５ is selected from Q or R; X ６ is selected from S or N; X ７ is selected from L, V, or I; X ８ is selected from Y or W; X ９ is selected from S or P; X １０ is selected from N, S, P, or A; X １１ is selected from N or H; X １２ is selected from Q, N, or Y; X １３ is selected from K or R; and X １４ is selected from Y or H; (ii) the amino acid sequence WX １５ SX １６ RX １７ X １８ LCVR CDR2 sequence having the following sequence: Here, X １５ is selected from A or G; X １６ is selected from T, N, M, or A; X １７ is selected from G or E; and X １８ is selected from S, E, Y, P, or T; and (iii) the amino acid sequence QQYYX １９ X ２０ LCVR CDR3 sequence having PFT (SEQ ID NO: 30), Here, X １９ is selected from S or F; and X ２０ is selected from Y or F; Including, the antibody or antigen-binding fragment thereof specifically binds to galectin-9 (Gal9); With the proviso that the antibody excludes antibodies comprising 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). An isolated monoclonal antibody or antigen-binding fragment thereof.

2. 2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, comprising: (a) HCVRs that include: (i) amino acid sequence GYX １ FX ２ X ３ HCVR CDR1 sequence having YTIH (SEQ ID NO: 1), Here, X １ is selected from T or E; and X ２ is selected from T or G; and X ３ is E; (ii) amino acid sequence WFYPGSGSTX ４ HCVR CDR2 sequence having the sequence: YAQKFQG (SEQ ID NO: 8); Here, X ４ is selected from W or V; and (iii) an HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) LCVR, including: (i) amino acid sequence KSSX ５ X ６ X ７ LX ８ X ９ X １０ X １１ X １２ X １３ NX １４ LCVR CDR1 sequence having: LA (SEQ ID NO: 13); Here, X ５ is Q; X ６ is S; X ７ is L; X ８ is Y; X ９ is selected from S or P; X １０ is N; X １１ is N; X １２ is Q; X １３ is K; and X １４ is Y; (ii) the amino acid sequence WX １５ SX １６ RX １７ X １８ LCVR CDR2 sequence having the following sequence: Here, X １５ is A; X １６ is T; X １７ is G; and X １８ is selected from S or E; and (iii) the amino acid sequence QQYYX １９ X ２０ LCVR CDR3 sequence having PFT (SEQ ID NO: 30), Here, X １９ is S; and X ２０ is selected from Y or F; Including, The antibody or antigen-binding fragment thereof specifically binds to Gal9. An isolated monoclonal antibody or antigen-binding fragment thereof.

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

4. 4. An isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising: 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; An isolated monoclonal antibody or antigen-binding fragment thereof.

5. 5. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 4, 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 Including, An isolated monoclonal antibody or antigen-binding fragment thereof.

6. 2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, comprising: (a) HCVRs that include: (i) amino acid sequence GYX １ FX ２ X ３ HCVR CDR1 sequence having YTIH (SEQ ID NO: 1), Here, X １ is selected from T, P, or A; X ２ is selected from T, S, or G; and X ３ is selected from S or D; (ii) amino acid sequence WFYPGSGSTX ４ HCVR CDR2 sequence having the sequence: YAQKFQG (SEQ ID NO: 8); Here, X ４ is E; and (iii) an HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) LCVR, including: (i) amino acid sequence KSSX ５ X ６ X ７ LX ８ X ９ X １０ X １１ X １２ X １３ NX １４ LCVR CDR1 sequence having: LA (SEQ ID NO: 13); Here, X ５ is selected from Q or R; X ６ is selected from S or N; X ７ is selected from L, V, or I; X ８ is selected from Y or W; X ９ is S; X １０ is selected from S, P, or A; X １１ is 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) the amino acid sequence WX １５ SX １６ RX １７ X １８ LCVR CDR2 sequence having the following sequence: Here, X １５ is selected from A or G; X １６ is selected from T, N, M, or A; X １７ is selected from G or E; and X １８ is selected from S, Y, P, or T; and (iii) the amino acid sequence QQYYX １９ X ２０ LCVR CDR3 sequence having PFT (SEQ ID NO: 30), Here, X １９ is F; and X ２０ is Y; Including, The antibody or antigen-binding fragment thereof specifically binds to Gal9. An isolated monoclonal antibody or antigen-binding fragment thereof.

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

8. 8. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, 6, or 7, comprising: 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; An isolated monoclonal antibody or antigen-binding fragment thereof.

9. 9. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, 6, 7, or 8, 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 Including, An isolated monoclonal antibody or antigen-binding fragment thereof.

10. 2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, comprising: (a) HCVRs that include: (i) amino acid sequence GYX １ FX ２ X ３ HCVR CDR1 sequence having YTIH (SEQ ID NO: 1), Here, X １ is selected from E or P; X ２ is selected from T or S; and X ３ is selected from E or S; (ii) amino acid sequence WFYPGSGSTX ４ HCVR CDR2 sequence having the sequence: YAQKFQG (SEQ ID NO: 8); Here, X ４ is selected from W or E; and (iii) an HCVR CDR3 sequence having the amino acid sequence HGGYDGFDY (SEQ ID NO: 12); and (b) LCVR, including: (i) amino acid sequence KSSX ５ X ６ X ７ LX ８ X ９ X １０ X １１ X １２ X １３ NX １４ LCVR CDR1 sequence having: LA (SEQ ID NO: 13); Here, X ５ is Q; X ６ is selected from S or N; X ７ is selected from L or V; X ８ is selected from Y or W; X ９ is selected from P or S; X １０ is selected from N or S; X １１ is N; X １２ is selected from Q or N; X １３ is K; and X １４ is Y; (ii) the amino acid sequence WX １５ SX １６ RX １７ X １８ LCVR CDR2 sequence having the following sequence: Here, X １５ is selected from A or G; X １６ is selected from T or N; X １７ is selected from G or E; and X １８ is S; and (iii) the amino acid sequence QQYYX １９ X ２０ LCVR CDR3 sequence having PFT (SEQ ID NO: 30), Here, X １９ is selected from S or F; and X ２０ is selected from Y or F; Including, The antibody or antigen-binding fragment thereof specifically binds to Gal9. An isolated monoclonal antibody or antigen-binding fragment thereof.

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

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

13. 13. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 12, The antigen-binding fragments thereof include Fab, Fab', F(ab') ２ , F ｄ , single chain Fv or scFv, disulfide bond F ｖ , V-NAR domain, IgNar, intrabody, IgG ACH ２ , minibody, F(ab') ３ , tetrabodies, triabodies, diabodies, domain antibodies (dAbs), DVD-Igs, Fcabs, mAbs ２ , (scFv) ２ , tandem scFv, DART®, TandAb, nanobody, or scFv-Fc; An isolated monoclonal antibody or antigen-binding fragment thereof.

14. 14. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 13, The monoclonal antibody or antigen-binding fragment thereof is ｄ binds to human Gal9 at 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; An isolated monoclonal antibody or antigen-binding fragment thereof.

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

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

17. 17. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 16, T cells (e.g., CD4 ＋ reverse Gal9-induced Th1 apoptosis in T cells; An isolated monoclonal antibody or antigen-binding fragment thereof.

18. 18. An isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, suppress Gal9-induced Treg proliferation; An isolated monoclonal antibody or antigen-binding fragment thereof.

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

20. 20. The method of claim 19, the cancer is a blood cancer or a solid tumor; method.

21. 21. The method of claim 20, The hematological cancers are 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, 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. Selected from: method.

22. 21. The method of claim 20, The solid tumor is breast cancer, head and neck cancer, lung cancer, melanoma, uveal melanoma, colorectal cancer, renal cancer, urothelial cancer, 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; method.

23. A method according to any one of claims 19 to 22, comprising: 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. method.

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

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

26. A polynucleotide encoding the heavy or light chain, or an antigen-binding portion thereof, of any one of claims 1 to 13.

27. 27. A vector comprising the polynucleotide of claim 26, 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. vector.

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

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

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

31. A method according to any one of claims 28 to 30, comprising: The determining step includes comparing the level of Gal9 in the sample to the reference level. method.

32. A method according to any one of claims 28 to 31, comprising: the cancer is a blood cancer or a solid tumor; method.

33. 33. The method of claim 32, further comprising: The hematological cancers are 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, 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. Selected from: method.

34. 33. The method of claim 32, further comprising: The solid tumor is breast cancer, head and neck cancer, lung cancer, melanoma, uveal melanoma, colorectal cancer, renal cancer, urothelial cancer, 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; method.

35. A method according to any one of claims 28 to 34, comprising: 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. method.

36. A method according to any one of claims 28 to 35, comprising: 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. method.

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

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

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