Anti-galectin-9 antibodies and their therapeutic uses

Abstract

For example, disclosed herein are methods for treating solid tumors (e.g., pancreatic ductal adenocarcinoma (PDAC), colorectal carcinoma (CRC), hepatocellular carcinoma (HCC), cholangiocarcinoma (CAA), renal cell carcinoma (RCC), urothelial carcinoma, head and neck cancer, breast cancer, lung cancer, or other gastrointestinal solid tumors) using anti-galectin-9 antibodies as monotherapy or in combination with immune checkpoint inhibitors.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under U.S. patent law of U.S. Provisional Application No. 63 / 182,521, filed April 30, 2021, U.S. Provisional Application No. 63 / 193,357, filed May 26, 2021, and U.S. Provisional Application No. 63 / 313,879, filed February 25, 2022, each of which is incorporated by reference in its entirety herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on April 26, 2022, is named 112174-0211-NP009WO1_SEQ.txt and is 89,119 bytes in size. [Background technology]

[0003] The immune system holds remarkable potential to recognize and destroy cancer cells, but the complex network that controls tumor immune evasion is a broad obstacle to effective immune regulation (Martinez-Bosch N, et al., Immune Evasion in Pancreatic Cancer: From Mechanisms to Therapy. Cancers (Basel). 2018; 10(1)). Approved immuno-oncology (IO) drugs have incrementally improved survival in many tumor types (e.g., melanoma, lung cancer, kidney cancer, bladder cancer, some colon cancers, etc.) and are rapidly being incorporated as standard of care in addition to and in combination with surgery, chemotherapy, and radiation therapy. However, there remain significant gaps in the treatment and survival of several other advanced malignancies. For example, metastatic pancreatic ductal adenocarcinoma (PDAC or PDA), cholangiocarcinoma (CCA), and colorectal cancer (CRC) still have 5-year survival rates of less than 9%, less than 5%, and less than 15%, respectively. These gastrointestinal tumors are highly aggressive, with many patients presenting with advanced stage disease, and the efficacy of approved immunotherapies is suboptimal (Rizvi, et al., Cholangiocarcinoma-evolving concepts and therapeutic strategies; Nat Rev Clin Oncol. 2018;15(2):95-111; Kalyan, et al., Updates on immunotherapy for colorectal cancer; J Gastrointest Oncol. 2018;9(1):160-169).

[0004] The success of first generation checkpoint inhibitors (anti-PD-1, anti-PD-L1, and anti-CTLA4) has led to an explosion of efficacy and differentiation in new IO clinical trials (Holl et al., Examining Peripheral and Tumor Cellular Immunome in Patients with Cancer; Front Immunol. 2019;10:1767). Unfortunately, alongside the successes, there have also been many development failures, and thus, there remains a need for newer and more effective treatments.

[0005] Galectin-9 is a tandem repeat lectin consisting of two carbohydrate recognition domains (CRDs) and was first discovered and described in 1997 in patients suffering from Hodgkin's lymphoma (HL) (Tureci et al., J. Biol. Chem. 1997, 272, 6416-6422). It exists in three isoforms and can be located intracellularly or extracellularly. Elevated levels of galectin-9 have been observed in a wide range of cancers, including melanoma, Hodgkin's lymphoma, hepatocellular carcinoma, pancreatic cancer, gastric cancer, colon cancer, and renal clear cell carcinoma (Wdowiak et al. Int. J. Mol. Sci. 2018, 19, 210). In renal cancer, patients with high galectin-9 expression showed larger tumor size and more advanced disease progression (Kawashima et al.; BJU Int. 2014; 113: 320-332). In melanoma, galectin-9 is expressed in 57% of tumors and was significantly increased in the plasma of patients with advanced melanoma compared to healthy controls (Enninga et al., Melanoma Res. 2016 Oct;26(5):429-441). Many studies have demonstrated the utility of galectin-9 as a prognostic marker and, more recently, as a potential drug target (Enninga et al., 2016; Kawashima et al. BJU Int 2014;113:320-332; Kageshita et al., Int J Cancer. 2002 Jun 20;99(6):809-16, and references therein).

[0006] Galectin-9 has been described to play an important role in many cellular processes, such as adhesion, cancer cell aggregation, apoptosis, and chemotaxis. Recent studies have shown a role for galectin-9 in tumor-supportive immune regulation, for example, through negative regulation of Th1-type responses, Th2 polarization, and polarization of macrophages toward the M2 phenotype. This includes studies showing that galectin-9 is involved in the direct inactivation of T cells through its interaction with the T cell immunoglobulin and mucin protein 3 (TIM-3) receptor (Dardalhon et al., J Immunol., 2010, 185, 1383-1392; Sanchez-Fueyo et al., Nat Immunol., 2003, 4, 1093-1101).

[0007] Galectin-9 also plays a role in polarizing T cell differentiation towards a tumor suppressor phenotype, as well as promoting tolerogenic macrophage programming and adaptive immune suppression (Daley et al., Nat Med., 2017, 23, 556-567). In mouse models of pancreatic ductal adenocarcinoma (PDAC), blocking the checkpoint interaction between galectin-9 and its receptor Dectin-1, found on innate immune cells in the tumor microenvironment (TME), has been shown to increase antitumor immune responses in the pancreatic TME and slow tumor progression (Daley et al., Nat Med., 2017, 23, 556-567). Galectin-9 has also been shown to bind to CD206, a surface marker of M2 macrophages, resulting in reduced secretion of CVL22 (MDC), a macrophage-derived chemokine that is associated with improved survival and reduced risk of recurrence in lung cancer (Enninga et al, J Pathol. 2018 Aug;245(4):468-477). Summary of the Invention

[0008] The present disclosure is based, at least in part, on the development of a treatment regimen for solid tumors (e.g., metastatic solid tumors), such as pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), cholangiocarcinoma (CAA), renal cell carcinoma (RCC), urothelial tumors, head and neck cancer, breast cancer, lung cancer, or other GI solid tumors, comprising an antibody capable of binding to human galectin-9, either alone or in combination with a checkpoint inhibitor, such as an anti-PD-1 antibody. Alternatively or additionally, the present disclosure is based, at least in part, on the unexpected discovery that the anti-galectin-9 antibody G9.2-17 (IgG4) has a faster clearance rate in human subjects compared to other antibody therapeutics. Thus, a treatment regimen has been developed that includes a weekly dosing schedule that ensures a suitable plasma concentration, e.g., therapeutic systemic exposure level, of the anti-galectin-9 antibody to achieve a therapeutic effect.

[0009] Accordingly, provided herein are methods for treating solid tumors, the methods comprising administering to a subject in need thereof (e.g., a human patient with a target solid tumor) an effective amount of an antibody that binds to human galectin-9 (anti-galectin-9 antibody). The anti-galectin-9 antibody may be administered to the subject at a dose of about 0.2 mg / kg to about 32 mg / kg, e.g., 0.2 mg / kg, 0.63 mg / kg, 2 mg / kg, 6.3 mg / kg, 10 mg / kg, or 16 mg / kg, once per week to once per six weeks. In some embodiments, any of the anti-galectin-9 antibodies disclosed herein may be administered to the subject by intravenous infusion.

[0010] In some embodiments, an anti-galectin-9 antibody (e.g., G9.2-17(IgG4)) may be administered to a subject at a dose of 0.2 mg / kg, 0.63 mg / kg, 2 mg / kg, 6.3 mg / kg, 10 mg / kg, or 16 mg / kg, once every two weeks to once every four weeks. In some examples, an anti-galectin-9 antibody may be administered to a subject once every two weeks. In certain embodiments, an anti-galectin-9 antibody (e.g., G9.2-17(IgG4)) is administered to a subject at a dose of 10 mg / kg or 16 mg / kg, once every two weeks to once every four weeks (e.g., once every two weeks).

[0011] Alternatively, an anti-Gal-9 antibody such as G9.2-17(IgG4) can be administered to a subject at a dose of about 650 mg to about 1120 mg once every 2 to 6 weeks, for example, once every 2 weeks, once every 3 weeks, or once every 4 weeks. In some examples, an anti-Gal-9 antibody is administered to a subject at a dose of about 650 mg to about 700 mg once every 2 to 6 weeks, for example, once every 2 weeks, once every 3 weeks, or once every 4 weeks. In other examples, an anti-Gal-9 antibody is administered to a subject at a dose of about 1040 mg to about 1120 mg once every 2 to 6 weeks, for example, once every 2 weeks, once every 3 weeks, or once every 4 weeks.

[0012] In some embodiments, an anti-galectin-9 antibody (e.g., G9.2-17(IgG4)) is administered to a subject at a dose of 0.2 mg / kg, 0.63 mg / kg, 2 mg / kg, 6.3 mg / kg, 10 mg / kg, or 16 mg / kg once a week. In specific embodiments, an anti-galectin-9 antibody (e.g., G9.2-17(IgG4)) is administered to a subject at a dose of 10 mg / kg or 16 mg / kg once a week. Alternatively, an anti-Gal-9 antibody disclosed herein, e.g., G9.2-17(IgG4), may be administered to a subject at a dose of about 650 mg to about 1120 mg once a week. For example, an anti-Gal-9 antibody may be administered to a subject at a dose of 10 mg / kg once a week, or at a flat dose of about 650 to 700 mg once a week. Alternatively, the anti-galectin-9 antibody can be administered to a subject at a dose of 16 mg / kg once weekly, or at a flat dose of about 1040-1120 mg once weekly.

[0013] In some embodiments, the anti-galectin-9 antibody may include: (a) a light chain variable region (V) comprising a light chain (LC) complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1, a LC complementarity determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 2, and a LC complementarity determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3; L ), and (b) a heavy chain variable region (V) comprising a heavy chain (HC) complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 4, a HC complementarity determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 5, and a HC complementarity determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO: 6; H ).

[0014] In some instances, the V L comprises the amino acid sequence of SEQ ID NO: 8. Alternatively or additionally, V of the anti-galectin-9 antibody H comprises the amino acid sequence of SEQ ID NO:7.

[0015] In some cases, the anti-galectin-9 antibody is a full-length antibody, e.g., an IgG1 or IgG4 molecule. In some examples, the anti-galectin-9 antibody is a human IgG4 molecule. Such an IgG4 molecule may have a modified Fc region compared to a wild-type human IgG4 counterpart. In some examples, the modified Fc region comprises the amino acid sequence of SEQ ID NO: 14. In a specific example, the anti-galectin-9 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 15. Such an anti-galectin-9 antibody may be G9.2-17(IgG4) as disclosed herein.

[0016] In some embodiments, the solid tumor to be treated by any of the methods disclosed herein may be pancreatic ductal adenocarcinoma (PDAC), colorectal carcinoma (CRC), hepatocellular carcinoma (HCC), cholangiocarcinoma (CAA), renal cell carcinoma (RCC), urothelial, head and neck cancer, breast cancer, lung cancer, or other GI solid tumors. In some cases, the solid tumor is a metastatic tumor. In some embodiments, the method comprises administering to a subject suffering from a solid tumor, e.g., PDAC, CRC, HCC, or CCA, an effective amount of an antibody that binds to human galectin-9 (referred to herein as an anti-Gal9 antibody or anti-galectin-9 antibody). In some cases, the subject has one or more of the following characteristics: (i) no resectable cancer; (ii) no SARS-CoV-2 infection; (iii) no active brain metastasis or leptomeningeal metastasis; (iv) unresectable metastatic cancer, which is an adenocarcinoma, and optionally, a squamous cell carcinoma.

[0017] In some embodiments, the subject has not received other anti-cancer therapies simultaneously with the anti-galectin-9 antibody. Alternatively, the method may further include administering to the subject an immune checkpoint inhibitor. In some examples, the immune checkpoint inhibitor is an antibody that binds to PD-1. Examples include pembrolizumab, nivolumab, tislelizumab, dostallimab, or cemiplimab. In some cases, the subject has not been exposed to an anti-PD-1 or anti-PD-L1 agent in any prior line of therapy, is free of microsatellite instability (MSI-H) and / or mismatch repair deficient (dMMR), or a combination thereof.

[0018] In one example, the antibody that binds to PD-1 is nivolumab. In some cases, nivolumab is administered to the subject at a dose of 240 mg once every two weeks. In another example, the antibody that binds to PD-1 is tislelizumab. In some cases, tislelizumab is administered intravenously at a dose of about 200 mg once every three weeks, or at a dose of about 400 mg every six weeks.

[0019] In some embodiments, the anti-galectin-9 antibody is administered to the subject at about 0.2 mg / kg to about 32 mg / kg (e.g., at a dosage level of about 3 mg / kg to about 15 mg / kg or about 2 mg / kg to about 16 mg / kg or more, or at a dosage level of about 0.2 mg / kg to about 15 mg / kg or about 0.2 to about 16 mg / kg or more) once every 2 to 3 weeks. In some embodiments, the anti-galectin-9 antibody is administered to the subject at a dosage level selected from 0.2 mg / kg, 0.63 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 6.3 mg / kg, 8 mg / kg, 10 mg / kg, 12 mg / kg, or 16 mg / kg, or more. In some embodiments, the anti-galectin-9 antibody is administered to the subject at a dose selected from a dose level of 2 mg / kg, 4 mg / kg, 8 mg / kg, 12 mg / kg, or 16 mg / kg, or more. In some embodiments, the anti-galectin-9 antibody is administered to the subject at a dose selected from a dose level of 0.2 mg / kg, 0.63 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 6.3 mg / kg, 10 mg / kg, or 16 mg / kg, or more. In some embodiments, the antibody is administered once every two weeks. In some embodiments, the anti-galectin-9 antibody is administered to the subject at a dose selected from a dose level of 2 mg / kg, 4 mg / kg, 8 mg / kg, 12 mg / kg, or 16 mg / kg, or more, once every two weeks. In some embodiments, the anti-galectin-9 antibody is administered to the subject once every two weeks at a dose selected from a dose level of 0.2 mg / kg, 0.63 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 6.3 mg / kg, 10 mg / kg, or 16 mg / kg or more. In some embodiments, the anti-galectin-9 antibody is administered once every two weeks for one cycle, once every two weeks for two cycles, once every two weeks for three cycles, once every two weeks for four cycles, or once every two weeks for more than four cycles. In some embodiments, the treatment period is 0-3 months, 3-6 months, 12-24 months, or more. In some embodiments, the treatment period is 12-24 months or more.In some embodiments, the cycle spans a period of 3 to 6 months, or 6 to 12 months, or 12 to 24 months, or longer. In some embodiments, the length of the cycle is modified, e.g., temporarily or permanently, to a longer period, e.g., 3 or 4 weeks. In some embodiments, the anti-galectin-9 antibody is administered to the subject by intravenous infusion. In some embodiments, the cancer is a metastatic cancer, including metastatic cancer of any of the cancers mentioned above. In some embodiments, the treatment method comprising administering an anti-galectin-9 antibody does not include any other concomitant anti-cancer therapy.

[0020] In some embodiments, the method of treatment using an anti-galectin-9 antibody includes another concurrent anti-cancer therapy. Thus, in some embodiments, the method of treatment using an anti-galectin-9 antibody further includes administering an immune checkpoint inhibitor to the subject. In some embodiments, the immune checkpoint inhibitor is an antibody that binds to PD-1, such as pembrolizumab, nivolumab, tislelizumab, dostarlimab, or cemiplimab. In some embodiments, the antibody that binds to PD-1 is nivolumab, which is administered to the subject at a dose of 240 mg every two weeks. In some embodiments, the antibody that binds to PD-1 is nivolumab, which is administered to the subject at a dose of about 240 mg every two weeks or about 480 mg every four weeks. In some embodiments, the antibody that binds to PD-1 is prembrolizumab, which is administered to the subject at a dose of 200 mg every three weeks. In some embodiments, the antibody that binds to PD-1 is cemiplimab, which is administered at a dose of about 350 mg once every three weeks. In some embodiments, the antibody that binds to PD-1 is tislelizumab, which is administered at a dose of about 200 mg once every three weeks or about 400 mg every six weeks. In some embodiments, the antibody that binds to PD-1 is dostallimab, which is administered at a dose of about 500 mg once every three weeks or about 1000 mg once every six weeks. In some embodiments, the immune checkpoint inhibitor is administered by intravenous infusion.

[0021] In some cases, the subject (v) has not been exposed to an anti-PD-1 agent or an anti-PD-L1 agent in any prior line of therapy and does not have microsatellite instability (MSI-H) and / or mismatch repair deficient (dMMR), or a combination thereof. In some cases, the subject has microsatellite instability (MSI-H) and / or mismatch repair deficient (dMMR), or a combination thereof.

[0022] In some examples, the checkpoint inhibitor is administered to the subject on the same day that the subject is administered the anti-galectin-9 antibody. Alternatively, the checkpoint inhibitor and the anti-galectin-9 antibody are administered to the subject on two consecutive days. For example, administration of the checkpoint inhibitor is performed before administration of the anti-galectin-9 antibody, or vice versa.

[0023] In some embodiments, the subject has undergone one or more prior anti-cancer therapies.In some examples, the one or more prior anti-cancer therapies include chemotherapy, immunotherapy, radiation therapy, therapy that includes biological agents, or combinations thereof.In some cases, the subject has disease progression from one or more prior anti-cancer therapies or is resistant to one or more prior therapies.

[0024] In some cases, the subject is a human patient with elevated levels of galectin-9 compared to a control value. For example, the human patient has elevated serum or plasma levels of galectin-9 compared to a control value. In some examples, the human patient has cancer cells that express galectin-9. Alternatively or additionally, the human patient has immune cells that express galectin-9. In some examples, the cancer cells are in tumor organoids derived from the human patient. In some embodiments, the control value is based on a value obtained from a healthy human subject.

[0025] Any of the methods disclosed herein may further include monitoring the occurrence of adverse effects in the subject. In some examples, the methods may further include reducing the dose of the anti-galectin-9 antibody, the dose of the checkpoint inhibitor, or both, if side effects are observed.

[0026] In some embodiments, the subject is administered multiple doses of anti-galectin-9 antibody, with the later doses being higher than the earlier doses.

[0027] Also within the scope of the disclosure are pharmaceutical compositions for use in the treatment of solid tumors (e.g., as described herein, including metastatic solid tumors), and the use of any of the anti-galectin-9 antibodies, administered alone or in combination with a checkpoint inhibitor, such as any of the anti-PD-1 antibodies disclosed herein, for the manufacture of a medicament for treating a solid tumor.

[0028] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the invention will be apparent from the following drawings and detailed description of certain embodiments, and from the appended claims.

[0029] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0030] [Figure 1] FIG. 1 is a schematic diagram showing an exemplary study scheme. CRM: reassessment method; RP2D: recommended second dose; PK: pharmacokinetics; PD: pharmacodynamics; PDAC: pancreatic ductal adenocarcinoma; CRC: colorectal cancer; CCA: cholangiocarcinoma; TBD: not determined. [Diagram 2] FIG. 1 is a graph showing a representative size-exclusion chromatography (SEC) profile of an anti-galectin-9 antibody, with the high molecular weight peaks labeled. [Figure 3A] Figure 1 includes bar graphs showing Galectin-9 expression levels measured in T cells (CD3+), macrophages (CD11b+), and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from pancreatic adenocarcinoma biopsies using anti-Galectin-9 G9.2-17 Fab fragment and a commercially available anti-Galectin-9 antibody (9M1-3). S2 fraction: organoids. S3 fraction: single cells. The corresponding isotype of G9.2-17 Fab ("Fab isotype") and "fluorescence minus one" (FMO) 9M1-3 ("Gal9 FMO") were used as controls for specificity, background staining, and fluorescence bleed-through from other channels. Levels of Galectin-9 in CD3+ cells measured in the S3 fraction are shown. [Figure 3B] Figure 1 includes bar graphs showing Galectin-9 expression levels measured in T cells (CD3+), macrophages (CD11b+), and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from pancreatic adenocarcinoma biopsies using anti-Galectin-9 G9.2-17 Fab fragment and a commercially available anti-Galectin-9 antibody (9M1-3). S2 fraction: organoids. S3 fraction: single cells. The corresponding isotype of G9.2-17 Fab ("Fab isotype") and "fluorescence minus one" (FMO) 9M1-3 ("Gal9 FMO") were used as controls for specificity, background staining, and fluorescence bleed-through from other channels. Levels of Galectin-9 in CD11b+ cells measured in the S3 fraction are shown. [Figure 3C]Figure 1 includes bar graphs showing Galectin-9 expression levels measured in T cells (CD3+), macrophages (CD11b+), and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from pancreatic adenocarcinoma biopsies using anti-Galectin-9 G9.2-17 Fab fragment and a commercially available anti-Galectin-9 antibody (9M1-3). S2 fraction: organoids. S3 fraction: single cells. The corresponding isotype of G9.2-17 Fab ("Fab isotype") and "fluorescence minus one" (FMO) 9M1-3 ("Gal9 FMO") were used as controls for specificity, background staining, and fluorescence bleed-through from other channels. Levels of Galectin-9 in Epcam+ cells measured in the S3 fraction are shown. [Figure 3D] Figure 1 includes bar graphs showing Galectin-9 expression levels measured in T cells (CD3+), macrophages (CD11b+), and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from pancreatic adenocarcinoma biopsies using anti-Galectin-9 G9.2-17 Fab fragment and a commercially available anti-Galectin-9 antibody (9M1-3). S2 fraction: organoids. S3 fraction: single cells. The corresponding isotype of G9.2-17 Fab ("Fab isotype") and "fluorescence minus one" (FMO) 9M1-3 ("Gal9 FMO") were used as controls for specificity, background staining, and fluorescence bleed-through from other channels. Levels of Galectin-9 in CD3+ cells measured in the S2 fraction are shown. [Figure 3E]Figure 1 includes bar graphs showing Galectin-9 expression levels measured in T cells (CD3+), macrophages (CD11b+), and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from pancreatic adenocarcinoma biopsies using anti-Galectin-9 G9.2-17 Fab fragment and a commercially available anti-Galectin-9 antibody (9M1-3). S2 fraction: organoids. S3 fraction: single cells. The corresponding isotype of G9.2-17 Fab ("Fab isotype") and "fluorescence minus one" (FMO) 9M1-3 ("Gal9 FMO") were used as controls for specificity, background staining, and fluorescence bleed-through from other channels. Levels of Galectin-9 in CD11b+ cells measured in the S2 fraction are shown. [Figure 3F] Figure 1 includes bar graphs showing Galectin-9 expression levels measured in T cells (CD3+), macrophages (CD11b+), and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from pancreatic adenocarcinoma biopsies using anti-Galectin-9 G9.2-17 Fab fragment and a commercially available anti-Galectin-9 antibody (9M1-3). S2 fraction: organoids. S3 fraction: single cells. The corresponding isotype of G9.2-17 Fab ("Fab isotype") and "fluorescence minus one" (FMO) 9M1-3 ("Gal9 FMO") were used as controls for specificity, background staining, and fluorescence bleed-through from other channels. Levels of Galectin-9 in Epcam+ cells measured in the S2 fraction are shown. [Figure 4A]1 includes a bar graph showing Galectin-9 expression levels measured in T cells (CD3+), macrophages (CD11b+), and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from colorectal cancer biopsies using anti-Galectin-9 G9.2-17 Fab fragment and a commercially available anti-Galectin-9 antibody (9M1-3). S2 fraction: organoids. S3 fraction: single cells. The corresponding isotype of G9.2-17 Fab ("Fab isotype") and FMO 9M1-3 ("Gal9 FMO") were used as controls for specificity, background staining, and fluorescence bleed-through from other channels. The levels of Galectin-9 in CD3+ cells measured in the S3 fraction are shown. [Figure 4B] 1 includes a bar graph showing Galectin-9 expression levels measured in T cells (CD3+), macrophages (CD11b+), and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from colorectal cancer biopsies using anti-Galectin-9 G9.2-17 Fab fragment and a commercially available anti-Galectin-9 antibody (9M1-3). S2 fraction: organoids. S3 fraction: single cells. The corresponding isotype of G9.2-17 Fab ("Fab isotype") and FMO 9M1-3 ("Gal9 FMO") were used as controls for specificity, background staining, and fluorescence bleed-through from other channels. The levels of Galectin-9 in CD11b+ cells measured in the S3 fraction are shown. [Figure 4C]1 includes a bar graph showing Galectin-9 expression levels measured in T cells (CD3+), macrophages (CD11b+), and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from colorectal cancer biopsies using anti-Galectin-9 G9.2-17 Fab fragment and a commercially available anti-Galectin-9 antibody (9M1-3). S2 fraction: organoids. S3 fraction: single cells. The corresponding isotype of G9.2-17 Fab ("Fab isotype") and FMO 9M1-3 ("Gal9 FMO") were used as controls for specificity, background staining, and fluorescence bleed-through from other channels. The levels of Galectin-9 in Epcam+ cells measured in the S3 fraction are shown. [Figure 4D] 1 includes a bar graph showing Galectin-9 expression levels measured in T cells (CD3+), macrophages (CD11b+), and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from colorectal cancer biopsies using anti-Galectin-9 G9.2-17 Fab fragment and a commercially available anti-Galectin-9 antibody (9M1-3). S2 fraction: organoids. S3 fraction: single cells. The corresponding isotype of G9.2-17 Fab ("Fab isotype") and FMO 9M1-3 ("Gal9 FMO") were used as controls for specificity, background staining, and fluorescence bleed-through from other channels. The levels of Galectin-9 in CD3+ cells measured in the S2 fraction are shown. [Figure 4E]1 includes a bar graph showing Galectin-9 expression levels measured in T cells (CD3+), macrophages (CD11b+), and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from colorectal cancer biopsies using anti-Galectin-9 G9.2-17 Fab fragment and a commercially available anti-Galectin-9 antibody (9M1-3). S2 fraction: organoids. S3 fraction: single cells. The corresponding isotype of G9.2-17 Fab ("Fab isotype") and FMO 9M1-3 ("Gal9 FMO") were used as controls for specificity, background staining, and fluorescence bleed-through from other channels. The levels of Galectin-9 in CD11b+ cells measured in the S2 fraction are shown. [Figure 4F] 1 includes a bar graph showing Galectin-9 expression levels measured in T cells (CD3+), macrophages (CD11b+), and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from colorectal cancer biopsies using anti-Galectin-9 G9.2-17 Fab fragment and a commercially available anti-Galectin-9 antibody (9M1-3). S2 fraction: organoids. S3 fraction: single cells. The corresponding isotype of G9.2-17 Fab ("Fab isotype") and FMO 9M1-3 ("Gal9 FMO") were used as controls for specificity, background staining, and fluorescence bleed-through from other channels. The levels of Galectin-9 in Epcam+ cells measured in the S2 fraction are shown. [Figure 5A]Galectin-9 Includes bar graphs showing Galectin-9 expression levels measured in T cells (CD3+), macrophages (CD11b+), and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from anti-pancreatic adenocarcinoma biopsies using G9.2-17 Fab fragment and a commercially available Galectin-9 antibody (9M1-3). S2 fraction: organoids. S3 fraction: single cells. The corresponding isotype of G9.2-17 Fab ("Fab isotype") and FMO 9M1-3 ("Gal9FMO") were used as controls for specificity, background staining, and fluorescence bleed-through from other channels. The levels of Galectin-9 in CD3+ cells measured in the S3 fraction are shown. [Figure 5B] Galectin-9 Includes bar graphs showing Galectin-9 expression levels measured in T cells (CD3+), macrophages (CD11b+), and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from anti-pancreatic adenocarcinoma biopsies using G9.2-17 Fab fragment and a commercially available Galectin-9 antibody (9M1-3). S2 fraction: organoids. S3 fraction: single cells. The corresponding isotype of G9.2-17 Fab ("Fab isotype") and FMO 9M1-3 ("Gal9FMO") were used as controls for specificity, background staining, and fluorescence bleed-through from other channels. The levels of Galectin-9 in CD11b+ cells measured in the S3 fraction are shown. [Figure 5C] Galectin-9 Includes bar graphs showing Galectin-9 expression levels measured in T cells (CD3+), macrophages (CD11b+), and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from anti-pancreatic adenocarcinoma biopsies using G9.2-17 Fab fragment and a commercially available Galectin-9 antibody (9M1-3). S2 fraction: organoids. S3 fraction: single cells. The corresponding isotype of G9.2-17 Fab ("Fab isotype") and FMO 9M1-3 ("Gal9FMO") were used as controls for specificity, background staining, and fluorescence bleed-through from other channels. The levels of Galectin-9 in Epcam+ cells measured in the S3 fraction are shown. [Figure 5D] Galectin-9 Includes bar graphs showing Galectin-9 expression levels measured in T cells (CD3+), macrophages (CD11b+), and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from anti-pancreatic adenocarcinoma biopsies using G9.2-17 Fab fragment and a commercially available Galectin-9 antibody (9M1-3). S2 fraction: organoids. S3 fraction: single cells. The corresponding isotype of G9.2-17 Fab ("Fab isotype") and FMO 9M1-3 ("Gal9FMO") were used as controls for specificity, background staining, and fluorescence bleed-through from other channels. The levels of Galectin-9 in CD3+ cells measured in the S2 fraction are shown. [Figure 5E] Galectin-9 Includes bar graphs showing Galectin-9 expression levels measured in T cells (CD3+), macrophages (CD11b+), and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from anti-pancreatic adenocarcinoma biopsies using G9.2-17 Fab fragment and a commercially available Galectin-9 antibody (9M1-3). S2 fraction: organoids. S3 fraction: single cells. The corresponding isotype of G9.2-17 Fab ("Fab isotype") and FMO 9M1-3 ("Gal9FMO") were used as controls for specificity, background staining, and fluorescence bleed-through from other channels. The levels of Galectin-9 in CD11b+ cells measured in the S2 fraction are shown. [Figure 5F]Galectin-9 Includes bar graphs showing Galectin-9 expression levels measured in T cells (CD3+), macrophages (CD11b+), and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from anti-pancreatic adenocarcinoma biopsies using G9.2-17 Fab fragment and a commercially available Galectin-9 antibody (9M1-3). S2 fraction: organoids. S3 fraction: single cells. The corresponding isotype of G9.2-17 Fab ("Fab isotype") and FMO 9M1-3 ("Gal9FMO") were used as controls for specificity, background staining, and fluorescence bleed-through from other channels. The levels of Galectin-9 in Epcam+ cells measured in the S2 fraction are shown. [Figure 6A] Photographs of immunohistochemical analysis of various tumors using anti-galectin-9 antibody 1G3 are included. All magnifications are 200X. Shown are chemotherapy-treated colorectal carcinomas with heterogeneous galectin-9 expression with intensity scores of 2 and 3 (moderate and high). Galectin-9 staining was observed especially at the cell membrane; in addition, intraglandular macrophages were moderately positive and the stromal reaction within the tumor shows multinucleated macrophage giant cells with moderately strong galectin-9 expression. [Figure 6B] Photographs of immunohistochemical analysis of various tumors using anti-galectin-9 antibody 1G3. All magnifications are 200X. Shown is a liver metastasis of colorectal cancer with high (intensity score 3) galectin-9 expression. Staining is membranous and cytoplasmic. [Figure 6C] Photographs of immunohistochemical analysis of various tumors using anti-galectin-9 antibody 1G3 are included. All magnifications are 200X. Galectin-9 positive (intensity score 2) trapped bile ducts and Galectin-9 negative carcinomas are shown. [Figure 7]Includes graphs showing the percentage of Annexin V and propidium iodide (PI) positive cells plotted as a function of antibody concentration used. MOLM-13 cells were co-incubated with various concentrations of G9.2-17 or human IgG4 isotype antibody and recombinant human galectin-9 for 16 hours. Cells were stained with Annexin V and propidium iodide prior to analysis by flow cytometry. Each condition was performed in triplicate. Analysis was performed with FlowJo software. [Figure 8A] Graphs showing the results of a study in which mice were treated with G9.2-17 mIgG2a alone or in combination with αPD-1 mAb. Mice (n=10 / group) implanted with orthotopically implanted KPC tumors were treated weekly for 3 weeks with commercial αPD-1 (200 μg) mAb or G9.2-17 mIg2a (200 μg), or a combination of G9.2-17 and αPD-1 or matching isotypes. Tumors were removed and weighed (FIG. 8A) and subsequently processed and stained for flow cytometry (FIG. 7B). Each point represents one mouse. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; by unpaired Student's t-test. [Figure 8B]Graphs showing the results of a study in which mice were treated with G9.2-17 mIgG2a alone or in combination with αPD-1 mAb. Mice (n=10 / group) implanted with orthotopically implanted KPC tumors were treated weekly for 3 weeks with commercial αPD-1 (200 μg) mAb or G9.2-17 mIg2a (200 μg), or a combination of G9.2-17 and αPD-1 or matching isotypes. Tumors were removed and weighed (FIG. 8A) and subsequently processed and stained for flow cytometry (FIG. 7B). Each point represents one mouse. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; by unpaired Student's t-test. Bar graphs showing that tumors were excised from control and treated animals at the end of the experiment (day 18) and processed for flow cytometry of intratumoral immune cells and associated activation and immunosuppression markers. Mouse tumors were digested prior to flushing. Flow cytometry was performed on an Attune NxT flow cytometer (ThermoFisher Scientific, Waltham, MA). Data were analyzed using FlowJov.10.1 (Treestar, Ashland, OR). [Figure 9A] 9A and 9B show graphs depicting the results of ADCC assays performed on the IgG1 form of G9.2-17 (FIG. 9A) and the IgG4 form of G9.2-17 (FIG. 9B). As expected for a human IgG4 mAb, G9.2-17 does not mediate ADCC (FIG. 9B). It was tested against its human counterpart IgG1 as a positive control, which, as expected, mediates ADCC and ADCP (FIG. 9A). [Figure 9B] 9A and 9B show graphs depicting the results of ADCC assays performed on the IgG1 form of G9.2-17 (FIG. 9A) and the IgG4 form of G9.2-17 (FIG. 9B). As expected for a human IgG4 mAb, G9.2-17 does not mediate ADCC (FIG. 9B). It was tested against its human counterpart IgG1 as a positive control, which, as expected, mediates ADCC and ADCP (FIG. 9A). [Figure 10A]Figure 1 shows a graph depicting the effect of 9.2-17 in a B16F10 subcutaneous syngeneic model. Tumors were implanted subcutaneously and treated with G9.2-17 IgG1 mouse mAb, anti-PD-1 antibody, or a combination of G9.2-17 IgG1 mouse mAb and anti-PD-1 antibody. Figure 2 shows the effect on tumor volume. [Figure 10B] A graph showing the effect of 9.2-17 in a B16F10 subcutaneous syngeneic model. Tumors were implanted subcutaneously and treated with G9.2-17 IgG1 mouse mAb, anti-PD-1 antibody, or a combination of G9.2-17 IgG1 mouse mAb and anti-PD-1 antibody. A graph showing intratumoral CD8 T cell infiltration. The results show that intratumoral effector T cells were enhanced in the combination treatment group. [Figure 11A] Includes charts showing ex vivo tumor cultures (organoids) from cholangiocarcinoma patients treated with G9.2-17. Patient-derived ex vivo tumor cultures (organoids) were treated with G9.2-17 or isotype control for 3 days. Expression of CD44 (Figure 11A) and TNFα (Figure 11B) in PDOTS-derived CD3+ T cells was evaluated. [Figure 11B] Includes charts showing ex vivo tumor cultures (organoids) from cholangiocarcinoma patients treated with G9.2-17. Patient-derived ex vivo tumor cultures (organoids) were treated with G9.2-17 or isotype control for 3 days. Expression of CD44 (Figure 11A) and TNFα (Figure 11B) in PDOTS-derived CD3+ T cells was evaluated. [Figure 12] Includes graphs showing the effect of G2.9-17 on TGF-β1 secretion measurements in whole blood of an exemplary healthy human donor. TGF-β1 release from cryopreserved macrophages of donors incubated in the presence of M2 polarizing cocktail. IgG4 isotype is a negative control antibody. Data represent mean + SEM of triplicate determinations. Significance was determined by two-way ANOVA with Dunnett's multiple comparison test. *p<0.05 [Figure 13]Includes graphs showing the effect of G2.9-17 on IL-10 secretion in whole blood of an exemplary healthy human donor. IL-10 release from cryopreserved macrophages of donors incubated in the presence of M2 polarizing cocktails (IL-4 / IL-13 or Gal-9). IgG4 isotype is a negative control antibody. Data represent the mean (±SEM) of triplicates. Significance was determined by two-way ANOVA with Tukey's multiple comparison test (*P<0.05). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Provided herein are methods of using anti-galectin-9 antibodies, e.g., G9.2-17(IgG4), to treat solid tumors, e.g., pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), cholangiocarcinoma (CAA), renal cell carcinoma (RCC), urothelial carcinoma, head and neck cancer, breast cancer, lung cancer, or other GI solid tumors. In some embodiments, the cancer is metastatic. In some embodiments, the methods disclosed herein provide a specific dose and / or dosing schedule, e.g., 0.2 mg / kg to 16 mg / kg of antibody once a week (e.g., 10 mg / kg or 16 mg / kg once a week). It has been discovered that G9.2-17(IgG4) exhibits an unexpectedly fast clearance rate in human subjects compared to conventional antibody therapeutics. Thus, a treatment regimen including a weekly dosing schedule has been developed to ensure systemic exposure levels of anti-galectin-9 antibodies that achieve therapeutic efficacy. In some cases, the methods disclosed herein target specific patient populations, such as patients who have undergone prior treatment and who show disease progression through prior treatment, or who are resistant (de novo or acquired) to prior treatment.

[0032] The tandem repeat lectin, galectin-9, is a β-galactoside-binding protein that has been shown to play a role in regulating cell-cell and cell-matrix interactions. It has been found to be strongly overexpressed in Hodgkin's disease tissues and other pathological conditions. In some cases, it has also been found circulating within the tumor microenvironment (TME).

[0033] Galectin-9 has been shown to interact with dectin-1, an innate immune receptor that is highly expressed on macrophages and cancer cells in PDAC (Daley, et al. Nat Med. 2017;23(5):556-6). Regardless of the source of galectin-9, disruption of its interaction with dectin-1 results in the upregulation of CD4 + and CD8 + Galectin-9 has been shown to reprogram cells into essential mediators of anti-tumor immunity. Thus, Galectin-9 serves as a valuable therapeutic target for blocking signaling mediated by Dectin-1. Thus, in some embodiments, the anti-Galectin-9 antibodies described herein disrupt the interaction between Galectin-9 and Dectin-1.

[0034] Galectin-9 has also been found to interact with TIM-3, a type I cell surface glycoprotein expressed on the surface of leukemic stem cells in all types of acute myeloid leukemia (except M3 (acute promyelocytic leukemia)), but not on normal human hematopoietic stem cells (HSCs). TIM-3 signaling resulting from ligation of galectin-9 has been found to have pleiotropic effects on immune cells, inducing apoptosis of Th1 cells (Zhu et al., Nat Immunol., 2005, 6:1245-1252) and stimulating the secretion of tumor necrosis factor alpha (TNF-α), which leads to maturation of monocytes into dendritic cells that trigger innate immune inflammation (Kuchroo et al., Nat Rev Immunol., 2008, 8:577-580). Furthermore, galectin-9 / TIM-3 signaling has been found to co-activate NF-κB and β-catenin signaling, two pathways that promote LSC self-renewal (Kikushige et al., Cell Stem Cell, 2015, 17(3):341-352). Anti-galectin-9 antibodies that interfere with galectin-9 / TIM-3 binding may have therapeutic value, particularly with respect to leukemia and other hematological malignancies. Thus, in some embodiments, the anti-galectin-9 antibodies described herein disrupt the interaction between galectin-9 and TIM-3.

[0035] Furthermore, galectin-9 has been found to interact with CD206, a mannose receptor highly expressed on M2-polarized macrophages, thereby promoting tumor survival (Enningaetal., JPathol. 2018Aug;245(4):468-477). Tumor-associated macrophages expressing CD206 are mediators of tumor immunosuppression, angiogenesis, metastasis, and recurrence (see, e.g., Scodeller et al., Sci Rep. 2017 Nov 7;7(1):14655, and references therein). Specifically, M1 (also called classically activated macrophages) are induced by Th1-associated cytokines and bacterial products, express high levels of IL-12, and are tumoricidal. In contrast, M2 (so-called alternatively activated macrophages) are activated with Th2-associated factors, express high levels of anti-inflammatory cytokines such as IL-10, and promote tumor progression (Biswas and Mantovani; Nat Immunol. 2010 Oct; 11(10): 889-96). The tumor-promoting effects of M2 include promoting angiogenesis, progression of invasion and metastasis, and protecting tumor cells from chemotherapy-induced apoptosis (Hu et al., Tumor Biol. 2015 Dec; 36(12): 9119-9126 and references therein). Tumor-associated macrophages are of an M2-like phenotype and are thought to have a pro-tumor role. Galectin-9 has been shown to mediate the differentiation of myeloid cells into the M2 phenotype (Enninga et al., Melanoma Res. 2016 Oct; 26(5): 429-41). Binding of galectin-9 to CD206 may potentially reprogram TAMs to an M2 phenotype, similar to what has previously been shown for dectin-1. Without wishing to be bound by theory, blocking the interaction of galectin-9 with CD206 may provide one mechanism by which anti-galectin-9 antibodies, such as the G9.2-17 antibody, may be therapeutically beneficial. Thus, in some embodiments, the anti-galectin-9 antibodies described herein disrupt the interaction between galectin-9 and CD206.

[0036] Galectin-9 has also been shown to interact with protein disulfide isomerase (PDI) and 4-1BB (Bi S, et al. Proc Natl Acad Sci US A. 2011; 108(26): 10650-5; Madireddi et al. J Exp Med. 2014; 211(7): 1433-48).

[0037] Anti-galectin-9 antibodies may function as therapeutic agents for treating diseases associated with galectin-9 (e.g., those involving galectin-9 signaling). Without being bound by theory, anti-galectin-9 antibodies may block signaling pathways mediated by galectin-9. For example, the antibodies may interfere with the interaction between galectin-9 and its binding partners (e.g., Dectin-1, TIM-3, or CD206), thereby blocking signaling caused by galectin-9 / ligand interactions. Alternatively or additionally, anti-galectin-9 antibodies may also exert their therapeutic effect by inducing blocking and / or cytotoxicity, e.g., ADCC, CDC, or ADCP, against pathological cells expressing galectin-9. Pathological cells refer to cells that directly or indirectly contribute to the initiation and / or development of a disease. See, e.g., WO2019 / 084553, WO2020 / 198390, WO2020 / 0223702, and WO2021022256, the relevant disclosures of each of which are incorporated by reference with respect to the subject matter and purposes referenced herein.

[0038] The anti-galectin-9 antibodies disclosed herein can inhibit galectin-9 mediated signaling (e.g., galectin-9 / Dectin-1 or galectin-9 / Tim-3 mediated signaling pathways) or eliminate pathological cells expressing galectin-9, e.g., by ADCC. Thus, the anti-galectin-9 antibodies described herein can be used to inhibit either galectin-9 signaling and / or eliminate galectin-9 positive pathological cells, thereby providing benefit in the treatment of diseases associated with galectin-9.

[0039] Anti-galectin-9 antibodies, such as G9.2-17 (e.g., G9.2-17(IgG4)), have been found to be effective in inducing apoptosis in cells expressing galectin-9. Furthermore, the anti-tumor efficacy of anti-galectin-9 antibodies, such as G9.2-17, alone or in combination with checkpoint inhibitors (e.g., anti-PD-1 antibodies), has been demonstrated in mouse models. As reported herein, the efficacy of G9.2-17 was tested in mouse models of PDAC and melanoma, as well as in patient-derived organ tumor models (PDOTs). The orthotopic PDAC KPC mouse model used (LSL-KrasG12D / +; LSL-Trp53R172H / +; Pdx-1-Cre) recapitulates many features of the human disease, including unresponsiveness to approved checkpoint inhibitors (Bisht and Feldmann G; Animal models for modeling pancreatic cancer and novel drug discovery; Expert Opin Drug Discov. 2019;14(2):127-142; Weidenhofer et al., Animal models of pancreatic cancer and their application in clinical research; Gastrointestinal Cancer: Targets and Therapy 2016;6). The B16F10 melanoma mouse model has been a long-standing standard for testing immunotherapies (Curran et al., PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors; Proc Natl Acad Sci USA. 2010; 107(9):4275-4280).

[0040] PDOTs isolated from fresh human tumor samples retain autologous lymphoid and myeloid cell populations (including antigen-experienced tumor-infiltrating CD4 and CD8 T lymphocytes) and respond to immunotherapy in short-term ex vivo cultures (Jenkins et al. Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids. Cancer Discov. 2018;8(2):196-215; Aref et al.,3D microfluidic ex vivo culture of organotypic tumor spheroids to model immune checkpoint blockade; Lab Chip. 2018;18(20):3129-3143). As reported herein, expression of galectin-9 on cancer cells was observed in patient-derived organoid assays.

[0041] In vivo studies were performed with G9.2-17 murine IgG1 (G9.2-17 mIgG1 contains exactly the same binding epitope as G9.2-17 human IgG4 and has the same effector function), which already achieved a significant reduction in tumor growth as a single agent in the orthotopic KPC model, where approved checkpoint inhibitors fail. In the B16F10 model, G9.2-17 significantly outperforms anti-PD-1 efficacy. In both models, modulation of the intratumoral immune microenvironment by upregulating effector T cell activity and inhibiting immunosuppressive signals using G9.2-17 mIgG1 was demonstrated, as well as enhancement of intratumoral CD8 T cell infiltration.

[0042] These results demonstrate that the anti-tumor methods disclosed herein, including anti-galectin-9 antibodies, optionally in combination with checkpoint inhibitors, will achieve superior therapeutic efficacy against targeted solid tumors.

[0043] Accordingly, described herein are therapeutic uses of anti-galectin-9 antibodies to treat certain cancers disclosed herein.

[0044] Antibodies that bind to galectin-9 The present disclosure provides the anti-galectin-9 antibody G9.2-17 and functional variants thereof for use in the treatment methods disclosed herein.

[0045] Antibodies (used interchangeably in the plural) are immunoglobulin molecules capable of specifically binding to targets, e.g., carbohydrates, polynucleotides, lipids, polypeptides, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody", e.g., anti-galectin-9 antibody, encompasses not only intact (e.g., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chain (scFv), variants thereof, antibody portions, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), as well as fusion proteins comprising immunoglobulin molecules of any other modified configuration that contains an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies, e.g., anti-galectin-9 antibodies, include antibodies of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and do not have to be of any particular class. Depending on the antibody amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the various classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known.

[0046] A typical antibody molecule consists of a heavy chain variable region (V H ) and the light chain variable region (V L), which are usually involved in antigen binding. H and V L The regions can be further subdivided into regions of hypervariability, also known as "complementarity determining regions" ("CDRs"), interspersed with regions that are more conserved, known as "framework regions" ("FRs"). H and V Lis typically composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of framework regions and CDRs can be precisely identified using methodologies known in the art, for example, by the Kabat definition, the Chothia definition, the AbM definition, the EU definition, the "Contact" numbering scheme, the "IMGT" numbering scheme, the "AHo" numbering scheme, and / or the contact definition, all of which are well known in the art. See, e.g., EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. al.(1987)J.Mol.Biol.196:901-917,Al-lazikani et al(1997)J.Molec.Biol.273:927-948;Edelman et al.,Proc Natl Acad Sci USA.1969 May;63(1):78-85; and Almagro, J.Mol.Recognit.17:132-143(2004);MacCallum et al., J. Mol. Biol. 262:732-745 (1996), Lefranc MP et al., Dev Comp Immunol, 2003 January;27(1):55-77; and Honegger A and Pluckthun A, J Mol Biol, 2001 June.8;309(3):657-70. See also: hgmp.mrc.ac.uk and bioinf.org.uk / abs).

[0047] In some embodiments, the anti-Galectin-9 antibodies described herein are full-length antibodies containing two heavy chains and two light chains, each of which comprises a variable domain and a constant domain. Alternatively, the anti-Galectin-9 antibodies may be antigen-binding fragments of full-length antibodies. Examples of binding fragments encompassed within the term "antigen-binding fragment" of a full-length antibody include: (i) a Fab fragment (V L , V H , C L , and C H (ii) F(ab')2 fragment (a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region); (iii) V H and C H (iv) a single-arm V of an antibody; L and V H (v) Fv fragment consisting of V domains; H (iv) isolated complementarity determining regions (CDRs) that retain function. In addition, two domains of the Fv fragment, the V L and V H Although the V and VL are encoded by separate genes, they can be joined using recombinant methods with a synthetic linker that can be made into a single protein chain. L Area and V H The regions pair to form monovalent molecules known as single-chain Fvs (scFvs). See, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883.

[0048] Any of the antibodies described herein, for example, anti-galectin-9 antibodies, can be either monoclonal or polyclonal. A "monoclonal antibody" refers to a homogeneous population of antibodies, whereas a "polyclonal antibody" refers to a heterogeneous population of antibodies. These two terms do not limit the source of the antibody or the method by which the antibody is made.

[0049] Reference antibody G9.2-17 refers to an antibody capable of binding to human galectin-9 and comprises a heavy chain variable region of SEQ ID NO: 7 and a light chain variable domain of SEQ ID NO: 8, both of which are provided below. In some embodiments, the anti-galectin-9 antibody used in the methods disclosed herein is a G9.2-17 antibody. In some embodiments, the anti-galectin-9 antibody used in the methods disclosed herein is an antibody that has the same heavy chain complementarity determining regions (CDRs) as the reference antibody G9.2-17 and / or the same light chain complementarity determining regions as the reference antibody G9.2-17. H and / or V L Two antibodies having CDRs mean that their CDRs are identical when determined by the same approach (e.g., the Kabat approach, the Chothia approach, the AbM approach, the Contact approach, or the IMGT approach known herein, see, e.g., bioinf.org.uk / abs / ).

[0050] The heavy and light chain CDRs of the reference antibody G9.2-17 are presented below in Table 1 (determined using the Kabat method). [Table 1]

[0051] In some examples, anti-galectin-9 antibodies used in the methods disclosed herein may comprise (according to the Kabat scheme) a heavy chain complementarity determining region 1 (CDR1) set forth in SEQ ID NO: 4, a heavy chain complementarity determining region 2 (CDR2) set forth in SEQ ID NO: 5, and a heavy chain complementarity determining region 3 (CDR3) set forth in SEQ ID NO: 6, and / or a light chain complementarity determining region 1 (CDR1) set forth in SEQ ID NO: 1, a light chain complementarity determining region 2 (CDR2) set forth in SEQ ID NO: 2, and a light chain complementarity determining region 3 (CDR3) set forth in SEQ ID NO: 3. Anti-galectin-9 antibodies, including the reference antibody G9.2-17, may be in any format disclosed herein, e.g., full length antibody or Fab. As used herein, the term "G9.2-17(IgG4)" refers to the G9.2-17 antibody, which is an IgG4 molecule. Similarly, the term "G9.2-17(Fab)" refers to the G9.2-17 antibody, which is a Fab molecule.

[0052] In some embodiments, the anti-galectin-9 antibody or binding portion thereof comprises a heavy chain variable region and a light chain variable region, and the CDR1, CDR2, and CDR3 amino acid sequences of the light chain variable region have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and any increment therein) sequence identity to the amino acid sequences of the light chain variable region CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, the anti-galectin-9 antibody or binding portion thereof comprises a heavy chain variable region and a light chain variable region, and the CDR1, CDR2, and CDR3 amino acid sequences of the heavy chain variable region have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and any increment therein) sequence identity to the amino acid sequences of the heavy chain variable region CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 4, 5, and 6, respectively.

[0053] Additional galectin-9 antibodies (e.g., those that bind to the CRD1 and / or CRD2 regions of galectin-9) are described in co-pending U.S. patent application Ser. No. 16 / 173,970 and co-pending international patent applications PCT / US18 / 58028 and PCT / US2020 / 024767, the contents of each of which are incorporated by reference in their entirety.

[0054] In some embodiments, the anti-galectin-9 antibodies disclosed herein have the corresponding V H Alternatively or additionally, in some embodiments, the anti-galectin-9 antibody comprises a light chain CDR that has, individually or collectively, at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the corresponding V CDRs of reference antibody G9.2-17. H The heavy chain CDRs have, individually or collectively, at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to the CDRs.

[0055] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the protein molecules of the invention. When gaps exist between the two sequences, gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0056] In other embodiments, the anti-galectin-9 antibodies described herein comprise a V CDR comprising HC CDR1, HC CDR2, and HC CDR3. H which collectively comprise up to 8 amino acid residue variations (8, 7, 6, 5, 4, 3, 2, or 1 variation(s)) including additions, deletions, and / or substitutions compared to the HC CDR1, HC CDR2, and HC CDR3 of the reference antibody G9.2-17. Alternatively or additionally, in some embodiments, the anti-galectin-9 antibodies described herein comprise a V CDR1, V CDR2, and V CDR3 comprising LC CDR1, LC CDR2, and LC CDR3. H which collectively contain up to 8 amino acid residue diversity (8, 7, 6, 5, 4, 3, 2, or 1 diversity(s) including additions, deletions, and / or substitutions) compared to the LC CDR1, LC CDR2, and LC CDR3 of reference antibody G9.2-17.

[0057] In one example, the amino acid residue diversity is a conservative amino acid residue substitution. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Diversity can be adjusted according to methods for modifying polypeptide sequences known to those skilled in the art, such as those found in references that summarize such methods, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FMAusubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0058] In some embodiments, the anti-galectin-9 antibodies disclosed herein having heavy chain CDRs disclosed herein are H The fragments include framework regions derived from a subclass of the germline V HRegions are well known in the art. See, for example, the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php. Examples include the IGHV1 subfamily (e.g., IGHV1-2, IGHV1-3, IGHV1-8, IGHV1-18, IGHV1-24, IGHV1-45, IGHV1-46, IGHV1-58, and IGHV1-69), the IGHV2 subfamily (e.g., IGHV2-5, IGHV2-26, and IGHV2-70), the IGHV3 subfamily (e.g., IGHV3-7, IGHV3-9, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-20, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3- 33, IGHV3-43, IGHV3-48, IGHV3-49, IGHV3-53, IGHV3-64, IGHV3-66, IGHV3-72, and IGHV3-73, IGHV3-74), IGHV4 subfamily (e.g., IGHV4-4, IGHV4-28, IGHV4-31, IGHV4-34, IGHV4-39, IGHV4-59, IGHV4-61, and IGHV4-B), IGHV subfamily (e.g., IGHV5-51, or IGHV6-1), and IGHV7 subfamily (e.g., IGHV7-4-1).

[0059] Alternatively or additionally, in some embodiments, anti-galectin-9 antibodies having light chain CDRs disclosed herein contain framework regions derived from germline VK fragments. Examples include IGKV1 frameworks (e.g., IGKV1-05, IGKV1-12, IGKV1-27, IGKV1-33, or IGKV1-39), IGKV2 frameworks (e.g., IGKV2-28), IGKV3 frameworks (e.g., IGKV3-11, IGKV3-15, or IGKV3-20), and IGKV4 frameworks (e.g., IGKV4-1). In other cases, anti-galectin-9 antibodies comprise light chain variable regions that contain frameworks derived from germline V fragments. Examples include IGλ1 frameworks (e.g., IGλV1-36, IGλV1-40, IGλV1-44, IGλV1-47, IGλV1-51), IGλ2 frameworks (e.g., IGλV2-8, IGλV2-11, IGλV2-14, IGλV2-18, IGλV2-23), IGλ3 frameworks (e.g., IGλV3-1, IGλV3-9, IGλV3-10, IGλV3-12, IGλV3-16, IGλV3-19, IGλV3-21, IGλV3-25, IGλV3-27), I Examples of such frameworks include IGλ4 frameworks (e.g., IGλV4-3, IGλV4-60, IGλV4-69), IGλ5 frameworks (e.g., IGλV5-39, IGλV5-45), IGλ6 frameworks (e.g., IGλV6-57), IGλ7 frameworks (e.g., IGλV7-43, IGλV7-46), IGλ8 frameworks (e.g., IGλV8-61), IGλ9 frameworks (e.g., IGλV9-49), or IGλ10 frameworks (e.g., IGλV10-54).

[0060] In some embodiments, the anti-galectin-9 antibody used in the methods disclosed herein has the same heavy chain variable region (V H ) and / or the same light chain variable region (V L ), and V H and V L The amino acid sequence of the region is provided below: VH : [ka] V L : [ka]

[0061] In some embodiments, the anti-Galectin-9 antibody has at least 80% sequence identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) with the heavy chain variable region of SEQ ID NO: 7. Alternatively or additionally, the anti-Galectin-9 antibody has at least 80% sequence identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) with the light chain variable region of SEQ ID NO:8.

[0062] In some cases, the anti-galectin-9 antibodies disclosed herein are functional variants of the reference antibody G9.2-17. The functional variants may be structurally similar to the reference antibody (e.g., including a limited number of amino acid residue variations in one or more of the heavy and / or light chain CDRs as G9.2-17 disclosed herein, or sequence identity with the heavy and / or light chain CDRs of G9.2-17 disclosed herein) that have substantially similar binding affinity to human galectin-9 (e.g., having KD values ​​in the same order).

[0063] In some embodiments, the anti-galectin-9 antibodies described herein are capable of binding to and inhibiting the activity of galectin-9 by at least 20% (e.g., 31%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein). The apparent inhibition constant (Ki) provides a measure of the potency of an inhibitor. app Or Ki ,app) is related to the concentration of inhibitor required to reduce enzyme activity and is independent of enzyme concentration. The inhibitory activity of the anti-galectin-9 antibodies described herein can be measured by routine methods known in the art.

[0064] Antibody K i, app The K value can be determined by measuring the inhibitory effect of different concentrations of antibody on the extent of the reaction (e.g., enzyme activity); fitting the change in the pseudo-first-order rate constant (v) as a function of inhibitor concentration to a modified Morrison equation (Equation 1) provides an estimate of the apparent K value. For competitive inhibitors, the K app is the K with respect to the substrate concentration i, app can be obtained from the y-intercept extracted from the linear regression analysis of the plot of

number

[0065] A is v o / E (initial velocity of enzyme reaction in the absence of inhibitor (I) divided by total enzyme concentration (E) (v o In some embodiments, the anti-galectin-9 antibodies described herein have a Ki of 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 pM or less for the target antigen or antigen epitope. app In some embodiments, the anti-galectin-9 antibody has a lower Ki value for a first target (e.g., CRD2 of galectin-9) compared to a second target (e.g., CRD1 of galectin-9). app Has. app The difference (e.g., in the case of specificity or other comparisons) is at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000, or 10 5In some examples, the anti-galectin-9 antibody is greater than or equal to the second antigen (e.g., the first protein or mimetic thereof in a second conformation; or the second protein) and inhibits the first antigen (e.g., the first protein or mimetic thereof in a first conformation). In some embodiments, any of the anti-galectin-9 antibodies further comprises a K app The antibody has been affinity matured to reduce

[0066] In some embodiments, the anti-galectin-9 antibody inhibits Dectin-1 signaling in tumor-infiltrating immune cells, such as macrophages. In some embodiments, the anti-galectin-9 antibody inhibits Galectin-9-induced Dectin-1 signaling by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein). Such inhibitory activity can be measured by conventional methods, such as routine assays. Alternatively or additionally, the anti-galectin-9 antibody inhibits Galectin-9-initiated T cell immunoglobulin mucin-3 (TIM-3) signaling. In some embodiments, the anti-galectin-9 antibody inhibits T cell immunoglobulin mucin-3 (TIM-3) signaling, e.g., in tumor infiltrating immune cells, e.g., in some embodiments, by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein). Such inhibitory activity can be measured by conventional methods, such as routine assays.

[0067] In some embodiments, the anti-galectin-9 antibody inhibits CD206 signaling, for example, in tumor-infiltrating immune cells. In some embodiments, the anti-galectin-9 antibody inhibits Galectin-9-induced CD206 signaling by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein). Such inhibitory activity can be measured by conventional methods, such as a routine assay. In some embodiments, the anti-galectin-9 antibody blocks or inhibits Galectin-9 binding to CD206 by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein). Such inhibitory activity can be measured by conventional methods, such as a routine assay.

[0068] In some embodiments, the anti-galectin-9 antibody induces cytotoxicity, such as ADCC, in a target cell expressing galectin-9, e.g., the target cell is a cancer cell or an immunosuppressive immune cell. In some embodiments, the anti-galectin-9 antibody induces apoptosis of an immune cell, such as a T cell, or a cancer cell by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein). Such inhibitory activity can be measured by conventional methods, such as routine assays. In some embodiments, any of the anti-galectin-9 antibodies described herein induces cytotoxicity, such as complement-dependent cytotoxicity (CDC), in a target cell expressing galectin-9.

[0069] Antibody-dependent cell-mediated phagocytosis (ADCP) is an important mechanism of action of antibodies that mediate some or all of their actions through phagocytosis, where antibodies mediate the uptake of specific antigens by antigen-presenting cells. ADCP can be mediated by monocytes, macrophages, neutrophils, and dendritic cells via FcγRIIa, FcγRI, and FcγRIIIa, with FcγRIIa (CD32a) on macrophages representing the major pathway.

[0070] In some embodiments, the anti-galectin-9 antibody induces cellular phagocytosis of target cells, e.g., cancer cells or immunosuppressive immune cells expressing galectin-9 (ADCP). In some embodiments, the anti-galectin-9 antibody increases phagocytosis of target cells, e.g., cancer cells or immunosuppressive immune cells, by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein).

[0071] In some embodiments, the anti-galectin-9 antibodies described herein induce cytotoxicity, such as complement dependent cytotoxicity (CDC), against target cells, e.g., cancer cells or immunosuppressed immune cells. In some embodiments, the anti-galectin-9 antibodies increase CDC against target cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein).

[0072] In some embodiments, the anti-galectin-9 antibody induces T cell activation, i.e., directly or indirectly suppresses Galectin-9-mediated inhibition of T cell activation, e.g., in tumor-infiltrating T cells. In some embodiments, the anti-galectin-9 antibody enhances T cell activation by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein). T cell activation can be determined by conventional methods, such as using well-known assays for measuring cytokines and checkpoint inhibitors (e.g., measuring CD44, TNF-alpha, IFN-gamma, and / or PD-1). In some embodiments, the anti-galectin-9 antibody enhances CD4+ cell activation by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein). In a non-limiting example, the anti-galectin antibody induces CD44 expression in CD4+ cells. In some embodiments, the anti-galectin-9 antibody increases CD44 expression in CD4+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein). In a non-limiting example, the anti-galectin antibody induces IFN-gamma expression in CD4+ cells. In some embodiments, the anti-galectin-9 antibody increases IFN-gamma expression in CD4+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein). In a non-limiting example, the anti-galectin antibody induces TNFα expression in CD4+ cells. In some embodiments, the anti-galectin-9 antibody increases IFN-alpha expression in CD4+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein).

[0073] In some embodiments, the anti-galectin-9 antibody enhances CD8+ cell activation by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein). In a non-limiting example, the anti-galectin antibody induces CD44 expression in CD8+ cells. In some embodiments, the anti-galectin-9 antibody increases CD44 expression in CD8+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein). In a non-limiting example, the anti-galectin antibody induces IFN-gamma expression in CD8+ cells. In some embodiments, the anti-galectin-9 antibody increases IFN-gamma expression in CD8+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein). In a non-limiting example, the anti-galectin antibody induces TNFα expression in CD8+ cells. In some embodiments, the anti-galectin-9 antibody increases IFN-alpha expression in CD8+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein).

[0074] In some embodiments, the anti-galectin-9 antibodies described herein have suitable binding affinity for a target antigen (e.g., galectin-9) or an antigenic epitope thereof. As used herein, "binding affinity" refers to the apparent binding constant or K A Refers to. A is the dissociation constant (K D The anti-galectin-9 antibodies described herein have a binding affinity of at least 10 to a target antigen or antigen epitope. -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 Binding affinity (K D The increase in binding affinity can be expressed as K DThis corresponds to a decrease in the binding affinity (or binding specificity). Binding affinity (or binding specificity) can be determined in a variety of ways, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for assessing binding affinity are in HBS-P buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 0.005% (v / v) surfactant P20).

[0075] These techniques can be used to measure the concentration of bound binding protein as a function of the concentration of target protein. Under certain conditions, the fractional concentration of bound binding protein ([bound] / [total]) is generally related to the concentration of total target protein ([target]) by the following formula: [Binding] / [Total]=[Target] / (Kd+[Target])

[0076] Not necessarily, K A Although it is not necessary to precisely determine K, it is sometimes sufficient to obtain a quantitative measure of affinity (e.g., as determined using methods such as ELISA or FACS analysis). A The affinity of the antibody can be proportional to the affinity of the antibody, and can be used to compare, e.g., to determine whether the affinity is higher, e.g., 2-fold higher, to obtain a qualitative measure of affinity or to obtain an estimate of affinity, e.g., by activity in a functional assay, e.g., in vitro or in vivo assay. In some cases, in vitro binding assays indicate in vivo activity. In other cases, in vitro binding assays do not necessarily indicate in vivo activity. In some cases, tight binding is beneficial, but in other cases, tight binding is not desirable in vivo and antibodies with lower binding affinity are more desirable.

[0077] In some embodiments, the heavy chain of any of the anti-galectin-9 antibodies described herein further comprises a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region can be of any suitable origin, e.g., human, mouse, rat, or rabbit. In one particular example, the heavy chain constant region is derived from human IgG (gamma heavy chain) of any of the IgG subfamilies described herein.

[0078] In some embodiments, the heavy chain constant region of the antibodies described herein comprises a single domain (e.g., CH1, CH2, or CH3), or any combination of single domains, of the constant region (e.g., SEQ ID NO: 4, 5, 6). In some embodiments, the light chain constant region of the antibodies described herein comprises a single domain (e.g., CL) of the constant region. Exemplary light and heavy chain sequences are described below. Exemplary light and heavy chain sequences are described below. The hIgG1 LALA sequence contains two mutations L234A and L235A (EU numbering) that inhibit FcgR binding, and a P329G mutation (EU numbering) that abolishes complement C1q binding, thereby abolishing all immune effector functions. The hIgG4 Fab arm replacement mutant sequence contains a mutation (S228P; EU numbering) that inhibits Fab arm replacement. The IL2 signal sequence (MYRMQLLSCIALSLALVTNS; SEQ ID NO: 9) may be located at the N-terminus of the variable region. This is used in expression vectors, where it is cleaved during secretion and therefore not in the mature antibody molecule. The mature proteins (after secretion) begin with "EVQ" for the heavy chain and "DIM" for the light chain. Exemplary heavy chain constant region amino acid sequences are provided below: hIgG1 heavy chain constant region (SEQ ID NO: 10) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* hIgG1 heavy chain constant region (SEQ ID NO: 12)

Chem.

[0079] In some cases, the heavy chain constant region of an anti-galectin-9 antibody disclosed herein (e.g., G9.2-17) may have the C-terminal lysine (K) residue removed, e.g., for manufacturing purposes. The corresponding amino acid sequence without the terminal K residue is provided below: hIgG1 heavy chain constant region without the C-terminal lysine (SEQ ID NO:24) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* hIgG1 LALA heavy chain constant region without the C-terminal lysine (SEQ ID NO:25) [ka] hIgG4 heavy chain constant region without the C-terminal lysine (SEQ ID NO:26) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG* hIgG4 heavy chain constant region without the C-terminal lysine (SEQ ID NO:27) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG* hIgG4 mutant heavy chain constant region without the C-terminal lysine (SEQ ID NO:28) [ka] hIgG4 mutant heavy chain constant region without the C-terminal lysine (SEQ ID NO:29) [ka]

[0080] In some embodiments, an anti-Galectin-9 antibody having any of the above light chain constant regions is paired with a light chain having the following light chain constant region: Light chain constant region (SEQ ID NO: 11) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0081] Exemplary full length anti-galectin-9 antibodies are provided below: G9.2-17 hIgG1 heavy chain (SEQ ID NO: 16) [ka] G9.2-17 hIgG1 heavy chain without the C-terminal lysine residue (SEQ ID NO:30) [ka] G9.2-17 hIgG1 LALA heavy chain (SEQ ID NO: 17) [ka] G9.2-17 hIgG1 LALA heavy chain without the C-terminal lysine residue (SEQ ID NO:31) [ka] G9.2-17 hIgG4 heavy chain (SEQ ID NO: 18) [ka] G9.2-17 hIgG4 heavy chain without the C-terminal lysine residue (SEQ ID NO:32) [ka] G9.2-17 hIgG4 heavy chain (SEQ ID NO:22) [ka] G9.2-17 hIgG4 heavy chain without the C-terminal lysine residue (SEQ ID NO:33) [ka] G9.2-17 hIgG4 Fab arm substitution mutant heavy chain (SEQ ID NO: 19) [ka] G9.2-17 hIgG4 Fab arm substitution mutant heavy chain without the C-terminal lysine residue (SEQ ID NO:34) [ka] G9.2-17 hIgG4 Fab arm substitution mutant heavy chain (SEQ ID NO: 23) [ka] G9.2-17 hIgG4 Fab arm substitution mutant heavy chain without the C-terminal lysine residue (SEQ ID NO:35) [ka]

[0082] Any of the above heavy chains may be combined with a light chain as shown below (SEQ ID NO:15). [ka]

[0083] In some embodiments, the anti-galectin-9 antibody comprises a heavy chain IgG1 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 10. In one embodiment, the constant region of the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO: 10. In one embodiment, the constant region of the anti-galectin-9 antibody comprises a heavy chain IgG1 constant region consisting of SEQ ID NO: 10.

[0084] In some embodiments, the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 20. In one embodiment, the constant region of the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO: 20. In one embodiment, the constant region of the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO: 20.

[0085] In some embodiments, the constant region is derived from human IgG4. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 13. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO: 13. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO: 13.

[0086] In some embodiments, the constant region is derived from human IgG4. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO:20. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO:20. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO:20.

[0087] In any of these embodiments, the anti-galectin-9 antibody comprises a light chain constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 11. In some embodiments, the anti-galectin-9 antibody comprises a light chain constant region comprising SEQ ID NO: 11. In some embodiments, the anti-galectin-9 antibody comprises a light chain constant region consisting of SEQ ID NO: 11.

[0088] In some embodiments, the IgG is a variant with minimal Fc receptor engagement. In one example, the constant region is derived from human IgG1 LALA. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG1 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 12. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG1 constant region comprising SEQ ID NO: 12. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG1 constant region consisting of SEQ ID NO: 12.

[0089] In some embodiments, the anti-galectin-9 antibody comprises a modified constant region. In some embodiments, the anti-galectin-9 antibody comprises a modified constant region that is immunologically inert, e.g., does not induce complement-mediated lysis or stimulate antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC activity can be assessed using the methods disclosed in U.S. Pat. No. 5,500,362. In other embodiments, the constant region is modified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCT Application No. PCT / GB99 / 01441; and / or UK Patent Application No. 9809951.8. In some embodiments, the IgG4 constant region is a mutant with reduced heavy chain substitution. In some embodiments, the constant region is derived from the human IgG4 Fab arm substitution mutant S228P.

[0090] In one embodiment, the constant region of the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 14. In one embodiment, the constant region of the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO: 14. In one embodiment, the constant region of the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO: 14.

[0091] In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 21. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO: 21. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO: 21.

[0092] In some embodiments, the anti-galectin-9 antibody has a light chain corresponding to SEQ ID NO: 15; exemplary heavy chain amino acid sequences correspond to SEQ ID NOs: 10 (hIgG1); 12 (hIgG1 LALA); 13 (hIgG4); 20 (hIgG4); 14 (hIgG4 mutant); and 21 (hIgG4 mutant).

[0093] In some embodiments, the anti-galectin-9 antibody has a light chain comprising, consisting essentially of, or consisting of SEQ ID NO: 15. In some embodiments, the anti-galectin-9 antibody has a heavy chain comprising, consisting essentially of, or consisting of any one of the sequences selected from the group consisting of SEQ ID NOs: 16-19, 22, and 23. In some embodiments, the anti-galectin-9 antibody has a light chain comprising, consisting essentially of, or consisting of SEQ ID NO: 15 and a heavy chain comprising, consisting essentially of, or consisting of any one of the sequences selected from the group consisting of SEQ ID NOs: 16-19. In some embodiments, the anti-galectin-9 antibody has a light chain comprising, consisting essentially of, or consisting of SEQ ID NO: 15 and a heavy chain comprising, consisting essentially of, or consisting of any one of the sequences selected from the group consisting of SEQ ID NOs: 16-19, 22, and 23. In some embodiments, the anti-galectin-9 antibody has a light chain comprising, consisting essentially of, or consisting of SEQ ID NO: 15 and a heavy chain comprising, consisting essentially of, or consisting of any one of the sequences selected from the group consisting of SEQ ID NOs: 16-19, 22, and 23. In some embodiments, the anti-galectin-9 antibody has a light chain consisting of SEQ ID NO: 15 and a heavy chain consisting of any one of the sequences selected from the group consisting of SEQ ID NOs: 16-19, 22, and 23. In one specific embodiment, the anti-galectin-9 antibody has a light chain consisting essentially of SEQ ID NO: 15 and a heavy chain consisting essentially of SEQ ID NO: 19. In another specific embodiment, the anti-galectin-9 antibody has a light chain consisting essentially of SEQ ID NO: 15 and a heavy chain consisting essentially of SEQ ID NO: 20.

[0094] In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 16. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO: 16. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 16.

[0095] In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 17. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO: 17. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 17.

[0096] In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 18. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO: 18. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 18.

[0097] In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 22. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO: 22. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 22.

[0098] In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 19. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO: 19. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 19.

[0099] In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 23. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO: 23. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 23.

[0100] In any of these embodiments, the anti-galectin-9 antibody comprises a light chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 15. In some embodiments, the anti-galectin-9 antibody comprises a light chain sequence comprising SEQ ID NO: 15. In some embodiments, the anti-galectin-9 antibody comprises a light chain sequence consisting of SEQ ID NO: 15.

[0101] In specific examples, the anti-galectin-9 antibody used in the treatment methods disclosed herein has a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 15. In some embodiments, the anti-galectin-9 antibody used in the treatment methods disclosed herein is G9.2-17 IgG4. In some examples, such anti-galectin-9 antibodies do not have a C-terminal lysine residue in the heavy chain.

[0102] Preparation of anti-galectin-9 antibody Antibodies capable of binding to galectin-9 as described herein can be made by any method known in the art, including but not limited to recombinant techniques, an example of which is provided below.

[0103] The nucleic acids encoding the heavy and light chains of the anti-galectin-9 antibody described herein can be cloned into one expression vector, with each nucleotide sequence operably linked to a suitable promoter. In one example, each of the nucleotide sequences encoding the heavy and light chains is operably linked to a separate promoter. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operably linked to a single promoter, such that both the heavy and light chains are expressed from the same promoter. If necessary, an internal ribosome entry site (IRES) can be inserted between the heavy and light chain coding sequences.

[0104] In some instances, the nucleotide sequences encoding the two chains of an antibody are cloned into two vectors, which can be introduced into the same or different cells. When the two chains are expressed in different cells, each of them can be isolated from a host cell expressing such a chain, and the isolated heavy and light chains can be mixed and incubated under suitable conditions to allow the formation of the antibody.

[0105] In general, the nucleic acid sequence encoding one or all chains of the antibody can be operably linked to a suitable promoter and cloned into a suitable expression vector using methods known in the art. For example, the nucleotide sequence and the vector can be contacted with a restriction enzyme to generate complementary ends on each molecule that can pair with each other and join with a ligase under suitable conditions. Alternatively, synthetic nucleic acid linkers can be ligated to the ends of the gene. These synthetic linkers contain nucleic acid sequences that correspond to specific restriction sites in the vector. The choice of expression vector / promoter will depend on the type of host cell used to produce the antibody.

[0106] A variety of promoters can be used to express the antibodies described herein, including, but not limited to, the cytomegalovirus (CMV) intermediate-early promoter, viral LTRs, such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Simian Virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and herpes simplex tk virus promoter.

[0107] Regulatable promoters can also be used, including those that use the lac repressor from E. coli as a transcriptional modulator to regulate transcription from mammalian cell promoters with the lac operator [Brown, M. et al., Cell, 49:603-612 (1987)], and those that use the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimers, VP16, or p65 using astroradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech, and Ariad.

[0108] Regulatable promoters containing repressors with operons can be used. In one embodiment, the lac repressor from E. coli can function as a transcription regulator to regulate transcription from mammalian cell promoters with lac operators (M. Brown et al., Cell, 49:603-612 (1987); Gossen and Bujard (1992); M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)). The tetracycline repressor (tetR) is combined with a transcription activator (VP16) to create a tetR mammalian cell transcription activator fusion protein, tTa (tetR-VP 16), in combination with a minimal promoter with tetO from the human cytomegalovirus (hCMV) major immediate early promoter to create a tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. The tetracycline repressor (tetR) instead of the tetR mammalian cell transcription factor fusion derivative can function as a powerful transmodulator to regulate gene expression in mammalian cells when the tetracycline operator is appropriately placed downstream of the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy, 10(16):1392-1399 (2003)). One particular advantage of this tetracycline-inducible switch is that it does not require the use of tetracycline repressor-mammalian cell transactivator or repressor fusion proteins, which in some cases may be toxic to cells, to achieve its regulatable effect (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)).

[0109] In addition, the vectors may contain, for example, some or all of the following: a selectable marker gene, such as a neomycin gene, for selection of stable or transient transfectants in mammalian cells; an enhancer / promoter sequence from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; an SV40 polyoma origin of replication and ColE1 for proper episomal replication; an internal ribosome binding site (IRES), a versatile multiple cloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for generating vectors containing transgenes are well known and available in the art.

[0110] Examples of polyadenylation signals useful in carrying out the methods described herein include, but are not limited to, the human collagen I polyadenylation signal, the human collagen II polyadenylation signal, and the SV40 polyadenylation signal.

[0111] One or more vectors (e.g., expression vectors) containing a nucleic acid encoding any of the antibodies can be introduced into a suitable host cell for producing the antibody. The host cells can be cultured under conditions suitable for expression of the antibody or any of its polypeptide chains. Such antibodies or their polypeptide chains can be recovered from the cultured cells (e.g., from the cells or culture supernatant) by conventional methods, e.g., affinity purification. If desired, the antibody polypeptide chains can be incubated under suitable conditions for a suitable period of time to allow for production of the antibody.

[0112] In some embodiments, the method for preparing the antibodies described herein includes a recombinant expression vector encoding both the heavy and light chains of an anti-galectin-9 antibody, as also described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods, for example, calcium phosphate-mediated transfection. Positively transformed host cells can be selected and cultured under suitable conditions that allow the expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or the medium. If necessary, the two chains recovered from the host cells can be incubated under suitable conditions that allow the formation of the antibody.

[0113] In one example, two recombinant expression vectors are provided, one encoding the heavy chain of an anti-galectin-9 antibody and the other encoding the light chain of an anti-galectin-9 antibody. Both of the two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Alternatively, each of the expression vectors can be introduced into a suitable host cell. Positive transformants can be selected and cultured under suitable conditions that allow the expression of the antibody polypeptide chains. When the two expression vectors are introduced into the same host cell, the antibody produced therein can be recovered from the host cell or medium. If necessary, the polypeptide chains can be recovered from the host cell or medium and then incubated under suitable conditions that allow the formation of the antibody. When the two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cell or the corresponding medium. The two polypeptide chains can then be incubated under suitable conditions for the formation of the antibody.

[0114] Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography using a Protein A or Protein G-bound matrix.

[0115] Any of the nucleic acids encoding the heavy chain, the light chain, or both, of the anti-galectin-9 antibodies described herein, vectors containing same (e.g., expression vectors), and host cells containing the vectors are within the scope of the disclosure.

[0116] Anti-galectin-9 antibodies thus prepared can be characterized using methods known in the art to detect and / or measure the reduction, amelioration, or neutralization of Galectin-9 biological activity. For example, in some embodiments, ELISA-type assays are suitable for the qualitative or quantitative measurement of Galectin-9 inhibition of Dectin-1 or TIM-3 signaling.

[0117] The biological activity of anti-galectin-9 antibodies can be verified by incubating the candidate antibody with Dectin-1 and Galectin-9 and monitoring any one or more of the following properties: (a) binding and inhibiting signal transduction mediated by the binding between Dectin-1 and Galectin-9; (b) preventing, ameliorating, or treating any aspect of solid tumors; (c) blocking or reducing Dectin-1 activation; (d) inhibiting (reducing) the synthesis, production, or release of Galectin-9. Alternatively, TIM-3 can be used to verify the biological activity of anti-galectin-9 antibodies using the above protocol. Alternatively, CD206 can be used to verify the biological activity of anti-galectin-9 antibodies using the above protocol.

[0118] In some embodiments, biological activity or efficacy is assessed in a subject, for example, by measuring peripheral and intratumoral T cell ratios, T cell activation, or by macrophage phenotyping.

[0119] Additional assays for determining the biological activity of anti-galectin-9 antibodies include measuring CD8+ and CD4+ (conventional) T cell activation (e.g., inflammatory cytokine levels, e.g., IFN gamma, TNF alpha, CD44, ICOS granzyme B, perforin, IL2 (upregulated), CD26L and IL-10 (downregulated)); measuring macrophage reprogramming (in vitro or in vivo), e.g., from an M2 to an M1 phenotype (e.g., increased MHCII, decreased CD206, increased TNF-α and iNOS), or assessing the level of ADCC, e.g., in an in vitro assay as described herein.

[0120] Treatment The present disclosure provides methods for treating solid tumors, including but not limited to PDAC, CRC, HCC, and cholangiocarcinoma, renal cell carcinoma, urothelial carcinoma, head and neck cancer, breast cancer, or other GI solid tumors, using an anti-galectin antibody, e.g., G9.2-17, e.g., G9.2-17 IgG4, alone or in combination with a checkpoint inhibitor, such as an anti-PD-1 antibody. Any of the anti-galectin-9 antibodies described herein can be used in any of the methods described herein. In some embodiments, the anti-galectin-9 antibody is G9.2-17 (e.g., G9.2-17(IgG4)). Such antibodies can be used to treat diseases associated with galectin-9. In some aspects, the present disclosure provides methods for treating cancer. In some embodiments, the methods of the present disclosure are methods for reducing, ameliorating, or eliminating one or more symptom(s) associated with cancer.

[0121] (A) Examples of target solid tumors In some embodiments, the present disclosure provides a method for treating a solid tumor in a subject, the method comprising administering to a subject in need thereof an effective amount of an anti-galectin-9 antibody described herein, including but not limited to G9.2-17 IgG4. In some examples, the methods disclosed herein are applied to a human patient suffering from pancreatic cancer, e.g., pancreatic ductal adenocarcinoma (PDAC). In some cases, the PDAC patient may suffer from metastatic cancer. In some examples, the methods disclosed herein are applied to a human patient suffering from colorectal cancer (CRC). In some embodiments, the colorectal cancer is metastatic. In some examples, the methods disclosed herein are applied to a human patient suffering from hepatocellular carcinoma. In some embodiments, the hepatocellular carcinoma is metastatic. In other examples, the methods disclosed herein are applied to a human patient suffering from cholangiocarcinoma. In some embodiments, the cholangiocarcinoma is metastatic.

[0122] Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with few long-term survivors (Yadav et al., Gastroenterology, 2013, 144, 1252-1261). Inflammation is paramount in PDAC progression, as oncogenic mutations alone are insufficient for tumorigenesis in the absence of concomitant inflammation (Guerra et al., Cancer Cell, 2007, 11, 291-302). Innate and adaptive immunity cooperate to drive tumor progression in PDAC. In particular, specific innate immune subsets within the tumor microenvironment (TME) are well suited to educate adaptive immune effector cells toward a tumor-permissive phenotype. Antigen-presenting cell (APC) populations, including M2-polarized tumor-associated macrophages (TAMs) and myeloid dendritic cells (DCs), induce the generation of immunosuppressive Th2 cells in favor of tumor-protective Th1 cells (Ochi et al., J of Exp Med., 2012, 209, 1671-1687; Zhu et al., Cancer Res., 2014, 74, 5057-5069). Similarly, myeloid-derived suppressor cells (MDSCs) mediate the generation of antitumor CD8+ / - cells in PDAC. +It has been shown to counteract cytotoxic T lymphocyte (CTL) responses and promote metastatic progression (Connolly et al., J Leuk Biol., 2010, 87, 713-725; Pylayeva-Gupta et al., Cancer Cell, 2012, 21, 836-847; Bayne et al., Cancer Cell, 2012, 21, 822-835).

[0123] Pancreatic cancer remains a difficult disease to treat due to its usually slow onset, relatively high resistance to chemotherapy, and lack of effective immunotherapy and targeted therapies. In 2018, approximately 455,000 new cases of pancreatic cancer were reported worldwide, and it is estimated that by 2040, 355,000 new cases of pancreatic cancer will occur annually, with roughly the same number of deaths reported each year as new cases. It is projected to become the second leading cause of cancer-related deaths in the United States by 2030. Despite interventions, life expectancy for patients with metastatic pancreatic cancer is less than one year with current treatments, although most patients (as much as 80%) are in the advanced / metastatic stage when the disease is at a point where curative resection is inevitable. Despite advances in the detection and management of pancreatic cancer, the 5-year survival rate for metastatic disease remains at 10%. The current standard of care for metastatic pancreatic cancer is primarily chemotherapy, but a distinct minority of patients (<10%) with BRCA1 / 2 mutated and mismatch repair deficient tumors may benefit from PARP inhibitors, primarily anti-PD-1 therapy, but for the vast majority of patients with this disease, currently approved immunotherapies are generally ineffective due to the highly immunosuppressive environment.

[0124] Colorectal cancer (CRC), also known as intestinal cancer, colon cancer, or rectal cancer, is any cancer that affects the colon and rectum. CRC is known to be caused by genetic alterations in tumor cells and is also influenced by tumor-host interactions. Recent reports have demonstrated a direct correlation between the density of specific T lymphocyte subpopulations and favorable clinical outcomes in CRC, supporting the key role of T cell-mediated immunity in suppressing tumor progression in CRC.

[0125] CRC presents one of the world's largest cancer burdens. It is currently the fourth most lethal cancer in the world, causing approximately 900,000 deaths annually worldwide. In the United States, 147,950 cases and 53,200 deaths are projected to occur in 2020 (Colorectal Cancer Statistics). Despite significant advances in standard treatment, the 5-year survival rate for metastatic colorectal cancer remains approximately less than 20%. Deaths from CRC are expected to nearly double within the next 20 years. The current standard of care for CRC is chemotherapy regimens combined and / or sequenced with antiangiogenic and anti-epidermal growth factor receptor therapies in selected patients. Furthermore, current immunotherapies are only effective in a small subset of patients whose tumors are mismatch repair deficient and microsatellite instability-high (dMMR / MSI-H) (albeit producing profound and durable responses) (Dekker et al., 2019). Immunotherapy outcomes in microsatellite-stable CRC, the majority of CRC patients, are suboptimal, representing a significant unmet medical need for aggressive immunotherapy. A small subset of CRCs that are dMMR / MSI-H will benefit from immunotherapy (Huygheeta l., 2019), but the majority of patients with mismatch repair proficient or microsatellite-stable CRC will not.

[0126] Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. HCC occurs most frequently in people with chronic liver disease, such as cirrhosis caused by hepatitis B or C infection. HCC is usually accompanied by cirrhosis with extensive lymphocytic infiltration due to chronic viral infection. Many studies have demonstrated that tumor-infiltrating effector CD8+ T cells and Th17 cells correlate with improved survival after surgical resection of tumors. However, tumor-infiltrating effector T cells fail to control tumor growth and metastasis (Pang et al., Cancer Immunol Immunother 2009;58:877-886).

[0127] Cholangiocarcinoma (CCA) is a group of cancers that arise in the bile duct. Cholangiocarcinomas are usually classified by their location in relation to the liver. For example, intrahepatic cholangiocarcinoma accounts for less than 10% of all cholangiocarcinoma cases and arises in the small bile ducts within the liver. In another example, perihilar cholangiocarcinoma (also known as Kratskin tumor), which accounts for more than half of cholangiocarcinoma cases, begins at the hilum where the two main bile ducts leave the liver after joining it. Others are classified as distal cholangiocarcinomas, which begin in bile ducts outside the liver.

[0128] CCA is an aggressive tumor, with most patients suffering from advanced stage disease at presentation. The incidence of CCA is increasing, and effective treatments are urgently needed. Gemcitabine and cisplatin remain the standard first-line systemic therapy for advanced CCA, but with a median survival of less than one year, much remains to be desired. Available evidence to guide therapeutic decisions beyond failure of first-line therapy is scarce. The Food and Drug Administration (FDA) only recently approved the first targeted therapy in this indication for patients with fibroblast growth factor receptor 2 gene fusions and other rearrangements within their tumors. Suboptimal response rates to immunotherapy in human clinical trials mean that the majority of CCA are immune "cold" tumors with a non-T cell infiltrated microenvironment. Indeed, immunotherapy to date has produced response rates not exceeding 17%, and as of the date of this prospectus, no immuno-oncology drugs have been approved (Zayac and Almhanna, 2020).

[0129] The silent presence of these tumors combined with their highly aggressive nature and resistance to chemotherapy causes alarming mortality (a dismal 2% 5-year survival rate for patients with distant disease) (Banales et al., 2020; Bile Duct Cancer Survival, 2020).

[0130] In some embodiments, methods are provided that increase anti-tumor activity (e.g., reduce cell proliferation, tumor growth, tumor volume, and / or tumor mass or weight, or reduce the number of metastatic lesions over time) by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to pre-treatment or control subject levels. In some embodiments, the reduction is measured by comparing the cell proliferation, tumor growth, and / or tumor volume of the subject before and after administration of the pharmaceutical composition. In some embodiments, methods are provided that improve one or more symptoms of cancer by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some embodiments, before, during, and after administration of the pharmaceutical composition, the subject's cancerous cells and / or biomarkers are measured in biological samples, such as blood, serum, plasma, urine, ascites, and / or biopsies from tissues or organs. In some embodiments, a method of reducing the volume, size, weight, or mass of a tumor in a subject to an undetectable size or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the tumor size, weight, or mass of the subject before treatment includes administering a composition of the invention. In other embodiments, a method is provided for reducing the cell proliferation rate or tumor growth rate in a subject to an undetectable rate or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the rate before treatment. In other embodiments, a method of reducing the incidence of metastatic lesions or the number or size of metastatic lesions in a subject to an undetectable rate or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the pre-treatment rate includes administering a composition of the invention.

[0131] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within an acceptable standard deviation, as practiced in the art. Alternatively, "about" can mean within a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and even more preferably up to ±1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. When a particular value is described in the application and claims, unless otherwise indicated, the term "about" is implicit in this context to mean within an acceptable error range of the particular value.

[0132] As used herein, the term "treating" refers to the application or administration of a composition comprising one or more active agents to a subject having a target disease or disorder, a symptom of a disease / disorder, or a predisposition to a disease / disorder, for the purpose of curing, curing, mitigating, alleviating, altering, treating, ameliorating, ameliorating, or affecting the disorder, the symptom of a disease or disorder, or the predisposition to a disease or disorder.

[0133] Alleviating the target disease / disorder includes delaying the onset or progression of the disease, or reducing the severity of the disease, or prolonging survival. Alleviating the disease or prolonging survival does not necessarily require a therapeutic effect. As used herein, "delaying" the onset of the target disease or disorder means postponing, hindering, slowing down, inhibiting, stabilizing, and / or postponing the progression of the disease. This delay can vary in time depending on the disease being treated and / or the medical history of the individual. A method of "delaying" or alleviating the onset of a disease, or a method of delaying the onset of a disease, is a method that reduces the probability of developing one or more symptoms of the disease within a given time frame and / or reduces the severity of symptoms in a given time frame compared to not using the method. Such comparisons are usually based on clinical studies using a sufficient number of subjects to obtain statistically significant results.

[0134] "Onset" or "progression" of a disease refers to the initial symptoms and / or subsequent progression of a disease. Onset of a disease can be detected and assessed using standard clinical techniques well known in the art. However, development also refers to progression that may be undetectable. For purposes of this disclosure, onset or progression refers to the biological course of a condition. "Onset" includes occurrence, recurrence, and onset. As used herein, "onset" or "onset" of a target disease or disorder includes initial onset and / or recurrence.

[0135] (B) Exemplary Patient Populations for Treatment Subjects with any of the above cancers can be identified by routine medical examination, e.g., clinical tests, organ function tests, genetic tests, interventional procedures (biopsy, surgery), any and all relevant imaging modalities.

[0136] In some embodiments, the subject to be treated with the methods described herein is a human cancer patient who has received or is receiving anti-cancer therapy regimens delivered systemically and / or locally, such as chemotherapy, radiation therapy, tumor treating fields (TTFields), immunotherapy, biologic therapy, small molecule inhibitors, anti-hormonal therapy, cell-based therapy, and / or surgery, in any combination or sequence of the therapies outlined. In some embodiments, the subject has previously received the immunomodulatory agents listed above or any other anti-tumor agent or treatment modality. Non-limiting examples of such immunomodulatory agents include, but are not limited to, anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-TIGIT, anti-PVRIG, anti-LAG-3, anti-CD47, anti-CD40, anti-CSFR1, anti-CD73, anti-SIRP, anti-A2AR, anti-OX40, anti-CD137, platinum-based agents, and the like. Non-limiting examples of platinum-based agents include cisplatin, carboplatin, oxaliplatin, nedaplatin, and lobaplatin. In some embodiments, the subject shows disease progression throughout treatment. In other embodiments, the subject is resistant to treatment (either de novo or acquired). In some embodiments, such a subject is documented to be suffering from an advanced malignant tumor (e.g., inoperable or metastatic). Alternatively or additionally, in some embodiments, the subject has no standard treatment options available or is ineligible for standard treatment options, which refers to therapies commonly used in clinical settings to treat the corresponding solid tumor.

[0137] Tumor Treating Electric Fields (TTFields) are a cancer treatment modality that uses alternating electric fields of intermediate frequency (approximately 100-500 kHz) and low intensity (1-3 V / cm) to disrupt cell division. In any of the embodiments described herein, an anti-Galectin-9 antibody, alone or in combination with a checkpoint inhibitor, such as an anti-PD-1 antibody, may be administered prior to, concurrently with, or following a Tumor Treating Electric Fields (TTFields) regimen.

[0138] In some cases, the subject may be a human patient suffering from a refractory disease, such as refractory PDAC, refractory CRC, refractory HCC, or refractory cholangiocarcinoma. As used herein, "refractory" refers to a tumor that does not respond to or becomes resistant to treatment. In some cases, the subject may be a human patient suffering from a recurrent disease, such as recurrent PDAC, recurrent CRC, recurrent HCC, or recurrent cholangiocarcinoma. As used herein, "recurrent" or "recurring" refers to a tumor that recurs or progresses after a period of improvement (e.g., partial or complete response) by treatment.

[0139] In some embodiments, a human patient to be treated with the methods disclosed herein meets one or more of the inclusion and exclusion criteria disclosed in Example 1 below. For example, the human patient may be 18 years of age or older; suffer from histologically unresectable metastatic or inoperable cancer (e.g., for which there is no standard treatment option), have a life expectancy of more than 3 months, have a recent archival tumor sample available for biomarker analysis (e.g., archived species for galectin-9 tumor tissue expression levels assessed by IHC); suffer from measurable disease with an Eastern Cooperative Oncology Group (ECOG) performance status of 0-1 or a Karnofsky score of >70 according to RECISTv 1.1; have no standard treatment options available, MSI-H (microsatellite instability high and MSS (microsatellite stable)); have received at least one line of systemic therapy in the advanced / metastatic setting; have adequate hematological and end-organ function (as defined in Example 1 below; e.g., for HCC in Part 1, ≥50×10 9 / L, neutrophil count ≥ 1 × 10 9 / L, platelet count ≥100×10 9 / L; without transfusion in the previous week, if hemoglobin ≥ 9.0 g / dL, creatinine ≤ 1.5 x ULN, AST (SGOT) ≤ 3 x ULN (≤ 5 x ULN if HCC or liver metastases are present), ALT (SGPT) ≤ 3 x ULN (≤ 5 x ULN if HCC or liver metastases are present), bilirubin ≤ 1.5 x ULN (patients with known Gilbert's disease may have bilirubin ≤ 3.0 x ULN), albumin ≥ 3.0 g / dL, INR and PTT ≤ 1.5 x ULN; and / or amylase and lipase ≦1.5×ULN); have completed treatment if brain metastases are present (see Example 1 below); have no evidence of active infection and no severe infection within the past month; have at least four (4) weeks or five half-lives (whichever is shorter) since the last dose of anti-cancer treatment before the first anti-Gal-9 antibody administration; have subsequent bisphosphonate treatment (zolendronic acid) or denosumab for bone metastases, if applicable. CCR or CCA patients eligible for immediate treatment may have at least one prior course of treatment required in the metastatic setting. In some embodiments, CCR or CCA patients eligible for this treatment have at least one prior course of treatment required in the metastatic setting.

[0140] Alternatively or additionally, subjects suitable for the treatments disclosed herein may not have one or more of the following: diagnosed with metastatic cancer of unknown primary; any patient with active uncontrollable bleeding and bleeding diathesis (e.g., active peptic ulcer disease); received any other investigational agent within 4 weeks or 5 half-lives of administration of anti-galectin-9 antibody; received radiation therapy within 4 weeks of the first administration of anti-galectin-9 antibody, excluding palliative radiation therapy to limited areas, e.g., for the treatment of bone pain or locally painful tumor masses; have a mycotic tumor mass; or, for PDAC patients, received gemcitabine-containing regimen within 6 months of initiating treatment. Patients with locally advanced PDAC who have previously received chemotherapy; have active clinically significant infections greater than grade 2 according to NCI-CTCAE version 5.0; have symptomatic or active brain metastases; have CTCAE grade 3 or greater cytotoxicity (see details and exceptions in Example 1); have a history of a secondary malignancy (see exceptions in Example 1); have severe or uncontrollable systemic disease, evidence of congestive heart failure; have critical non-healing wounds, ongoing ulcers, or untreated fractures; have uncontrollable pleural, pericardial, or ascites necessitating recurrent drainage procedures; have spinal cord compression not definitively treated with surgery and / or radiation therapy. leptomeningeal disease, ongoing or previously treated; significant vascular disease; active autoimmune disorder (see exception in Example 1); requiring systemic immunosuppressive treatment; tumor-related pain (greater than grade 3) unresponsive to extensive analgesic interventions (oral and / or patch); uncontrolled hypercalcemia despite the use of bisphosphonates; any history of immune-related grade 4 adverse events due to prior checkpoint inhibitor therapy (CIT); have undergone organ transplant(s); and / or are undergoing dialysis; for HCC and / or CCA patients, have undergone resection therapy prior to treatment; hepatic encephalopathy or severe hepatic adenoma; and / or Child-Pugh score ≧7.In some cases, the human patient may have advanced metastatic hepatocellular carcinoma while receiving at least one prior line of systemic therapy; may have rejected or not tolerated sorafenib; or may have received a standard treatment that is deemed ineffective, intolerable or inappropriate, or for which an effective standard treatment is not available.

[0141] In some embodiments, a human patient to be treated with the methods disclosed herein meets one or more of the inclusion and exclusion criteria disclosed in Example 1 below. For example, the human patient may be over 18 years old and may have histologically unresectable metastatic cancer (e.g., adenocarcinoma and squamous cell carcinoma). The patient may have measurable disease according to RECIST v.1.1. In some cases, the human patient may have a recent archival tumor sample (e.g., obtained within the last 5 years) available for biomarker analysis (e.g., galectin-9 tumor tissue expression, which may be assessed by IHC). In some cases, the human patient is a PDAC patient who has undergone at least one series of systemic therapy in the metastatic cancer setting. In some cases, the human patient is a PDAC patient who may or may not have undergone systemic therapy prior to receiving an anti-galectin-9 containing regimen. Such patients may be gemcitabine-containing regimen naive or at least 6 months after being treated with a gemcitabine-containing regimen in a prior disease stage setting. Patients may have an Eastern Cooperative Oncology Group (ECOG) performance status of 0-1 and / or a Karnofsky score of >70. Patients may have adequate hematologic and end-organ function, e.g., neutrophil count >1×10 9 / L, platelet count ≥100×10 9 / L, HCC in part 1 ≥ 50 × 10 9 / L; without transfusion in the previous week, hemoglobin ≥ 9.0 g / dL, creatinine ≤ 1.5 x ULN, AST (SGOT) ≤ 3 x ULN (≤ 5 x ULN if HCC or liver metastases present), ALT (SGPT) ≤ 3 x ULN (≤ 5 x ULN if HCC or liver metastases present), bilirubin ≤ 1.5 x ULN (patients with known Gilbert's disease may have bilirubin ≤ 3.0 x ULN), albumin ≥ 3.0 g / dL, INR and PTT ≤ 1.5 x ULN; and / or amylase and lipase ≤ 1.5 x ULN. In some cases, human patients show no evidence of active infection or infection requiring parenteral antibiotics, nor any severe infection within 4 weeks prior to the start of treatment. Pancreatic, biliary, or enteric fistulas are permitted if they are appropriately non-infectious and controlled with patent drains.

[0142] Alternatively or additionally, human patients receiving any of the treatments disclosed herein may be free of: (i) metastatic cancer of unknown primary site; (ii) clinically significant active uncontrollable bleeding, any bleeding diathesis (e.g., active peptic ulcer disease); (iii) radiation therapy within 4 weeks of the first dose of treatment; (iv) the presence of a fungal tumor mass; (v) CTCAE grade 3 or higher toxicity from prior cancer treatment (excluding alopecia and vitiligo); (v) history of a second malignancy; (vi) evidence of severe or uncontrollable systemic disease, New York Heart Association (NYHA) class 2 or greater congestive heart failure, or myocardial infarction (MI) within 6 months; (vii) severe non-healing wounds, active ulcers, or untreated fractures; (viii) uncontrollable pleural effusion, pericardial effusion, or ascites requiring frequent drainage procedures; (ix) a chimeric antibody or (x) history of severe allergic, anaphylactic, or other hypersensitivity reactions to the humanized antibody or fusion protein; (x) history of significant vascular disease within 6 months of the procedure (e.g., aortic aneurysm or recent arterial thrombosis requiring surgical repair), history of pulmonary embolism, stroke, or transient ischemic attack within 3 months prior to the procedure, and / or history of abdominal fistula or gastrointestinal perforation within 6 months prior to the procedure; (xi) active autoimmune disease (excluding type I diabetes mellitus, hypothyroidism requiring hormone replacement only, vitiligo, psoriasis, or alopecia); (xii) requiring systemic immunosuppressive treatment; (xii) tumor-related pain (greater than grade 3) unresponsive to extensive analgesic interventions (oral and / or patch); (xiii) uncontrollable hypercalcemia despite the use of bisphosphonates; (xiv) has undergone organ transplant(s).

[0143] In some cases, the subject is a human patient with an elevated level of galectin-9 compared to a control level. The level of galectin-9 can be the plasma or serum level of galectin-9 in the human patient. In other examples, the level of galectin-9 is the level of galectin-9 in cancer cells in a tumor. In other examples, the level of galectin-9 is the level of galectin-9 in immune cells in a tumor. In other examples, the level of galectin-9 can be the level of cell surface galectin-9, e.g., the level of galectin-9 on cancer cells. In one example, the level of galectin-9 can be the level of galectin-9 expressing cancer cells, e.g., on the surface of cancer cells, or the level of galectin-9 expressed on immune cells, measured in patient-derived organotypic tumor spheroids (PDOT), which can be prepared, e.g., by the methods disclosed in the Examples below. The control level can refer to the level of galectin-9 in a corresponding sample of a homogenous subject (e.g., human) without a solid tumor. In some examples, the control level represents the level of galectin-9 in a healthy subject, hi some embodiments, the control level may be a baseline level before treatment.

[0144] To identify such subjects, a suitable biological sample can be obtained from a subject suspected of suffering from a solid tumor, and the biological sample can be analyzed to determine the level of galectin-9 contained therein (e.g., free, cell surface expressed, or total) using conventional methods, such as ELISA or FACS. In some embodiments, organoid cultures are prepared, for example, as described herein, and used to evaluate the level of galectin-9 in a subject. Single cells derived from a particular fraction obtained as part of the organoid preparation process are also suitable for evaluating the level of galectin-9 in a subject. In some cases, an assay for measuring the level of free form or cell surface expressed galectin-9 includes the use of an antibody that specifically binds to galectin-9 (e.g., specifically binds to human galectin-9). Any of the anti-galectin-9 antibodies known in the art can be tested for suitability in any of the above assays and then used in such assays in a predetermined manner. In some embodiments, the antibodies described herein (e.g., G9.2-17 antibody) can be used in such assays. In some embodiments, the antibody is described in U.S. Pat. No. 10,344,091 and WO 2019 / 084553, the relevant disclosures of each of which are incorporated by reference for the purposes and subject matter referenced herein. In some examples, the anti-galectin-9 antibody is a Fab molecule. Assay methods for determining galectin-9 levels as disclosed herein are also within the scope of this disclosure.

[0145] (C) Exemplary treatment conditions In some embodiments, an antibody described herein, e.g., G9.2-17, is administered to a subject in need of treatment in an amount sufficient to suppress Galectin-9 (and / or Dectin-1 or TIM-3 or CD206) activity in immunosuppressive immune cells of a tumor in vivo by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more). In other embodiments, an antibody described herein, e.g., G9.2-17, is administered in an amount effective to reduce Galectin-9 (and / or Dectin-1 or TIM-3 or CD206) activity levels in immunosuppressive immune cells by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) (compared to levels before treatment or in a control subject). In some embodiments, an antibody described herein, e.g., G9.2-17, is administered to a subject in need of treatment in an amount sufficient to promote M1-like programming in TAMs in vivo (compared to pre-treatment or control subject levels) by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more).

[0146] Conventional methods known to those skilled in the art can be used to administer the pharmaceutical composition to a subject, depending on the type of disease or site of disease to be treated, hi some embodiments, the anti-galectin-9 antibody may be administered to the subject by intravenous infusion.

[0147] Injectable compositions may contain various carriers, such as vegetable oils, dimethylactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol). For intravenous infusion, water-soluble antibodies may be administered by drip infusion, in which a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is injected. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the antibody, may be administered by dissolving it in a pharmaceutical excipient, such as water for injection, 0.9% saline, or 5% glucose solution.

[0148] An effective amount of the pharmaceutical compositions described herein can be administered to a subject (e.g., a human) in need of treatment via a suitable route, either systemically or locally. In some embodiments, the anti-galectin-9 antibody is administered intravenously, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-arterial, intra-articular, intrasynovial, intrathecal, intratumoral, suburothelial, oral, inhalation, or topical routes. In one embodiment, the anti-galectin-9 antibody is administered to the subject by intravenous infusion. In one embodiment, the anti-galectin-9 antibody is administered to the subject intraperitoneally.

[0149] As used herein, an "effective amount" refers to the amount of each active agent required to confer a therapeutic effect on a subject, alone or in combination with one or more other active agents. In some embodiments, the therapeutic effect is a decrease in Galectin-9 activity and / or amount / expression, a decrease in Dectin-1 signaling, a decrease in TIM-3 signaling, a decrease in CD206 signaling, or an increase in an anti-tumor immune response in the tumor microenvironment. Non-limiting examples of an increase in an anti-tumor response include an increase in activation levels of effector T cells, or a switch of TAMs from an M2 phenotype to an M1 phenotype. In some cases, the anti-tumor response includes an increase in an ADCC response. Determining whether an amount of antibody has achieved a therapeutic effect will be apparent to one of skill in the art. As will be recognized by those of skill in the art, an effective amount will vary depending on the particular condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex, and weight, duration of treatment, the nature of the combination therapy (if any), the particular route of administration, and similar factors within the knowledge and expertise of the medical practitioner. These factors are well known to those of skill in the art and can be addressed with no more than routine experimentation. In general, it is preferred that maximum doses of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.

[0150] Generally, empirical considerations such as half-life contribute to the determination of dosage. For example, antibodies compatible with the human immune system, such as humanized antibodies or fully human antibodies, are used in some cases to extend the half-life of the antibody and prevent the antibody from being attacked by the host's immune system. The frequency of administration can be determined and adjusted over the course of treatment, and is generally, but not necessarily, based on the treatment and / or suppression and / or improvement and / or delay of the target disease / disorder. Alternatively, a sustained release formulation of the antibody may be appropriate. A variety of formulations and devices for achieving sustained release are known in the art.

[0151] In one example, the dosage of the antibody described herein is empirically determined in an individual who is administered one or more doses of the antibody. The individual is given increasing doses of the antagonist. To assess the effectiveness of the antagonist, indicators of the disease / disorder can be tracked.

[0152] (D) Anti-galectin-9 antibody treatment In some embodiments, the anti-galectin-9 antibodies described herein are used to treat the target cancers disclosed herein, i.e., no other anti-cancer therapies are administered simultaneously with the therapy using the anti-galectin-9 antibodies. In some cases, the anti-galectin-9 antibodies, such as G9.2-17(IgG4) disclosed herein, may be used in monotherapy (i.e., the anti-galectin-9 antibodies are the only active agents). In other cases, the anti-galectin-9 antibodies, such as G9.2-17(IgG4) disclosed herein, may be used in combination therapy, e.g., in combination with a PD-1 inhibitor as disclosed herein.

[0153] In some embodiments, the disclosure provides a method for treating a solid tumor in a subject, the method comprising administering to a subject in need thereof an effective amount of an anti-galectin-9 antibody, or a pharmaceutical composition comprising a galectin-9 antibody or antigen-binding fragment thereof described herein. Any of the anti-galectin-9 antibodies disclosed herein, for example, antibody G9.2-17 (IgG4), can be used in the methods disclosed herein (e.g., having a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 15).

[0154] In some embodiments, the antibody is administered, for example, by intravenous infusion, once every 2-6 weeks. In some examples, the antibody may be administered once every 2-4 weeks, for example, once every 2 weeks. In other examples, the antibody may be administered once a week. In some embodiments, the anti-galectin-9 antibody disclosed herein (e.g., G9.2-17 IgG4) is administered intravenously via an infusion period of 30 minutes to 6 hours. In some examples, the intravenous infusion of the anti-galectin-9 antibody may be performed for 30 minutes to 2 hours. In other examples, the anti-galectin-9 antibody may be administered via a longer infusion period, for example, about 2-6 hours, for example, about 2-4 hours or about 4-6 hours. In specific examples, the anti-galectin-9 antibody may be intravenously infused for a period of about 3 hours, about 4 hours, about 5 hours, or about 6 hours.

[0155] In some embodiments, an anti-galectin-9 antibody disclosed herein (e.g., G9.2-17(IgG4)) used to treat a solid tumor (e.g., those disclosed herein) can be administered to a subject at a dose of 0.2 mg / kg to about 32 mg / kg, e.g., the dose can be selected from dose levels of 0.2 mg / kg, 0.63 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 6.3 mg / kg, 8 mg / kg, 10 mg / kg, 12 mg / kg, and 16 mg / kg, or more. In some embodiments, an anti-galectin-9 antibody can be administered to a subject at a dose of about 1 mg / kg to about 32 mg / kg, e.g., the dose can be selected from dose levels of 2 mg / kg, 4 mg / kg, 8 mg / kg, 12 mg / kg, and 16 mg / kg, or more. In some examples, the anti-galectin-9 antibody may be administered to a subject at a dose of about 0.2 mg / kg to about 32 mg / kg, for example, the dose may be selected from dose levels of 0.2 mg / kg, 0.63 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 6.3 mg / kg, 10 mg / kg, or 16 mg / kg, or more.

[0156] In some embodiments, the anti-galectin-9 antibody is administered once every two weeks. In some embodiments, the anti-galectin-9 antibody is administered once every two weeks for one cycle, once every two weeks for two cycles, once every two weeks for three cycles, once every two weeks for four cycles, or once every two weeks for more than four cycles. In some embodiments, the anti-galectin-9 antibody is administered once every two weeks for four cycles. In some embodiments, the anti-galectin-9 antibody is administered once every four or six weeks. In some embodiments, the treatment period is 12 to 24 months or longer. In some embodiments, the cycle spans a period of three to six months, or six to twelve months, or 12 to 24 months, or longer. In some embodiments, the length of the cycle is modified, e.g., temporarily or permanently, to a longer period, e.g., three or four weeks. In some embodiments, the use further comprises administering to the subject an immune checkpoint inhibitor, e.g., an anti-PD-1 antibody, as described herein, e.g., administered according to a regimen described herein. In some embodiments, the interval or cycle is 1 week. In some embodiments, the interval or cycle is 2 weeks. In certain embodiments, the interval or cycle is 2 weeks. In certain embodiments, the interval or cycle is 3 weeks. In certain embodiments, the interval or cycle is 4 weeks.

[0157] The solid tumor is selected from pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), cholangiocarcinoma (CAA), renal cell carcinoma (RCC), urothelial carcinoma, head and neck cancer, breast cancer, lung cancer, and other GI solid tumors, and in some embodiments, the regimen or dosing schedule is once every two weeks for 1 cycle, once every two weeks for 2 cycles, once every two weeks for 3 cycles, once every two weeks for 4 cycles, or once every two weeks for 4 cycles. In some embodiments, the treatment is once every two weeks for 1-3 months, once every two weeks for 3-6 months, once every two weeks for 6-12 months, or once every two weeks for 12-24 months, or more. In some embodiments, the antibody is administered by intravenous infusion.

[0158] In some embodiments, the regimen or dosing schedule is once every 3 or 4 weeks for 1 cycle, once every 3 or 4 weeks for 2 cycles, once every 3 or 4 weeks for 3 cycles, once every 3 or 4 weeks for 4 cycles, or once every 3 or 4 weeks for more than 4 cycles. In some embodiments, the treatment is once every 3 or 4 weeks for 1-3 months, once every 3 or 4 weeks for 3-6 months, once every 3 or 4 weeks for 6-12 months, or once every 3 or 4 weeks for 12-24 months, or more. In some embodiments, the treatment is once every 3 or 4 weeks for 1-3 months, once every 6 weeks for 3-6 months, once every 3 or 4 weeks for 6-12 months, or once every 3 or 4 weeks for 12-24 months, or more. In some embodiments, the treatment is for longer than 24 months, as clinically indicated. In some embodiments, the antibody is administered by intravenous infusion.

[0159] In other embodiments, an anti-galectin-9 antibody such as G9.2-17(IgG4) may be administered to a human patient once a week at a suitable dose (e.g., a dose disclosed herein). For example, 2.0 mg / kg of G9.2-17(IgG4) may be administered to a human patient once a week. For example, 6.3 mg / kg of G9.2-17(IgG4) may be administered to a human patient once a week. In another example, 10 mg / kg of G9.2-17(IgG4) may be administered to a human patient once a week. Alternatively, 12 mg / kg of G9.2-17(IgG4) may be administered to a human patient once a week. In yet another example, 16 mg / kg of G9.2-17(IgG4) may be administered to a human patient once a week.

[0160] In some cases, the anti-galectin-9 antibody may be administered to the human patient for at least 2 cycles, at least 3 cycles, at least 4 cycles, at least 5 cycles, at least 6 cycles, or more. In some cases, the treatment period may be 6 months to 12 months. In other cases, the treatment period may be 12 months to 24 months. In other cases, the treatment period may be more than 24 months.

[0161] In some cases, an anti-Gal-9 antibody, such as G9.2-17(IgG4) disclosed herein, may be administered to a subject at a fixed dose, e.g., about 650 mg to about 1120 mg, once a week to once every four weeks. In some examples, an anti-Gal-9 antibody is administered to a subject at about 650 mg to about 700 mg once a week. In some examples, an anti-Gal-9 antibody is administered to a subject at about 650 mg to about 700 mg once every two weeks. In some examples, an anti-Gal-9 antibody is administered to a subject at about 1040 mg to about 1120 mg once a week. In some examples, an anti-Gal-9 antibody is administered to a subject at about 1040 mg to about 1120 mg once every two weeks.

[0162] In some embodiments, the dosage(s) are adjusted according to the patient's response to treatment. In some embodiments, the dosage is altered between treatment intervals. In some embodiments, the treatment may be temporarily stopped. In some embodiments, the treatment may be temporarily stopped. In some embodiments, the anti-galectin-9 therapy is temporarily stopped. In some embodiments, the checkpoint inhibitor therapy used in combination with the anti-galectin-9 antibody is temporarily stopped. In some embodiments, both are temporarily stopped.

[0163] Alternatively, human patients may be started on a low dose of an anti-galectin-9 antibody, such as G9.2-17(IgG4), disclosed herein, e.g., 0.2 mg / kg, 0.63 mg / kg, or 2 mg / kg. If no side effects are observed, the dose of the antibody may be increased, e.g., to 6.3 mg / kg, 10 mg / kg, or 16 mg / kg.

[0164] Considering that the tumor-promoting effects of galectin-9 are mediated through interactions with immune cells (e.g., with lymphoid cells via TIM-3, CD44, and 41BB, and with macrophages via Dectin-1 and CD206), and considering that galectin-9 is expressed on a large number of tumors, targeting galectin-9, for example, using galectin-9-binding antibodies to inhibit interactions with its receptors, provides a therapeutic approach that can be applied to a variety of different tumor types.

[0165] (E) Combination Therapy In some embodiments, any of the anti-galectin-9 antibodies described herein (e.g., a G9.2-17 antibody, such as G9.2-17(IgG4) disclosed herein) can be used in any of the methods described herein and is administered in combination with a second therapeutic agent, e.g., a checkpoint inhibitor, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody. Non-limiting examples of checkpoint inhibitors and administration regimens are provided elsewhere.

[0166] Thus, the treatment methods disclosed herein may further include administering to the subject an inhibitor of a checkpoint molecule, e.g., PD-1. Examples of PD-1 inhibitors include anti-PD-1 antibodies, e.g., pembrolizumab, nivolumab, tislelizumab, dostarlimab, and cemiplimab. Such checkpoint inhibitors can be administered simultaneously or sequentially (in any order) with the anti-galectin-9 antibody according to the present disclosure. In some embodiments, the checkpoint molecule is PD-L1. Examples of PD-L1 inhibitors include anti-PD-L1 antibodies, such as durvalumab, avelumab, and atezolizumab. In some embodiments, the checkpoint molecule is CTLA-4. An example of a CTLA-4 inhibitor is the anti-CTLA-4 antibody ipilimumab. In some embodiments, the inhibitor targets a checkpoint molecule selected from CD40, GITR, LAG-3, OX40, TIGIT, and TIM-3.

[0167] In some embodiments, the anti-galectin-9 antibody improves overall response, e.g., at 3 months, compared to a regimen including an inhibitor of a checkpoint molecule (e.g., anti-PD-1, e.g., nivolumab) alone.

[0168] In some embodiments, the anti-PD-1 antibody is PD-1 nivolumab, and the methods described herein comprise administering nivolumab intravenously to a subject at a dose of 240 mg once every two weeks.

[0169] In some embodiments, the antibody that binds PD-1 is combined with an anti-galectin-9 antibody disclosed herein (e.g., G9.2-17(IgG4)). In some cases, the anti-PD-1 antibody can be administered using a fixed dose. In some embodiments, the antibody that binds PD-1 is nivolumab, which can be administered to the subject at a dose of about 240 mg every two weeks or about 480 mg every four weeks. In some embodiments, the antibody that binds PD-1 is prembrolizumab, which can be administered at a dose of 200 mg once every three weeks. In some embodiments, the antibody that binds PD-1 is cemiplimab, which can be administered intravenously at a dose of about 350 mg once every three weeks. In some embodiments, the antibody that binds PD-1 is tislelizumab, which can be administered intravenously at a dose of about 200 mg once every three weeks or at a dose of about 400 mg once every six weeks. In some embodiments, the antibody that binds to PD-1 is dostarlimab, which can be administered at a dose of about 500 mg every three weeks or about 1000 mg every six weeks.

[0170] In some embodiments, an antibody that binds PD-L1 (anti-PD-L1 antibody) is combined with an anti-galectin-9 antibody disclosed herein (e.g., G9.2-17(IgG4)). In some cases, the antibody that binds PD-L1 is administered using a fixed dose. In some examples, the anti-PD-L1 antibody is atezolizumab, which may be administered intravenously at a dose of 1200 mg once every three weeks. In some examples, the anti-PD-L1 antibody is avelumab, which may be administered intravenously at a dose of 10 mg / kg every two weeks. In some embodiments, the anti-PD-L1 antibody is durvalumab, which may be administered intravenously at a dose of 1500 mg every four weeks.

[0171] In a specific example, any of the methods disclosed herein include (i) administering to a human patient having a target solid tumor disclosed herein (e.g., pancreatic ductal adenocarcinoma (PDAC or PDAC), CRC, HCC, CCA, RCC, urothelial carcinoma, head and neck cancer, breast cancer, lung cancer, or other GI solid tumor) any of the anti-galectin-9 antibodies disclosed herein (e.g., G9.2-17, such as an antibody having a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 5) at a dose of about 0.2 to about 32 mg / kg (e.g., about 3 mg / kg or about 15 mg / kg) once every two weeks; and (ii) administering to the human patient an effective amount of an anti-PD-1 antibody (e.g., nivolumab, prembrolizumab, tislelizumab, or cemiplimab, dostallimab, durvalumab, avelumab, and atezolizumab).

[0172] In other specific examples, any of the methods disclosed herein include (i) administering to a human patient having a target solid tumor disclosed herein (e.g., pancreatic ductal adenocarcinoma (PDAC or PDAC), CRC, HCC, CCA, RCC, urothelial carcinoma, head and neck cancer, breast cancer, lung cancer, or other GI solid tumor) any of the anti-galectin-9 antibodies disclosed herein (e.g., G9.2-17, such as an antibody having a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 5) at a dose of about 0.2 to about 32 mg / kg (e.g., about 10 mg / kg or about 16 mg / kg) once a week; and (ii) administering to the human patient an effective amount of an anti-PD-1 or anti-PD-L1 antibody (e.g., nivolumab, prembrolizumab, tislelizumab, or cemiplimab, dostallimab, durvalumab, avelumab, and atezolizumab).

[0173] Without wishing to be bound by theory, it is believed that anti-galectin-9 antibodies can reprogram the immune response against tumor cells, e.g., through inhibition of Dectin-1 activity, by inhibiting the activity of Dectin-1, by inhibiting the activity of γδ T cells infiltrating the tumor microenvironment and / or enhancing immune surveillance against tumor cells, e.g., by activating CD4+ and / or CD8+ T cells. Thus, the combination of anti-galectin-9 antibodies and immunomodulatory agents as described herein is expected to significantly enhance anti-tumor efficacy.

[0174] In some embodiments, methods are provided in which an anti-galectin-9 antibody is administered simultaneously with a checkpoint inhibitor. In some embodiments, the anti-galectin-9 antibody is administered before or after the checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is administered systemically. In some embodiments, the checkpoint inhibitor is administered locally. In some embodiments, the checkpoint inhibitor is administered intravenously, e.g., as a bolus or by continuous infusion over a period of time, intramuscularly, intraperitoneally, intracerebrospinal, subcutaneously, intra-arterial, intra-articular, intravesically, intrasynovial, intrathecal, intratumoral, or suburothelial routes. In one embodiment, the checkpoint inhibitor is administered to the subject by intravenous infusion.

[0175] In some cases, a checkpoint inhibitor, such as any of the anti-PD-1 antibodies disclosed herein and any of the anti-galectin-9 antibodies disclosed herein, such as G9.2-17 (e.g., G9.2-17(IgG4)), may have the same day administration. In some examples, the checkpoint inhibitor may be administered to the subject prior to administration of the anti-galectin-9 antibody. In other examples, the administration of the checkpoint inhibitor, e.g., the anti-PD-1 antibody, and the administration of the anti-galectin-9 antibody are performed on two consecutive days. The checkpoint inhibitor, e.g., the anti-PD-1 antibody, may be administered to the subject on the first day of administration, and the anti-galectin-9 antibody may be administered to the subject the following day.

[0176] In other examples, a checkpoint inhibitor, such as any of the anti-PD-1 antibodies disclosed herein, may be administered about 1 to 7 days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days) prior to administration of an anti-galectin-9 antibody disclosed herein, such as G9.2-17.

[0177] In some instances, the anti-galectin-9 antibody can be administered to the subject prior to administration of a checkpoint inhibitor, such as an anti-PD-1 antibody. In other instances, the administration of the anti-galectin-9 antibody and the administration of the checkpoint inhibitor, such as an anti-PD-1 antibody, are performed on two consecutive days. The anti-galectin-9 antibody can be administered to the subject on the first day of administration, and the checkpoint inhibitor, such as an anti-PD-1 antibody, can be administered to the subject on the following day.

[0178] In other cases, an anti-galectin-9 antibody disclosed herein, such as G9.2-17, may be administered about 1 to 7 days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days) prior to administration of a checkpoint inhibitor, such as any of the anti-PD-1 antibodies disclosed herein.

[0179] In any of the embodiments of the methods described herein, the anti-galectin-9 antibody can be administered once every two weeks for one cycle, once every two weeks for two cycles, once every two weeks for three cycles, once every two weeks for four cycles, or once every two weeks for more than four cycles (alone or in combination with an anti-PD-1 antibody). In some embodiments, the treatment is for 1-3 months, 3-6 months, 6-12 months, 12-24 months, or more. In some embodiments, the treatment is once every two weeks for 1-3 months, once every two weeks for 3-6 months, once every two weeks for 6-12 months, or once every two weeks for 12-24 months, or more.

[0180] Alternatively or additionally, the anti-galectin-9 antibody may be used in combination with a regimen including UGN-102, UGN-201, or UGN-302. In one embodiment, UGN-102, UGN-201, or UGN-302 is formulated into a hydrogel, e.g., a hydrogel based on reverse thermal hydrogel technology. In some examples, the anti-galectin-9 antibody may be administered prior to UGN-102, UGN-201, or UGN-302. In some examples, the anti-galectin-9 antibody may be administered simultaneously with UGN-102, UGN-201, or UGN-302. In some examples, the anti-galectin-9 antibody may be administered after UGN-102, UGN-201, or UGN-302.

[0181] (F) Monitoring Treatment Response Response to a treatment, e.g., a treatment of a solid tumor described herein, can be evaluated according to the RECIST or RECIST 1.1 criteria and / or irRC, irRECIST, iRECIST, imRECIST PDAC described below: Example 1 below and Eisenhower et al., New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1); European Journal Of Cancer 45 (2009) 228-247; or Borcoman et al., Annals of Oncology 30: 385-396, 2019; Nishino et al., Clin Cancer Res 2013; 19 (14): 3936-3943, the contents of each of which are incorporated herein by reference in their entirety.

[0182] In some embodiments, methods are provided for improving and / or managing overall response / tumor burden / tumor size (e.g., at about 2, 3, 6, or 12 months or later), comprising administering an anti-galectin-9 antibody described herein, e.g., compared to baseline levels obtained before initiation of a G9.2-17 IgG4 treatment regimen. In some embodiments, the methods are for improving and / or managing overall response / tumor burden / tumor size at about 2 months. In some embodiments, when the anti-galectin-9 antibody is administered in a combination regimen with a checkpoint inhibitor, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody, the treatment may improve or manage overall response / tumor burden / tumor size (e.g., at about 2, 3, 6, or 12 months or later), e.g., compared to baseline levels obtained before initiation of treatment. In some embodiments, methods are provided that result in a complete response, partial response, or stable disease (e.g., measured at about 2 months, 3 months, 6 months, or 12 months, or thereafter, or any other time point as clinically indicated), the methods comprising administering an anti-galectin-9 antibody as described herein. Such a response may be temporary or permanent over a period of time.

[0183] In some embodiments, the method improves the likelihood of a complete response, partial response, or stable disease, for example, compared to baseline levels obtained before the initiation of the G9.2-17 IgG4 treatment regimen (e.g., measured at about 2, 3, 6, or 12 months, or thereafter, or any other time point as clinically indicated). Such a response may be temporary or permanent over a period of time. In some embodiments, the treatment may result in a reduction or attenuation of progressive disease, for example, compared to baseline levels obtained before the initiation of the G9.2-17 IgG4 treatment regimen (e.g., measured at about 2, 3, 6, or 12 months, or thereafter, or any other time point as clinically indicated). Such attenuation may be temporary or permanent. In any of these embodiments, the anti-galectin-9 antibody may be administered in combination with a checkpoint inhibitor, for example, an anti-PD-1 antibody.

[0184] In some embodiments, the present disclosure provides methods for ameliorating disease progression or reducing progressive disease (e.g., measured at about 3 months, 6 months, or 12 months, or thereafter, or any other time point as clinically indicated). The methods include administering to a subject a therapeutically effective amount of an anti-galectin-9 antibody disclosed herein. In any of these embodiments, the anti-galectin-9 antibody may be administered in combination with a checkpoint inhibitor, e.g., an anti-PD-1 antibody.

[0185] In any of the methods described herein, partial response, stable disease, complete response, partial response, stable disease, progressive disease, ongoing disease (e.g., measured at about 2 months, 3 months, 6 months, or 12 months, or thereafter, or any other time point as clinically indicated) can be assessed according to irC criteria, RECIST criteria, RECIST1.1, irRECIST or iRECIST, or imRECIST criteria, or other criteria known in the art (see, e.g., Borcoman et al., Annals of Oncology 30:385-396, 2019'iRC: Hoos et al., J. Immunother. 30(1):1-15).

[0186] A partial response is a reduction in the size of a tumor or the extent of cancer in the body, i.e., tumor burden, in response to treatment, compared to the baseline level before treatment begins. For example, according to the RECIST response criteria, a partial response is defined as a reduction of at least 30% in the sum of the diameters of the target lesions, based on the baseline sum diameter. Progressive disease is disease that is progressing, expanding, or worsening. For example, according to the RECIST response criteria, progressive disease includes disease in which an increase of at least 20% in the sum of the diameters of the target lesions is observed, and the sum must also show an absolute increase of at least 5 mm. In addition, the appearance of one or more new lesions is also considered to be progression. A tumor that has neither decreased nor increased in extent or severity, compared to the baseline level before treatment begins, is considered to have stable disease. For example, according to the RECIST response criteria, stable disease occurs when there is neither sufficient shrinkage to qualify as a partial response, nor sufficient increase to qualify as progressive disease, based on the minimum sum diameter under study.

[0187] In some embodiments, the present disclosure provides a method for reducing or maintaining tumor size in a subject, including a human subject, for a permanent or minimal period of time compared to a baseline tumor size prior to the initiation of treatment in the subject (e.g., measured at about 2 months, 3 months, 6 months, or 12 months, or thereafter, or any other time point as clinically indicated), the method comprising administering to the subject a therapeutically effective amount of an anti-galectin-9 antibody, alone or in combination with a checkpoint inhibitor, e.g., an anti-PD-1 antibody. Tumor size, e.g., tumor diameter, can be measured according to methods known in the art, including measurements from CT and MRI images combined with various software tools, according to specific measurement protocols, as described, e.g., in Eisenhower et al., cited above. Thus, in some embodiments, tumor size is measured at regularly scheduled restaging scans (e.g., CT with / without contrast, MRI with / without contrast, PET-CT (diagnostic CT) and / or X-ray, ultrasound and / or other related imaging modalities). In some embodiments, reduction in tumor size, maintenance of tumor size refers to the size of the target lesion. In some embodiments, tumor size reduction, tumor size maintenance refers to the size of non-target lesions.According to RECIST1.1, if multiple measurable lesions are present at baseline, a total of up to 5 total lesions (and up to 2 lesions per organ) representing all involved organs should be identified as target lesions.All other lesions (or disease sites), including pathological lymph nodes, should be identified as non-target lesions.

[0188] In some embodiments, the present disclosure provides methods for increasing the likelihood of reducing or maintaining tumor burden (e.g., measured at about 2 months, 3 months, 6 months, or 12 months, or thereafter, or any other time point as clinically indicated), the methods comprising administering to a subject a therapeutically effective amount of an anti-galectin-9 antibody disclosed herein, alone or in combination with a checkpoint inhibitor, e.g., an anti-PD-1 antibody. In some embodiments, the treatment may result in a greater likelihood of reducing tumor burden, or maintaining tumor burden (e.g., measured at about 2 months, 3 months, 6 months, or 12 months, or thereafter, or any other time point as clinically indicated). As used herein, tumor burden refers to the amount of cancer, tumor size, or tumor volume in a subject's body, occupying all disease sites. Tumor burden can be measured using methods known in the art, including, but not limited to, FDG positron emission tomography (FDG-PET), magnetic resonance imaging (MRI), and optical imaging, including bioluminescence imaging (BLI) and fluorescence imaging (FLI).

[0189] In some embodiments, the methods described herein extend the time to disease progression or progression-free survival (e.g., measured at about 2 months, 3 months, 6 months, or 12 months, or thereafter, or at any other time point clinically indicated after treatment initiation). Progression-free survival can be either permanent or progression-free survival over a period of time. In some embodiments, the methods provide a higher likelihood of progression-free survival (permanent progression-free survival, or for a period of time, e.g., 3, 6, or 12 months, or measured at about 2 months, 3 months, 6 months, or 12 months, or thereafter, or at any other time point clinically indicated after treatment initiation). Progression-free survival (PFS) is defined as the time from random assignment in a clinical trial, e.g., from the start of treatment, to disease progression or death from any cause. In some embodiments, the methods achieve longer survival or a higher likelihood of survival in a particular period of time, e.g., at 6 months or 12 months.

[0190] Response to treatment, for example, treatment of solid tumors as described herein, can be evaluated according to iRECIST criteria, as described in Seymour et al., iRECIST: guidelines for response criteria for use in trials; The Lancet, Vol18, March 2017, the contents of which are incorporated herein by reference in their entirety. To ensure consistent design and data collection, particularly in cancer immunotherapy trials, iRECIST was developed to use modified RECIST1.1 criteria, and can be used as a guideline for a standard approach to measurement and definition of solid tumors for objective changes in tumor size used in trials in which immunotherapy is used. iRECIST is based on RECIST1.1. Responses assigned using iRECIST have the prefix "i" (i.e., immune) to distinguish them from responses assigned using RECIST 1.1 - e.g., "immune" complete response (iCR) or partial response (iPR) and unconfirmed progressive disease (iUPD) or confirmed progressive disease (iCPD) or stable disease (iSD), all as defined by Seymour et al. RECIST 1.1. In some embodiments, comparisons can be made to baseline levels before the start of treatment. In any of these embodiments, the anti-galectin-9 antibody can be administered alone or in combination with a checkpoint inhibitor, e.g., an anti-PD-1 antibody disclosed herein.

[0191] Thus, in some embodiments, the disclosure provides methods for improving overall response (iOR) or achieving an "immune" complete response (iCR), partial response (iPR), or stable disease (iSD) (e.g., measured at about 2 months, 3 months, 6 months, or 12 months, or any time thereafter or other clinically indicated) compared to the baseline level of disease before the start of treatment. For example, the reduction in the "immune" response of iCR, iPR, or iSD can be temporary or permanent over a period of time. In some embodiments, the treatment may improve the likelihood of a complete response (iCR), partial response (iPR), or stable disease (iSD) (e.g., measured at about 2 months, 3 months, 6 months, or 12 months, or thereafter, or any other time point as clinically indicated), e.g., in some embodiments, the present disclosure provides a method for ameliorating disease progression or reducing progressive disease, e.g., reducing unconfirmed progressive disease (iUPD) or reducing confirmed progressive disease (iCPD) (e.g., measured at about 2 months, 3 months, 6 months, or 12 months, or thereafter, or any other time point as clinically indicated), the method comprising administering to the subject a therapeutically effective amount of an anti-galectin-9 antibody disclosed herein. Any of these above iRECIST criteria may be compared to baseline levels before the start of treatment. In any of these methods, the anti-galectin-9 antibody may be administered alone or in combination with a checkpoint inhibitor, e.g., an anti-PD-1 antibody.

[0192] The reduction in iUPD or iCPD may be temporary or permanent over a period of time. In some embodiments, the treatment may result in a higher likelihood of an overall reduction in unconfirmed progressive disease (iUPD) or confirmed progressive disease (iCPD) (e.g., measured at about 2 months, 3 months, 6 months, or 12 months, or thereafter, or any other time point as clinically indicated), and in some embodiments, according to iRECIST criteria (e.g., measured at about 2 months, 3 months, 6 months, or 12 months, or thereafter, or any other time point as clinically indicated), provides a method for reducing the number of new lesions in a subject, including a human subject, comprising administering to the subject a therapeutically effective amount of an anti-galectin-9 antibody disclosed herein. The reduction in the number of lesions is compared to baseline levels before the start of treatment, and the reduction may be temporary or permanent over a period of time. In any of these embodiments, the anti-galectin-9 antibody may be administered in combination with a checkpoint inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0193] Additional criteria can be used to measure treatment response. For example, tumor burden can be measured according to irRC criteria (Hoos et al., 2007). In irRC, tumor burden is measured by combining "index" lesions with new lesions; that is, new lesions are considered as changes in tumor burden. In irRC, immune-related complete response (irCR) is disappearance of all lesions, measured or unmeasured, and no new lesions; immune-related partial response (irPR) is a 50% decrease in tumor burden from irRC-defined baseline; immune-related progressive disease (irPD) is a 25% increase in tumor burden from the lowest level recorded. All others are considered immune-related stable disease (irSD).

[0194] Immune-related RECIST (irRECIST) is based on one-dimensional measurements of RECIST, and certain immune-related criteria have been further redefined in irRECIST. Recently, new criteria have been evaluated based on atezolizumab in NSCLC, and immune-modified RECIST (imRECIST) requires confirmation of disease progression at least 4 weeks after the first evaluation (Hodi et al., JCO 2018;36(9):850-858). For comparisons of RECIST1.1., irRC, irRECIST, iRECIST, and imRECIST, see, for example, Borcoman et al., Annals of Oncology 30:385-396, 2019; Nishino et al., Clin Cancer Res 2013;19(14):3936-3943, Figure 4 (the contents of which are incorporated herein by reference in their entirety). Any of these criteria are suitable for determining the response rate in any of the methods described herein.

[0195] Subjects being treated with any of the anti-galectin-9 antibodies disclosed herein (e.g., G9.2-17), alone or in combination with a checkpoint inhibitor disclosed herein (e.g., an anti-PD-1 or anti-PD-L1 antibody), may be monitored for the occurrence of adverse effects (e.g., serious side effects). Exemplary adverse effects to monitor are provided in Example 1 below. If the occurrence of an adverse effect is observed, treatment conditions may be modified for that subject. For example, the dose of the anti-galectin-9 antibody may be reduced and / or the administration interval may be extended. The suitability and extent of reduction may be evaluated by a qualified clinician. In one embodiment, a reduction level of 30 or 50% of the previous dose level is implemented. In one specific example, the reduction level as assessed by the clinician, or at least 30%, is implemented (dose level 1, to the level at the time of the first dose reduction). If necessary, another dose reduction by 30% of dose level-1 is performed (dose level-2, the level of the second dose reduction). In another example, another dose reduction by 50% of dose level-1 is performed (dose level-2). In some embodiments, one or more dose reductions of about 10% to about 80% of the previous dose level are performed. In some embodiments, one or more dose reductions of about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, or about 70% to about 80% of the previous dose level are performed. In some embodiments, one or more dose reductions of 10% to 20%, 20% to 30%, 30% to about 40%, 40% to 50%, 50% to about 60%, or 70% to about 80% of the previous dose are performed. In some embodiments, one or more dose reductions of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% of the previous dose are performed. In some embodiments, one or more dose reductions of 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the previous dose level are performed. Alternatively or additionally, the dose of the checkpoint inhibitor can be reduced and / or the administration interval of the checkpoint inhibitor can be extended. In some cases (e.g., the occurrence of life-threatening adverse effects), treatment can be discontinued.

[0196] (G) Regulation of immune responses Response to treatment may also be characterized by one or more of blood and tumor immunophenotype, cytokine profile (serum), soluble galectin-9 levels in blood (serum or plasma), immunohistochemistry (tumor, stromal, immune cells), tumor mutational burden (TMB), PD-L1 expression (e.g., by immunohistochemistry), mismatch repair status, or galectin-9 tumor tissue expression levels and patterns by disease-associated tumor markers (e.g., measured at about 3 months, 6 months, or 12 months, or thereafter, or at any other time point as clinically indicated). Examples of such tumor markers include, but are not limited to, CA15-3, CA-125, CEA, CA19-9, alpha-fetoprotein. These parameters may be compared to baseline levels before the initiation of treatment. In any of these embodiments, the anti-galectin-9 antibody may be administered alone or in combination with a checkpoint inhibitor, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0197] In any of the methods disclosed herein, the subject may be tested for one or more of the following features before, during, and / or after treatment: (a) one or more tumor markers in a blood sample from the subject, optionally the one or more tumor markers include CA15-3, CA-125, CEA, CA19-9, and / or alpha fetoprotein, as well as any other tumor type specific tumor marker; (b) a cytokine profile; and (c) Galectin-9 serum / plasma levels; (d) peripheral blood mononuclear cell immunophenotyping, (e) multiplexed immunophenotyping of tumor tissue biopsy / resection specimens, (f) Galectin-9 expression levels and patterns of tumor tissue biopsy / resection specimens, (g) any other immunoscore tests, such as PD-L1 immunohistochemistry, tumor mutation burden (TMB), tumor microsatellite instability status, and panels, such as Immunoscore®-HalioDx, ImmunoSeq-Adaptive Biotechnologies, NanoString TIS developed based on the nCounter® gene expression system, 18-gene signature, PanCancer IO 360™ assay (NanoString Technologies), etc. Other suitable biomarkers specific to target tumors such as PDAC may also be used. In one non-limiting example, PD-L1 (SP263) (Roche, Ventana) can be used to detect PD-L1 in cancer tissues using immunohistochemistry.

[0198] In some embodiments, methods are described herein for altering the levels of immune cells and immune cell markers in blood or tumors, the methods including administering an anti-Gal-9 antibody alone or in combination with a checkpoint inhibitor, e.g., an anti-PD-1 antibody. Such alterations can be measured in patient blood and tissue samples using methods known in the art, such as multiplex flow cytometry and multiplex immunohistochemistry. For example, a panel of phenotypic and functional PBMC immune markers can be assessed at baseline before the start of treatment and at various time points during treatment. Table 2 lists non-limiting examples of markers useful for these assessment methods. Flow cytometry (FC) is a technique of choice that provides fast and informative information for analyzing cellular phenotype and function, and has gained attention in immune phenotypic surveillance. It allows characterization of many subsets of cells, including rare subsets, in complex mixtures such as blood, and represents a method to rapidly obtain large amounts of data. The advantages of FC are its speed, sensitivity, and specificity. Standardized antibody panels and procedures can be used to analyze and classify immune cell subtypes. Multiplex IHC is a powerful investigative tool that provides objective quantitative data describing the immune landscape of a tumor in terms of both number and location of immune subsets, allowing the assessment of multiple markers in a single tissue section. Computer algorithms can be used to combine chromogenic IHC methods and staining with digital pathology approaches to quantify IHC-based biomarker content from whole slide images of patient biopsies.

[0199] Thus, in some embodiments, methods for modulating immune responses, such as those in Table 2, are described herein, which include administering an anti-gal9 antibody alone or in combination with a checkpoint inhibitor therapy. In some embodiments, the modulation includes one or more of the following: (1) an increase in more CD8 cells in plasma or tumor tissue, (2) a decrease in regulatory T cells (Treg) in plasma or tumor tissue, (3) an increase in M1 macrophages in plasma or tumor tissue, and (4) a decrease in MDSCs in plasma or tumor tissue, and (5) a decrease in M2 macrophages in plasma or tumor tissue (e.g., measured after about 2 months, 3 months, 6 months, or 12 months, or thereafter, or at any other time point as clinically indicated). In some embodiments, the markers evaluated using the techniques described above or known in the art are selected from CD4, CD8, CD14, CD11b / c, and CD25. These parameters can be compared to baseline levels before the start of treatment. [Table 2]

[0200] In some embodiments, methods are described herein that include administering anti-gal9 alone or in combination with checkpoint inhibitor therapy to regulate pro-inflammatory and anti-inflammatory cytokines. In some embodiments, the methods provide for one or more of the following: (1) increasing the level of IFN gamma in plasma or tumor tissue; (2) increasing the level of TNF alpha in plasma or tumor tissue; (3) decreasing the level of IL-10 in plasma or tumor tissue (e.g., measured at about 3 months, 6 months, or 12 months, or thereafter, or at any other time point as clinically indicated). These parameters can be compared to baseline levels before the start of treatment.

[0201] In some embodiments, cytokine or immune cell levels may be assessed between a tumor biopsy prior to dose 1 and a repeat biopsy performed when feasible. In some embodiments, cytokine or immune cell levels may be assessed between two repeat biopsies. In some embodiments, methods are provided for modulating one or more of soluble galectin-9 levels in blood (serum or plasma) or galectin-9 tumor tissue expression levels and expression patterns by immunohistochemistry (tumor, stroma, immune cells) (e.g., measured at about 3 months, 6 months, or 12 months, or thereafter, or any other time point as clinically indicated). In some embodiments, the methods reduce soluble galectin-9 levels in blood (serum or plasma) or galectin-9 tumor tissue expression levels or expression patterns by immunohistochemistry (tumor, stroma, immune cells) (e.g., measured at about 3 months, 6 months, or 12 months, or thereafter, or any other time point as clinically indicated). Galectin-9 levels may be compared to baseline levels prior to the initiation of treatment. In some embodiments, galectin-9 levels may be compared to untreated controls or healthy subjects. In any of these embodiments, the anti-galectin-9 antibody may be administered alone or in combination with a checkpoint inhibitor, e.g., an anti-PD-1 antibody. In some embodiments, a method is provided for modulating PD-L1 expression, e.g., as assessed by immunohistochemistry, comprising administering an anti-galectin-9 antibody alone or in combination with a checkpoint inhibitor, e.g., an anti-galectin-9 antibody. In some embodiments, the method modulates (increases or decreases) one or more tumor markers associated with the disease (e.g., measured at about 2 months, 3 months, 6 months, or 12 months, or thereafter, or at any other time point as clinically indicated). Examples of such tumor markers include, but are not limited to, CA15-3, CA-125, CEA, CA19-9, alpha-fetoprotein. These parameters may be compared to baseline levels before the start of treatment.In any of these embodiments, the anti-galectin-9 antibody may be administered alone or in combination with a checkpoint inhibitor, such as an anti-PD-1 antibody.

[0202] In some embodiments, the present disclosure provides a method of modulating an immune response in a subject. As used herein, the term "immune response" includes T cell-mediated and / or B cell-mediated immune responses that are affected by modulation of immune cell activity, e.g., T cell activation. In one embodiment of the present disclosure, the immune response is T cell-mediated. As used herein, the term "modulate" means to change or alter, and includes both upregulation and downregulation. For example, "modulating an immune response" means to change or alter the state of one or more immune response parameter(s). Exemplary parameters of a T cell-mediated immune response include levels of T cells (e.g., increased or decreased effector T cells) and levels of T cell activation (e.g., increased or decreased production of a particular cytokine). Exemplary parameters of a B cell-mediated immune response include increased levels of B cells, B cell activation, and B cell-mediated antibody production.

[0203] When the immune response is modulated, some immune response parameters may decrease and others may increase. For example, in some cases, modulating the immune response increases (or upregulates) one or more immune response parameters and decreases (or downregulates) one or more other immune response parameters, resulting in an overall increase in the immune response, such as an overall increase in the inflammatory immune response. In another example, modulating the immune response causes an increase (or upregulation) of one or more immune response parameters and a decrease (or downregulation) of one or more other immune response parameters, resulting in an overall decrease in the immune response, such as an overall decrease in the inflammatory response. In some embodiments, the increase in the overall immune response, i.e., an increase in the overall inflammatory immune response, is determined by a decrease in tumor weight, tumor size, or tumor burden, or any of the RECIST or iRECIST criteria described herein. In some embodiments, an increase in the overall immune response is determined by an increase in the level of one or more of the most potent proinflammatory cytokines (e.g., one or more, including two or more, three or more, etc., or the majority of the proinflammatory cytokines (one or more, including two or more, etc., or the majority of the anti-inflammatory and / or immunosuppressive cytokines, and / or one or more of the most potent anti-inflammatory or immunosuppressive cytokines are decreased or remain constant). In some embodiments, an increase in the overall immune response is determined by an increase in the level of one or more of the most potent proinflammatory cytokines (one or more of the anti-inflammatory and / or immunosuppressive cytokines, including one or more of the most potent cytokines are decreased or remain constant). In some embodiments, an increase in the overall immune response is determined by a decrease in the level of one or more, including the majority of the immunosuppressive and / or anti-inflammatory cytokines (e.g., the level of one or more, or the majority of the proinflammatory cytokines, including the most potent proinflammatory cytokines are increased or remain constant).In some embodiments, the increase in overall immune response is an increase in one or more of the most potent anti-inflammatory and / or immunosuppressive cytokines (e.g., one or more, or a majority of, the proinflammatory cytokines, including the most potent proinflammatory cytokines, increase or remain constant). In some embodiments, the increase in overall immune response is determined by any combination of the above. Also, an increase (or upregulation) of one type of immune response parameter may cause a corresponding decrease (or downregulation) of another type of immune response parameter. For example, an increase in the production of a particular proinflammatory cytokine may lead to the downregulation of a particular anti-inflammatory and / or immunosuppressive cytokine, and vice versa.

[0204] In some embodiments, the present disclosure provides methods for modulating an immune response in a subject, including a human subject (e.g., measured at about 2 months, 3 months, 6 months, or 12 months, or thereafter, or any other time point as clinically indicated), the method comprising administering to the subject a therapeutically effective amount of an anti-galectin-9 antibody disclosed herein. In some embodiments, the present disclosure provides methods for modulating levels of immune cells and immune cell markers, including but not limited to those set forth in Table 2 herein, in the blood or tumor of a subject, including a human subject, e.g., compared to baseline levels obtained prior to the initiation of an anti-Gal9 antibody treatment regimen, compared to baseline levels prior to the initiation of treatment, the method comprising administering to the subject a therapeutically effective amount of an anti-galectin-9 antibody disclosed herein. In some embodiments, the overall result of the modulation is upregulation of proinflammatory immune cells and / or downregulation of immunosuppressive immune cells. In some embodiments, the present disclosure provides a method of modulating levels of immune cells, where modulating includes one or more of: (1) increasing CD8 cells in plasma or tumor tissue, (2) decreasing Tregs in plasma or tumor tissue, (3) increasing M1 macrophages in plasma or tumor tissue, and (4) decreasing MDSCs in plasma or tumor tissue, and (5) decreasing M2 macrophages in plasma or tumor tissue, the method comprising administering to a subject a therapeutically effective amount of an anti-galectin-9 antibody disclosed herein. In some embodiments, markers for assessing the levels of such immune cells include, but are not limited to, CD4, CD8, CD14, CD11b / c, and CD25. In some embodiments, the present disclosure provides methods for modulating levels of pro-inflammatory and immunosuppressive cytokines in the blood or tumor of a subject, including a human subject, e.g., compared to baseline levels before the start of treatment (e.g., measured at about 2 months, 3 months, 6 months, or 12 months, or thereafter, or any other clinically indicated time point), the methods comprising administering to the subject a therapeutically effective amount of an anti-galectin-9 antibody disclosed herein.In some embodiments, the overall result of the modulation is upregulation of proinflammatory cytokines and / or downregulation of immunosuppressive cytokines. In some embodiments, the disclosure provides methods for modulating levels of cytokines, where modulating includes one or more of the following: (1) increasing the level of IFN-gamma in plasma or tumor tissue; (2) increasing the level of TNF-alpha in plasma or tumor tissue; (3) decreasing the level of IL-10 in plasma or tumor tissue.

[0205] In some embodiments, the present disclosure provides methods for altering one or more of soluble galectin-9 levels in blood (serum or plasma) or galectin-9 tumor tissue expression levels and expression patterns by immunohistochemistry (tumor, stroma, immune cells) (e.g., measured at 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, or 12 months, or thereafter, or any other time point as clinically indicated), the methods comprising administering to a subject a therapeutically effective amount of an anti-galectin-9 antibody disclosed herein. In some embodiments of the methods, one or more of soluble galectin-9 levels in blood (serum or plasma) or galectin-9 tumor tissue expression levels and expression patterns by immunohistochemistry (tumor, stroma, immune cells) remain unchanged. In some embodiments, the methods provided herein reduce one or more of soluble galectin-9 levels in blood (serum or plasma) or galectin-9 tumor tissue expression levels and expression patterns by immunohistochemistry (tumor, stromal, immune cells) (e.g., measured at 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, or 12 months, or thereafter, or any other time point as clinically indicated). Galectin-9 levels can be compared to baseline levels before the start of treatment. In some embodiments, Galectin-9 levels can be compared to healthy subjects. In some embodiments, treating results in a change in PD-L1 expression, e.g., by immunohistochemistry. Dose levels of 16 mg / kg or higher, Dose levels of 16 mg / kg or higher, Dose levels of 16 mg / kg or higher.

[0206] In some embodiments, the present disclosure provides a method for altering PD-L1 expression (e.g., measured at 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, or 12 months, or thereafter, or any other time point as clinically indicated), e.g., as assessed by immunohistochemistry, comprising administering to a subject a therapeutically effective amount of an anti-galectin-9 antibody disclosed herein. In some embodiments of the method, PD-L1 expression, e.g., as assessed by immunohistochemistry, remains unchanged. PD-L1 levels can be compared to baseline levels before the start of treatment. In some embodiments, the methods provided herein reduce PD-L1 expression, e.g., as assessed by immunohistochemistry. PD-L1 levels can be measured using routine methods known in the art. In one non-limiting example, PD-L1 (SP263) (Roche, Ventana) can be used to detect PD-L1 in cancer tissues using immunohistochemistry.

[0207] In some embodiments, the present disclosure provides a method for altering (increasing or decreasing) one or more tumor markers associated with a disease (e.g., measured at 2 weeks, 4 weeks, 1 month, 3 months, 6 months, or 12 months, or thereafter, or any other time point as clinically indicated), the method comprising administering to a subject a therapeutically effective amount of an anti-galectin-9 antibody disclosed herein. In some embodiments of the method, the one or more tumor markers associated with the disease (increased or decreased) remain unchanged. Examples of such tumor markers include, but are not limited to, CA15-3, CA-125, CEA, CA19-9, alpha-fetoprotein. The level of the tumor marker can be compared to a baseline level before the start of treatment. In some embodiments, the method provided herein reduces the occurrence of one or more tumor markers associated with the disease.

[0208] In some embodiments, the present disclosure provides a method for altering (increasing or decreasing) one or more biomarkers associated with a disease (e.g., measured at 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, or 12 months, or thereafter, or any other time point as clinically indicated), the method comprising administering to a subject a therapeutically effective amount of an anti-galectin-9 antibody disclosed herein. The level of biomarkers in clinical tissue from a patient can be measured using routine methods, such as multiplex immunofluorescence (mIF) techniques, as described in the Examples herein. An exemplary panel of biomarkers can include CD3, CD4, CD8, CD45RO, FoxP3, CD11b, CD14, CD15, CD16, CD33, CD68, CD163, HLA-DR, Arginase 1, Granzyme B, Ki67, PD-1, PD-L1, F4 / 80, Ly6G / C, and PanCK.

[0209] Any of the anti-galectin-9 antibodies described herein may be used in any of these methods described herein for modulating immune responses, cytokines, biomarkers, e.g., galectin-9 or PD-L1 levels, or tumor markers. In some embodiments, the antibody comprises a light chain complementarity determining region 1 (CDR1) set forth in SEQ ID NO: 1, a light chain complementarity determining region 2 (CDR2) set forth in SEQ ID NO: 2, and a light chain complementarity determining region 3 (CDR3) set forth in SEQ ID NO: 3, and / or a heavy chain complementarity determining region 1 (CDR1) set forth in SEQ ID NO: 4, a heavy chain complementarity determining region 2 (CDR2) set forth in SEQ ID NO: 5, and a heavy chain complementarity determining region 3 (CDR3) set forth in SEQ ID NO: 6. In some embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 7. In some embodiments, the antibody comprises a light chain variable region comprising SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain comprising SEQ ID NO: 19. In some embodiments, the antibody comprises a light chain comprising SEQ ID NO: 15.

[0210] In some embodiments, the antibody is G9.2-17 IgG4. In some embodiments, the anti-galectin-9 antibody is administered to the subject at a dose of about 0.2 mg / kg to about 32 mg / kg, e.g., the dose may be selected from 0.2 mg / kg, 0.63 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 6.3 mg / kg, 8 mg / kg, 10 mg / kg, 12 mg / kg, and 16 mg / kg, or more. In some embodiments, the anti-galectin-9 antibody is administered to the subject at a dose of about 1 mg / kg to about 32 mg / kg, e.g., the dose may be selected from 2 mg / kg, 4 mg / kg, 8 mg / kg, 12 mg / kg, and 16 mg / kg, or more. In some embodiments, the anti-galectin-9 antibody is administered to the subject at a dose of about 0.2 mg / kg to about 32 mg / kg, for example, the dose may be selected from 0.2 mg / kg, 0.63 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 6.3 mg / kg, 10 mg / kg, or 16 mg / kg, or higher dose levels. In some embodiments, the antibody is administered, for example, by intravenous infusion, once every two weeks.

[0211] In some embodiments, the method further comprises administering to the subject an immune checkpoint inhibitor, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the solid tumor is pancreatic ductal adenocarcinoma (PDAC), colorectal carcinoma (CRC), hepatocellular carcinoma (HCC), cholangiocarcinoma (CAA), renal cell carcinoma (RCC), urothelial carcinoma, head and neck cancer, breast cancer, lung cancer, and other GI solid tumors, in some embodiments, the solid tumor is a metastatic tumor.

[0212] In some embodiments, the present disclosure provides methods for improving quality of life and / or improving symptom control in a subject, including a human subject (e.g., measured at 1 month, 3 months, 6 months, or 12 months, or thereafter, or any other time point as clinically indicated), the methods comprising administering to the subject a therapeutically effective amount of an anti-galectin-9 antibody disclosed herein. The improved quality of life and symptom control may be compared to a baseline before the start of treatment. In some embodiments, the improvement may be measured on the ECOG scale.

[0213] Kits for use in treating diseases associated with galectin-9 The present disclosure also provides kits for use in treating or ameliorating a disease associated with galectin-9, e.g., a disease associated with binding of galectin-9 to cell surface glycoproteins (e.g., Dectin-1, TIM3, CD206, etc.), or a pathological cell (e.g., cancer cell) that expresses galectin-9. Examples include solid tumors, such as PDAC, CRC, HCC, cholangiocarcinoma and other GI solid tumors, as well as others described herein. Such kits may include one or more containers containing an anti-galectin-9 antibody, e.g., any of the antibodies described herein, and optionally, a second therapeutic agent (e.g., a checkpoint inhibitor, such as an anti-PD-1 antibody disclosed herein) (also described herein) to be used in combination with the anti-galectin-9 antibody.

[0214] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include instructions for administration of the anti-galectin-9 antibody, and optionally a second therapeutic agent, to treat, delay the onset of, or alleviate a target disease described herein. In some embodiments, the kit further includes instructions for selecting an individual suitable for treatment based on identifying whether the individual is afflicted with a target disease, e.g., by applying a diagnostic method described herein. In still other embodiments, the instructions include instructions for administration of the antibody to an individual at risk for a target disease.

[0215] The instructions for use of the anti-galectin-9 antibody will typically include information regarding dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. The instructions provided in the kits of the invention will typically be written instructions on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions recorded on a magnetic or optical memory disk) are also acceptable.

[0216] The label or package insert indicates that the composition is used for treating, delaying the onset of, and / or alleviating a disease associated with Galectin-9 (e.g., Dectin-1, TIM-3, or CD206 signaling). In some embodiments, instructions for performing any of the methods described herein are provided.

[0217] The kit of the present invention is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Packages for use in combination with certain devices, such as inhalers, nasal administration devices (e.g., atomizers), or injection devices, such as mini-pumps, are also contemplated. In some embodiments, the kit has a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). In some embodiments, the container also has a sterile access port (e.g., the container is an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an anti-galectin-9 antibody as described herein.

[0218] The kit may optionally provide additional components, such as buffers and interpretive information. Typically, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the invention provides an article of manufacture comprising the contents of the kit described above.

[0219] Common methods The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are fully described in, for example, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Elis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Oberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987); Current Protocols in Molecular Biology (FMAusubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology (JEColigan et al., eds.,1991);Short Protocols in Molecular Biology(Wiley and Sons,1999);Immunobiology(CAJaneway and P.Travers,1997);Antibodies(P.Finch,1997);Antibodies: a practical approach(D.Catty.,ed.,IRL Press,1988-1989);Monoclonal antibodies: a practical approach(P.Shepherd and C.Dean, eds.,Oxford University Press,2000);Using antibodies: a laboratory manual(E.Harlow and D.Lane(Cold Spring Harbor Laboratory Press,1999);The Antibodies(M.Zanetti and JDCapra,eds.,Harwood Academic Publishers,1995).

[0220] Without further elaboration, it is believed that one skilled in the art can utilize the present invention to its fullest extent based on the above description. Accordingly, the following specific embodiments are to be construed as merely illustrative, and not limiting in any way to the remainder of the disclosure. All publications cited herein are incorporated by reference for the purpose or subject matter referenced herein. EXAMPLES

[0221] Example 1: A Phase 1 / 2, Open-Label, Multicenter Study of the Safety, Pharmacokinetics, and Antitumor Activity of an Anti-Galectin-9 Monoclonal Antibody, Alone and in Combination with an Anti-PD-1 Antibody in Patients with Metastatic Solid Tumors Galectin-9 is a molecule that is overexpressed in many solid tumors, including pancreatic, colorectal, and hepatocellular carcinoma solid tumors. In addition, galectin-9 is also expressed on tumor-associated macrophages and intratumoral immunosuppressive gamma delta T cells, thereby acting as a potent mediator of cancer-associated immunosuppression. As described herein, monoclonal antibodies targeting galectin-9 (e.g., G9.2-17, IgG4) have been developed. Data demonstrate that G9.2-17 inhibits pancreatic tumor growth by 50% in an orthotopic KPC model and extends survival of KPC animals by more than two-fold. Without wishing to be bound by theory, anti-galectin-9 antibodies reverse the M2 phenotype to an M1 phenotype and upregulate intratumoral CD8 + It is believed to promote T cell activation. Furthermore, the antibody G9.2-17 (IgG4) (having a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 15) has been found to synergize with anti-PD-1.

[0222] G9.2-17(IgG4) is a fully human IgG4 monoclonal antibody (mAb) that targets the galectin-9 (-gal-9) protein. Gal-9 functions as an immunosuppressant, conferring immune privilege to tumor cells and negating immune-mediated cancer attack by controlling the susceptibility of cancer cells to macrophage, T cell, myeloid-derived suppressor cells, and cytotoxic T cell-induced cell death. Based on available data, blockade of gal-9 by G9.2-17(IgG4) interferes with the immunosuppressive function of gal-9, thereby resulting in effective immune activation and tumor growth inhibition across multiple preclinical models.

[0223] Gal-9 can be overexpressed and / or secreted in many solid tumor types, including pancreatic adenocarcinoma, cholangiocarcinoma (CCA), colorectal carcinoma (CRC), breast cancer, bladder cancer, ovarian cancer, non-small cell and small cell lung cancer, nasopharyngeal carcinoma, melanoma, ovarian cancer, etc., and high levels of tissue and / or circulating Gal-9 correlate with aggressive tumor characteristics and adverse survival outcomes.

[0224] Thus, the target indication for G9.2-17(IgG4) is recurrent or refractory metastatic solid tumors, and G9.2-17(IgG4) is being investigated as a single agent and / or in combination with checkpoint inhibitors (e.g., programmed cell death 1 [PD1] antibodies, e.g., nivolumab, pembrolizumab, cemiplimab, dostallimab, or tislelizumab).

[0225] Dose escalation (Part 1) will be performed in all solid tumor types to establish the safety and tolerability profile of G9.2-17 (IgG4), evaluate its immunogenic potential, establish the pharmacokinetic (PK) and pharmacodynamic (PD) profile, and arrive at a recommended Phase 2 dose (RP2D), which may be the maximum tolerated dose (MTD). Expansion cohorts (Part 2) are planned in first-line metastatic pancreatic ductal adenocarcinoma (PDAC), as well as CRC and CCA, both as single agents and in combination with anti-PD1 antibodies.

[0226] There are no other therapies that target gal-9 that are currently known to be approved or in clinical trials for any indication.

[0227] In preclinical studies conducted to date, no significant toxicity has been observed at doses approximately 500 times greater than those intended for administration to humans. Furthermore, G9.2-17(IgG4) has been shown to be highly specific for gal-9 and has been demonstrated to be effective in multiple animal models of cancer. The patient population enrolled is at a late stage of disease and has not received standard treatment prior to enrollment in this study. G9.2-17(IgG4) would be expected to provide benefit in the treatment of malignancies such as malignant solid tumors when taken alone or in combination with checkpoint inhibitors such as anti-PD-1 antibodies (e.g., nivolumab, pembrolizumab, tislelizumab, dostallimab, or cemiplimab).

[0228] Objectives and Endpoints [Table 3] [Table 4]

[0229] research design This is an open-label, non-controlled, multicenter Phase 1 / 2 study (dose escalation phase (part 1) and cohort expansion phase (part 2)) in patients with relapsed / refractory metastatic solid tumors. The study will be conducted at up to 20 centers in the United States. The study duration is estimated to be 12-24 months. Survival follow-up will continue for up to 2 years. The study scheme is presented in Figure 1.

[0230] The study includes both monotherapy with G9.2-17 (IgG4) and combination therapy with G9.2-17 and an anti-PD-1 antibody, such as nivolumab. The dose of G9.2-17 may range from about 3 mg / kg to 15 mg / kg once every two weeks. In another embodiment, the dose of G9.2-17 may range from about 0.2 mg / kg to 16 mg / kg, or higher dose levels, once every two weeks. The antibody is administered by intravenous infusion.

[0231] Treatment period and duration Treatment period Administration of the study drug will continue until disease progression, unacceptable toxicity, or withdrawal from the study. Patients who discontinue the study drug before disease progression and are not being treated with other systemic anti-cancer therapy(ies) will be followed in the study until the time of disease progression.

[0232] Treatment period The study consisted of the following periods in both Part 1 and Part 2: Screening period: Up to 4 weeks prior to first dose (day -28 to day -1) Treatment duration: 28-day treatment cycle as proposed in the evaluation schedule (SoA; Tables 5-6 below) Post-treatment period: 30 days after last treatment (end of treatment visit / early discontinuation visit) IMAR follow-up: 90 days after the last treatment (G9.2-17 (IgG4) + anti-PD-1 antibody treatment group) Follow-up: Long-term follow-up (visits every 3 months) for up to 2 years for patients who discontinued treatment for reasons other than disease progression and did not receive additional systemic anticancer therapy.

[0233] Part 1: Dose Escalation Phase A dose-finding study will be conducted using the continuous reassessment method (CRM) (O'Quigley et al., 1990) to establish DLT and RP2D. Two to six patients per treatment cohort 1-6 will be assigned to receive IV injections of successively higher concentrations of G9.2-17 (IGG4) every 2 weeks (Q2W) on days 1 and 15 of each 28-day cycle, starting with a dose of 0.2 mg / kg. Patients assigned to a particular dose escalation cohort will receive the study dose corresponding to that cohort. They will receive the study drug at one of eight dose levels until disease progression, unacceptable toxicity, or withdrawal from the study for other reasons. Only patients who withdraw during the first treatment cycle for reasons other than toxicity or tolerability issues will be replaced.

[0234] Cohorts 1-6 will be dosed two patients at a time based on CRM design. Dose escalation is based on analysis of patient safety data focusing on occurrence of DLTs at previous dose levels and other relevant safety and dosing data from previous cohorts. Dose escalation may occur after at least 28 days (1 cycle). Skipping dose levels is not permitted.

[0235] After completion of cohort 6 under the CRM design, the once weekly (QW) G9.2-17 (IGG4) dosing scheme will be evaluated within the CRM design, provided that the RP2D has not been reached. Cohorts 7 and 8 will not be evaluated in the CRM design. Patients will only be allowed to enter cohort 7 if no DLTs have been identified.

[0236] Cohorts 7 and 8 will be dosed four patients at a time per cohort. Four patients per dose level in cohorts 7 and 8 will be assigned to receive sequentially higher concentration IV injections of G9.2-17 (IGG4) on days 1, 8, 15, and 22 of each 28-day cycle every week (QW). Starting with the first four patients in cohort 7, dose escalation to the next cohort will occur only if no DLTs are identified. If a single DLT is recorded in cohort 7, no further patients will be dosed within that cohort and cohort 8 will not be activated.

[0237] Approximately 36 patients will be enrolled in cohorts 1-8 of Part 1. Within the CRM design, a total of six dosage levels will be evaluated: Dose escalation cohort 1 = 0.2 mg / kg Q2W Dose escalation cohort 2 = 0.63mg / kg Q2W Dose Escalation Cohort 3 = 2mg / kg Q2W Dose Escalation Cohort 4 = 6.3mg / kg Q2W Dose Escalation Cohort 5 = 10mg / kg Q2W Dose Escalation Cohort 6 = 16mg / kg Q2W Two additional dosage levels are included to account for the RP2D. Dose Escalation Cohort 7 = 10mg / kg QW Dose Escalation Cohort 8 = 16mg / kg QW

[0238] Patients treated in the initial cohort before the RP2D is identified will be allowed to dose escalate to the highest dose level cleared. Dose escalation may occur at least 28 days (1 cycle) after a complete cycle. Dose escalation may not occur mid-cycle. Patients may continue to escalate to the highest approved dose level until discontinued due to toxicity or disease progression, or other reasons (e.g., the patient chooses to discontinue the study).

[0239] Dose escalation is based on the occurrence of DLT in patients treated at the prior dose level. For each dose cohort, a prior DLT probability is identified from GLP-compliant toxicity studies and preclinical models. For a given target DLT rate and total number of dose levels, a skeleton of the power model d^exp(a) is generated following the approach of Lee and Cheung using the prior MTD adjusted with PK / PD data, with dose level median and interval measures at delta = 0.05 (Lee and Cheung, 2011). The prior distribution of parameter "a" has a mean zero normal distribution with the least information of prior variance. If the lower limit of Agresti and Coull's binomial confidence interval (CI) for the lowest studied dose level exceeds the target DLT rate, the study is stopped for safety (Agresti and Coull, 1998). RP2D is the MTD dose derived from part 1.

[0240] If a DLT occurs in any patient during the first 28 days of treatment, the patient will be permanently discontinued from receiving the study drug.

[0241] In patients who experience toxicity (including IMAR) outside the DLT window, dose reductions are permitted and may be obtained continuing at a lower dose of G9.2-17(IgG4) only if clinical benefit is anticipated. The dose of G9.2-17(IgG4) will be initially reduced by 50% and then further reduced by 50% as defined in the dose modification guidance presented in Table 3. No further dose reductions are permitted. [Table 5-1] [Table 5-2]

[0242] Part 1 Complete Part 1 will be completed when up to six patients have received the dose identified as the RP2D, which is based in part on the continuous reevaluation method (CRM) study design, PK and PD data parameters, additional safety and efficacy data, and any other factors considered.

[0243] Backfill Cohort The purpose of the backfill cohort is to evaluate the safety, tolerability, and biological effects of G9.2-17(IGG4) in patients whose tumors are gal-9 positive. The gal-9 status of the RP2D cohort will be determined retrospectively. If there are fewer than six patients with gal-9 positive tumors treated at the RP2D, patients assigned to the backfill cohort will require prospective evaluation of gal-9 tumor status by IHC. Up to six additional patients whose tumors are gal-9 positive may be enrolled to enroll the cohort at the RP2D dose level.

[0244] Part 2: Cohort expansion phase The second part of the protocol will employ the Simon two-stage optimal design, which will include approximately 223 patients. Based on the implementation of expansion cohorts and tumor-specific considerations for clinical trial endpoints, it is planned to expand the cohorts to PDAC, CRC, and CCA and / or potentially other solid tumor types. The rationale behind this approach is to ensure feasibility of recruitment and capture clinical needs for specific indications.

[0245] CRC and CCA patients will receive one of two treatments (four treatment arms in total): LYT200 as a single agent -G9.2-17(IGG4) + anti-PD-1 antibody as combination therapy.

[0246] Anti-PD-1 antibodies should be administered before G9.2-17(IgG4). If for some reason it is not possible to administer them on the same day, anti-PD-1 antibodies should be administered on the first day and G9.2-17(IgG4) should be administered on the next day.

[0247] In some cases, the study may investigate the use of the anti-galectin-9 antibody G9.2-17 (IgG4) alone (in the single-agent arm of the study) or in combination with nivolumab (e.g., administered at a fixed dose of 240 mg once every two weeks).

[0248] CRC and CCA patients Treatment of single agent or combination agent cohorts for CRC and CCA patients may be performed in parallel.

[0249] G9.2-17 (IgG4) monotherapy The starting dose of G9.2-17 (IgG4) in the single-agent treatment is the RP2D specified in Part 1. In the CRC and CCA single-agent treatment arms, an optimal two-stage design (Stage I and II) will be used to test the null hypothesis that ORR3 is ≤5% versus the alternative hypothesis that ORR3 is ≥15% in the single-agent treatment arms.

[0250] After testing the investigational agent in 23 patients in stage I, this arm will be discontinued if 1 or fewer patients respond. If the trial progresses to stage II of the Simon optimal design, approximately 33 additional patients will be treated in each of the single-agent arms. If the total number of responding patients is 5 or fewer, the investigational agent in that arm will be rejected. If 6 or more patients have a confirmed ORR3, a part 3 expansion cohort for that arm will be activated and described in the protocol amendment.

[0251] Dose reductions may be expected if clinical benefit is anticipated and followed by lower doses of G9.2-17(IgG4). The dose of G9.2-17(IgG4) will be initially reduced by 50% and may be further reduced by 50% as defined in the dose modification guidance provided in the protocol. No further dose reductions will be permitted.

[0252] G9.2-17 (IgG4) + anti-PD-1 antibody combination treatment The dose of G9.2-17(IGG4) in combination treatment with an anti-PD-1 antibody (e.g., nivolumab or pembrolizumab) is RP2D-1, which is the dose immediately preceding the RP2D dose identified in Part 1. The optimal two-stage design will also be used to test the null hypothesis that ORR3 is ≤10% and the alternative hypothesis that ORR3 is ≥25%.

[0253] To ensure patient safety, a safety run-in will be conducted in which the first eight patients will be dosed. This treatment arm will continue to enroll only if two or fewer patients develop DLTs below the 25% target toxicity level (TTL). If three or more patients develop DLTs, this combination treatment arm will be discontinued for the cancer type being treated. In this combination treatment run-in cohort, patients who drop out for reasons other than toxicity or tolerability issues will only be replaced during the first treatment cycle. If a DLT occurs during any of the eight safety runs for a patient during the first 28 days of treatment, the patient will be permanently discontinued from receiving the study drug.

[0254] In patients who experience toxicity outside the DLT window, dose reduction is permitted only if clinical benefit is anticipated and may be obtained by continuing with a lower dose of G9.2-17(IgG4). The dose of G9.2-17(IgG4) will be initially reduced by 50% and may be further reduced by 50% as defined in the dose modification guidance provided in the protocol. No further dose reductions are permitted. Dose modifications of the anti-PD-1 antibody are permitted.

[0255] If IMAR occurs / recurs and cannot be managed by dose reduction of either drug, both study drugs must be discontinued.

[0256] After testing the combination in 18 patients in the first phase, each arm will be stopped if two or fewer patients respond. If the trial progresses to Stage II of the Simon optimal design, approximately 25 additional patients will be treated within each combination arm. If the total number of responding patients is seven or fewer, the combination within that arm will be rejected. If eight or more patients have confirmed ORR3, an expansion cohort for that arm will be enabled and is described in a protocol amendment.

[0257] PDAC patient Part 2 cohort of patients with metastatic PDAC will require combination treatment with G9.2-17 (IgG4) in first-line metastatic cases.

[0258] The dose of G9.2-17 (IGG4) is the RP2D-1 dose, which is the dose level in the cohort immediately preceding the RP2D dose identified in Part 1. To ensure patient safety, a run-in will be conducted in which the first 8 patients will be dosed and the treatment arm will continue if 2 or less patients develop DLTs below the 25% target toxicity level (TTL). If 3 or more patients develop DLTs, the combination treatment arm will be discontinued. In this combination treatment run-in cohort, patients who withdraw for reasons other than toxicity or tolerability issues will only be replaced during the first treatment cycle. If a DLT occurs during any of the 8 safety runs for a patient during the first 28 days of treatment, the patient will be permanently discontinued from receiving the study drug.

[0259] In patients who experience toxicity outside the DLT window, dose reductions are permitted only if clinical benefit is anticipated and continued treatment with a lower dose of G9.2-17(IgG4) may be achieved. The dose of G9.2-17(IgG4) will be initially reduced by 50% and may be reduced by a further 50%. No further dose reductions are permitted.

[0260] If IMAR occurs / recurs and cannot be managed by dose reduction of either drug, both study drugs must be discontinued.

[0261] The primary efficacy endpoint was PFS6 in patients.

[0262] Part 2 Complete Completion of part 2 will depend on patients achieving ORR3 for CRC and CCA patients and PFS6 for PDAC.

[0263] Part 3: Expansion If promising efficacy signals are identified within one or more of the trial arms, expansion cohorts will be initiated to confirm the findings as described above. Sample sizes for each expansion arm will be determined based on the point estimates determined in Part 2, combined with predefined levels of precision for the 95% CIs around ORR / OS and PFS. A protocol amendment will be submitted with details on the expansion population, treatment regimen, and statistical analysis plan prior to initiating Part 3.

[0264] Dose-limiting toxicity criteria Dose-limiting toxicities evaluated in this study were defined as clinically significant hematologic and / or nonhematologic AEs or abnormal laboratory values ​​assessed as unrelated to metastatic tumor disease progression, intercurrent illness, or concomitant medications that were possibly or were related to the study drug and occurred during the first cycle (28 days) of the study. Patients who experience a DLT in Part 1 or Part 2 during the first 28 days of treatment will be permanently discontinued from receiving study drug.

[0265] A DLT is a toxicity that meets any of the following criteria: Any death without an underlying disease or external cause Signs of potential drug-induced liver injury (Highe's Law): ALT or AST >3x the upper limit of normal (ULN) confirmed by repeat testing after 24 hours, and Serum total bilirubin (TBL) > 2 × ULN (confirmed by repeat testing after 24 hours) No other explanation can be found for the elevation of TBL and / or AT, e.g., viral hepatitis (A, B, or C), alcoholic or autoimmune hepatitis, pre-existing or acute liver disease, gallbladder obstruction or bile duct disease, Gilbert's syndrome, disease progression, or another drug that may be causing the observed effects. Any grade 4 non-hematologic or hematologic toxicity of any duration All grade 3 non-hematologic and hematologic toxicities. Exceptions include: Grade 3 nausea, vomiting, and diarrhea that can be managed to ≤ Grade 2 within 48 hours with supportive care without requiring hospitalization or total parenteral nutrition support. Grade 3 electrolyte abnormalities are corrected to Grade 2 or less within 24 hours. o Grade 3 electrolyte abnormalities that last less than 24-72 hours, are clinically uncomplicated, and resolve spontaneously or respond to conventional medical intervention. Amylase or lipase grade 3 or higher without symptoms or clinical signs of pancreatitis.

[0266] Definition of end of research The end of study part 1 will be defined as the time point when all patients are treated with G9.2-17(IGG4) until RP2D is identified and disease progression is confirmed.

[0267] Study end-of-study for part 2 of the study was defined for each of the three tumor types following completion of the Simon two-stage optimal design, with all enrolled patients being treated with G9.2-17(IGG4) (alone or in combination) until confirmed disease progression.

[0268] In both Part 1 and Part 2, patients will be followed for OS for up to 2 years after their last dose of G9.2-17(IgG4), if they discontinue treatment for reasons other than disease progression and are not receiving additional systemic anti-cancer therapy.

[0269] The end of the study will be defined as the date of the last patient's last visit.

[0270] Clinical trial suspension rules Part 1 If the lower limit of Agresti and Coull's CI for the lowest studied dose level exceeds the target DLT rate, the trial will be stopped for safety ( Agresti and Coull, 1998 ).

[0271] Part 2 After testing the investigational drug in 23 patients in Stage I of the Simon optimal design for the single agent treatment arms in CRC and CCA, each arm is stopped if 1 or fewer patients respond. If the trial proceeds to Stage II of the Simon optimal design, an arm is stopped if the total number of patients in that arm who respond is 5 or fewer.

[0272] Similarly, for the combination of G9.2-17 (IgG4) + anti-PD-1 antibody in CRC and CCA, Simon's optimal design also induces a trial stop. After testing this combination in 18 patients in stage I, each trial arm is stopped if there are 2 or fewer responding patients. If the trial proceeds to stage II, a trial arm is stopped if the total number of responding patients in that treatment arm is 7 or fewer.

[0273] A safety run-in will be performed to ensure patient safety in both combination treatment arms, with the first 8 patients dosed. For each cancer type (e.g., CCA, CRC, and / or PDAC), enrollment will continue only if 2 or fewer patients develop DLTs, which are below the target toxicity level (TTL) of 25%. If 3 or more patients with a given cancer type develop DLTs in the combination treatment arm, enrollment will be halted for that cancer type within that arm.

[0274] Study population Inclusion criteria Participants were eligible to be included in the study only if they met all of the following criteria: Part 1 and Part 2 1. Written informed consent (mentally normal patients, able to understand and willing to sign the informed consent form) 2. Age 18 or older, male or non-pregnant female 3. Histologically confirmed unresectable metastatic carcinoma (adenocarcinoma and squamous cell carcinoma are acceptable). Patients with resectable disease will be excluded. 4. Ability to comply with research protocols Life expectancy of over 5.3 months 6. Eastern Cooperative Oncology Group (ECOG) performance status 0–1 7. Coronavirus SARS-CoV-2 (COVID-19) negative patients 8. Patients able and willing to undergo pre- and intra- / post-procedure biopsies. Planned biopsies should not expose patients to significantly increased risk of complications. Every effort will be made to biopsy the same lesions during repeat biopsies. 9. Measurable disease according to Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. It should be noted that the lesion intended for biopsy should not be the target lesion. 10. Adequate hematologic and end-organ function as defined by the following laboratory test results obtained prior to the first dose of investigational drug treatment: A neutrophil count ≥ 1 x 10 9 / L b. Platelet count ≧100×10 9 / L; Part 1 hepatocellular carcinoma (HCC) ≥ 50 × 10 9 For / L C. Hemoglobin ≥ 9.0 g / dL without transfusion in the previous week Creatinine ≤ 1.5 x upper limit of normal (ULN) e. Aspartate aminotransferase (AST) ≤ 3 × ULN (≤ 5 × ULN if HCC or liver metastases are present) f. Alanine aminotransferase (ALT [SGPT]) ≤ 3 × ULN (≤ 5 × ULN if HCC or liver metastasis is present) g. Bilirubin ≦1.5×ULN (patients with known Gilbert's disease may have bilirubin ≦3.0×ULN) h. Albumin ≥ 3.0 g / dL i. International normalized ratio (INR) and partial thromboplastin time (PTT) ≦ 1.5 x ULN j. Amylase and lipase ≦1.5 x ULN 11. No evidence of active infection or infection requiring parenteral antibiotics and no serious infection within 4 weeks prior to study initiation. 12. Females of childbearing potential must have a negative pregnancy test result within 72 hours prior to starting treatment. For females of childbearing potential: Agree to remain abstinent (abstain from heterosexual intercourse) or use a method of contraception with a failure rate of less than 1% per year during treatment and for at least 180 days after the last study treatment. A woman is of childbearing potential if she has had her first menstrual period, has not yet reached postmenopausal status (amenorrhea for 12 or more consecutive months with no identified cause other than menopause), and has not been surgically sterilized (removal of the ovaries and / or uterus). Examples of contraceptive methods with failure rates of less than 1% per year include bilateral tubal ligation, male sterilization, hormonal contraceptives that inhibit ovulation, hormone-releasing intrauterine devices, and copper intrauterine devices. The reliability of sexual abstinence should be evaluated in relation to the duration of the clinical trial and the patient's preferred usual lifestyle. Periodic abstinence (e.g., calendar, ovulation, symptom-temperature, or postovulatory methods) and withdrawal are not acceptable contraceptive methods. Males of reproductive potential must practice an effective method of contraception during the study unless evidence of infertility is present. 13. Four (4) weeks or five half-lives (whichever is shorter) since the last dose of anticancer therapy prior to the first dose of G9.2-17(IgG4) 14. Continuation of bisphosphonate treatment (e.g., zoledronic acid) or denosumab for bone metastases that have been stable for at least 6 months prior to C1D1 is permitted. 15. Biliary or gastric outlet obstruction is permitted, provided it can be effectively evacuated by endoscopic, surgical, or interventional means. 16. Pancreatic, biliary, or enteric fistulas are permitted provided they are managed with an appropriate, patent, non-infected drain (if a drain or stent is in situ, patency must be confirmed prior to study initiation). Additionally, for part 1 only: 17.Patient: a. Have already received at least one prior line of systemic therapy for metastatic disease, or b. Those with tumor types for which there are no available standard treatment options. Additionally, for part 2 only: 18. PDAC expansion cohort: 1st-line metastatic patients who have not received a gemcitabine-containing regimen or have been previously treated with a gemcitabine-containing regimen in the neoadjuvant or adjuvant / local advanced care setting for at least 3 months 19. CRC and CCA expansion cohort - Patients who have received at least one prior line of therapy in the metastatic setting.

[0275] Exclusion criteria Participants will be excluded from the study if they meet any of the following criteria: 1. Patient is unwilling or unable to comply with the requirements of the protocol 2.Patients diagnosed with metastatic cancer of unknown primary site 3. Prior or current illegal drug addiction (medical and recreational cannabis / cannabidiol (CBD) / tetrahydrocannabinol (THC) would not be considered "illegal"). 4. Any patient with clinically significant active uncontrolled bleeding and a bleeding diathesis (e.g., active peptic ulcer disease). Prophylactic or therapeutic use of anticoagulants is permitted. 5. Pregnant and / or breastfeeding women 6. Receiving any other investigational drug, or participating in any other clinical trial involving another investigational drug for the treatment of solid tumors within 4 weeks prior to Cycle 1, Day 1 of the trial, or within 5 half-lives of the administered drug (whichever is shorter), or other investigational therapy or major surgery within 4 weeks from the date of consent, or surgery scheduled within 4 weeks of the anticipated start of the trial (this includes dental surgery). 7. Radiation therapy within 4 weeks of first dose of investigational drug, except palliative radiation therapy to limited areas such as bone pain or treatment of locally painful tumor masses, that does not jeopardize measurable lesions necessary for response evaluation (RECIST v1.1). 8.Patients with fungal tumor masses 9. Patients with locally advanced PDAC without distant organ metastatic deposits 10. Grade 4 immune-mediated toxicity from prior checkpoint inhibitor. Grade 2 or Grade 3 pneumonitis or other Grade 3 checkpoint inhibitor-related toxicity that led to discontinuation of immunotherapy treatment. Low-grade (<grade 3) toxicities, e.g., neuropathy, manageable electrolyte abnormalities and lymphopenia, alopecia, and vitiligo from prior treatment, are acceptable. 11. History of secondary malignancies (excluding those previously treated with curative intent for ≥5 years with no or low chance of recurrence (e.g., nonmelanotic skin cancer, cervical carcinoma in situ, early (or localized) prostate cancer, or superficial bladder cancer)) 12. Active brain or leptomeningeal metastases. Patients with brain metastases are eligible if they have clinically and radiographically stable disease (SD) at least 4 weeks after definitive treatment and have not used steroids (≥10 mg / day prednisone or equivalent) for at least 4 weeks prior to the first dose of study drug. 13. Severe or uncontrolled systemic disease, congestive heart failure > New York Heart Association (NYHA) class 2, myocardial infarction (MI) within 6 months, or evidence of laboratory findings that would preclude the patient from participating in the trial. 14. Any significant medical condition compromising patient safety or compromising the interpretation of the G9.2-17 (IgG4) toxicity assessment 15. Serious non-healing wounds, ongoing ulcers, or untreated fractures 16. Uncontrolled pleural, pericardial, or ascites requiring repeated drainage procedures. For the purposes of this trial, "recurrence" is defined as 3 or more drains in the past 30 days. 17. History of severe allergic, anaphylactic, or other hypersensitivity reactions to chimeric or humanized antibodies or fusion proteins 18. Significant vascular disease within 6 months of Cycle 1, Day 1 (e.g., aortic aneurysm requiring surgical repair, or recent arterial thrombosis) 19. History of pulmonary embolism, stroke, or transient ischemic attack within 3 months prior to Cycle 1, Day 1 20. History of abdominal fistula or gastrointestinal perforation within 6 months prior to Cycle 1, Day 1 21. Active autoimmune disease (excluding diabetes mellitus type I / II, hypothyroidism requiring hormone replacement only, vitiligo, psoriasis, or alopecia areata) 22. Requires systemic immunosuppressive therapy, including but not limited to cyclophosphamide, azathioprine, methotrexate, thalidomide, and anti-TNF agents. Patients who have received or are receiving acute low-dose systemic immunosuppressants (e.g., 10 mg / day or less of prednisone or equivalent) may be enrolled. Replacement therapy (e.g., thyroxine, insulin, physiological corticosteroid replacement therapy [e.g., 10 mg / day or less of prednisone equivalent] for adrenal or pituitary insufficiency) is not considered a form of systemic treatment. Use of inhaled corticosteroids and mineralocorticoids (e.g., fludrocortisone), topical steroids, intranasal steroids, intra-articular steroids, and ophthalmic steroids is permitted. 23. Severe tumor-related pain (grade 3 or higher according to Common Terminology Criteria for Adverse Events (CTCAE) v. 5.0) unresponsive to extensive analgesic interventions (oral and / or patch) 24. Hypercalcemia (grade 3 according to CTCAE v5.0) despite use of bisphosphonates 25. Any other disease, metabolic dysfunction, physical examination finding, or clinical laboratory finding that contraindicates the use of the investigational product or that gives reasonable suspicion of a disease or condition that may affect the interpretation of the results or place the patient at high risk of complications of treatment. 26. Administered organ transplant(s) 27. Patients undergoing dialysis 28. Patients enrolled in the anti-PD-1 antibody combination cohort must have had no prior exposure to any anti-PD-1 or anti-PD-L1 agents in any line of prior therapy. Additionally, patients diagnosed with dMMR / MSI-H will be excluded. 29. In Part 1, hormonal androgen deprivation therapy will be permitted to continue for men with metastatic castration-resistant prostate cancer. 30. Any ablative therapy (radiofrequency ablation or percutaneous ethanol injection) for HCC within 6 weeks prior to study entry 31. Hepatic encephalopathy or severe hepatic adenoma 32. Child-Pugh score ≥ 7 Additionally, for part 2 only: 33. Hypersensitivity to any of the active substances or excipients listed in the components of the anti-PD-1 antibody Additionally, in the Part 2 combination (G9.2-17(IGG4) + anti-PD-1 antibody) arm only: 34. Have received a live vaccine within 28 days of starting treatment. Inactivated vaccines (i.e., influenza and Covid-19) are permitted.

[0276] Investigational Drugs and Other Interventions Investigational intervention(s) are defined as the investigational drug(s), marketed product(s), placebo, or medical device(s) intended to be administered / used to study participants according to the study protocol.

[0277] G9.2-17 Drugs administered in combination with IgG4 Nivolumab Nivolumab (OPDIVO®) is a programmed death receptor 1 (PD-1) blocking antibody indicated for the treatment of multiple tumor types. Nivolumab can be used as an exemplary anti-PD-1 antibody in combination with an anti-galectin-9 antibody disclosed herein, such as G9.2-17 IgG4.

[0278] Nivolumab may be administered as an intravenous infusion over 30 minutes at a dose of 240 mg every 2 weeks in a 28-day cycle (unless otherwise indicated). Per FDA labeling, there are no contraindications to the administration of nivolumab.

[0279] Nivolumab AEs are presented in the following table according to their frequency of occurrence. [Table 6] [Table 7]

[0280] Clinical Trial Intervention Management All patients will receive G9.2-17(IgG4), administered by IV infusion weekly or every 2 weeks until disease progression, unacceptable toxicity, or withdrawal of consent.

[0281] In Part 1, patients will receive G9.2-17 (IgG4) alone at escalating doses starting at 0.2 mg / kg.

[0282] In Part 2, patients will receive G9.2-17(IgG4)RP2D (determined in Part 1) as a single agent or G9.2-17(IgG4)RP2D-1 in combination with an anti-PD-1 antibody as follows: Patients with CRC or CCA G9.2-17 (IgG4) in CRC G9.2-17 (IgG4) in CCA ○G9.2-17 (IgG4) + anti-PD-1 antibody in CRC ○G9.2-17 (IgG4) + anti-PD-1 antibody in CCA Other solid tumor types (based on data from Part 1) G9.2-17 (IgG4) as a single agent and / or in combination with checkpoint inhibitors or chemotherapy as determined based on each tumor type

[0283] See Table 4 for a summary description of each study intervention.

[0284] Patients who experience a DLT in Part 1 will not resume treatment. Patients who experience a DLT in Part 2 will have treatment discontinued. If clinical benefit is achieved, treatment may be resumed with the same or reduced dose of G9.2-17(IgG4). [Table 8]

[0285] Preparation of G9.2-17 (IgG4) The manufacture and packaging of Investigational Medicinal Product (IMP) G9.2-17 (IgG4) follows applicable current Good Manufacturing Practice (cGMP) and the product meets the standards applicable for human use.

[0286] The G9.2-17 (IgG4) formulation is diluted to the target dose prior to administration. All dilutions should be performed in a controlled, sterile environment (patient doses are prepared and administered by IV infusion over approximately 60 minutes).

[0287] G9.2-17(IgG4) is a sterile liquid and should be stored at 2°C to 8°C, protected from light.

[0288] Dose escalation If a patient experiences clinical benefit with G9.2-17(IgG4) and protocol efficacy criteria, and the patient experiences an adverse reaction not attributable to G9.2-17(IgG4), then treatment may continue with G9.2-17(IgG4) alone. G9.2-17(IgG4) may be continued if: The patient's clinical condition is not rapidly deteriorating; and ● The concomitant medication is discontinued due to an AE attributable solely to the concomitant medication.

[0289] If IMAR occurs / recurs and cannot be managed by dose reduction of either drug, both study drugs must be discontinued.

[0290] Nab-paclitaxel is not recommended for patients with total bilirubin >5xULN or AST >10xULN. In addition, Nab-paclitaxel is not recommended for patients with metastatic adenocarcinoma of the pancreas with moderate to severe hepatic impairment (total bilirubin >1.5xULN and AST <10xULN). For patients with moderate or severe hepatic impairment, the starting dose must be reduced.

[0291] Dose Modifications for Specific AEs Associated with Nivolumab Administration Recommendations for nivolumab modifications based on specific AEs are provided below. No dose modifications of nivolumab are recommended for hypothyroidism or hyperthyroidism.

[0292] Recommended dose modifications of nivolumab for AEs other than IMAR are presented below: [Table 9]

[0293] Dose modification of IMAR If IMAR occurs, see guidance provided herein regarding dose management of G9.2-17 IgG4 and / or nivolumab.

[0294] Discontinuation of study intervention In rare cases, it may be necessary for a patient to permanently discontinue the investigational intervention. If the investigational intervention is permanently discontinued for reasons other than disease progression and the patient is not being treated with other anti-cancer therapy(ies), the patient will continue to be evaluated for disease progression for up to 2 years. See SoA for data to be collected at the time of discontinuation of investigational intervention and follow-up, and any further optional evaluations that need to be completed.

[0295] Investigators must make every effort to continue patients on study treatment until one of the reasons for discontinuing study treatment (disease progression, study drug-related toxicity, withdrawal of consent) is met. If a patient has radiographic progression, the patient may continue study treatment if there is no clear clinical progression and no alternative treatment is initiated. However, if a patient has clear clinical progression in the absence of radiographic progression, study treatment should be stopped and the patient has been advised of available treatment options.

[0296] Patients may be discontinued prior to disease progression for any of the following reasons: • DLT per definition in Section 3.4.4. The AE occurs / recurs outside the DLT window requiring discontinuation of the study treatment(s). Occurrence / recurrence of an IMAR requiring discontinuation of the study treatment(s). ●Completion of research by PureTech Health, LLC Any intercurrent illness or medical condition that may prevent further administration of treatment or jeopardize the patient's safety if continued on investigational treatment. Pregnancy Use of non-protocol anticancer therapy

[0297] Patients may also be discontinued prior to disease progression for any of the following reasons: -Significant deviation from protocol on the part of the patient (including lack of compliance)

[0298] An explanation of why a patient discontinues study treatment should be documented in the Case Report Form (CRF). If a patient discontinues study treatment due to toxicity, "dose-limiting toxicity" or "adverse event" will be recorded as the primary reason for withdrawal. If a patient is prematurely discontinued from the study at any time due to an AE or SAE, the patient should be followed until recovery to Grade 2 or less, unless improvement is unlikely due to the underlying condition.

[0299] Combination therapy Any medications or vaccines (including over-the-counter or formulated drugs, recreational drugs, vitamins, and / or herbal supplements) that participants are receiving at the time of enrollment or will be receiving during the study must be recorded with the following information: ●Reason for use Date of administration, including start and end dates Dosage information, including dosage and frequency

[0300] Permitted medicines The following concomitant medications are permitted: • Standard of care premedication for patients on combination treatment regimens. • Continuation of bisphosphonate treatment (e.g., zoledronic acid) or denosumab for bone metastases, stable for at least 6 months prior to treatment (C1D1). • Use of inhaled corticosteroids and mineralocorticoids (e.g., fludrocortisone), topical steroids, intranasal steroids, intra-articular steroids, and ophthalmic steroids. Prophylactic or therapeutic use of anticoagulants Vaccination against COVID-19, common influenza, and / or other common clinically indicated indications (e.g., tetanus, pneumococcal, HBV, etc.) is permitted prior to or during the study period. Timing and type of vaccination must be recorded.

[0301] Prohibited Substances The following medications are not permitted during this study: - Concomitant administration of other investigational drugs other than G9.2-17 (IGG4) for any indication. Systemic immunosuppressive treatments, including but not limited to cyclophosphamide, azathioprine, methotrexate, thalidomide, and anti-TNF agents. However, patients are permitted to take acute low doses of systemic immunosuppressants (e.g., up to 10 mg / day of prednisone or equivalent). • Replacement therapy (e.g., thyroxine, insulin, or physiologic corticosteroid replacement therapy [e.g., prednisone equivalent 10 mg / day or less] for adrenal or pituitary insufficiency) is not considered a form of systemic treatment.

[0302] supportive care Patients should receive full supportive care during the study, including transfusion of blood and blood products; treatment with antibiotics, antiemetics, antidiarrheals, and analgesics; and other treatments as deemed appropriate and in accordance with institutional guidelines.

[0303] Evaluation schedule [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6] [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4] [Table 11-5] [Table 11-6]

[0304] Study assessment and procedures A signed, written ICF approved by the Institutional Review Board (IRB) must be obtained prior to potential patients participating in any study-specific procedures, including study-specific screening procedures.

[0305] Patients are enrolled in the study if they complete all screening procedures and are determined to meet all eligibility criteria. Part 1, Cohorts 1-6 study procedures and respective timing are summarized in the SoA (Table 8). Part 1, Cohorts 7 and 8 study procedures and respective timing are summarized in the SoA (Table 9). Protocol waivers or exemptions will not be permitted. • Compliance with all study requirements, including those specified in the SoA, is essential and mandatory for the conduct of the study. • Immediate safety concerns must be discussed as soon as they arise or are recognized to determine the need for intervention or study discontinuation. All screening assessments must be completed and outlined to ensure potential participants meet all eligibility criteria. A screening log will be maintained to record details of all participants screened and, where applicable, confirm eligibility or record the reasons for screening failure. • Procedures performed as part of the participant's routine clinical management (e.g., blood counts) and obtained prior to signing the ICF may be utilized for screening or baseline purposes only if they meet the criteria specified in the protocol and the procedure was performed within the time frame defined in the SoA.

[0306] Evaluation for each visit The SoA (Tables 5-6) present a list of assessments to be performed during the screening period (maximum 28 days), the treatment period (shown as 28-day cycles), the end-of-treatment / early discontinuation period, the IMAR follow-up, and the long-term follow-up period. If medically indicated, optional visits are allowed during each treatment cycle, during which study assessments may be performed.

[0307] During the COVID-19 pandemic, many governments have mandated social distancing for their citizens and more vulnerable populations have been advised to self-isolate. These types of restrictions may impact our ability to conduct this clinical study as originally intended. Planned site visits may be adjusted to allow the study to continue safely during the pandemic. Possible modifications may include: Postponement of visits and / or clinical trial procedures ● Substitute by phone / video call(s) Alternative to home visits Visits conducted at alternative clinics Visits by health care providers other than the study team ● Visits and / or study procedures have been cancelled entirely.

[0308] Screening Period (between Day 28 and Day 1) The following procedures must be performed within 4 weeks of starting treatment: Study Procedures and Exams ● Written informed consent ● Check inclusion and exclusion criteria for patient eligibility Patient demographics Medical history Pre- and concomitant medications ●ECHO / Multi-Gated Acquisition Scan (MUGA) ● 12-lead ECG (QT interval corrected using Fridericia's formula [QTcF]) Physical Examination – For patients with stable, treated brain metastases, a neurological examination should be performed. ECOG performance status Vital signs Tumor imaging evaluation (computed tomography [CT] or magnetic resonance imaging [MRI] with or without contrast, or positron emission tomography [PET]-CT, with CT with contrast preferred) Clinical Laboratory Pregnancy testing for women of childbearing potential (WOCBP) ●Hematology Serum Chemistry Thyroid stimulating hormone (TSH), free T4 or thyroxine (fT4), serum lipase, amylase, parathyroid hormone (PTH), follicle stimulating hormone (FSH), luteinizing hormone (LH), free cortisol ●Blood coagulation Urine tests Pharmacodynamics and Pharmacokinetics Tumor biopsy o If a biopsy is deemed dangerous to the patient, the biopsy may be omitted. If a biopsy is not available, the institution will make every effort to obtain an archival tumor tissue specimen available as a formalin-fixed, paraffin-embedded (FFPE) block. Acceptable archival specimens include specimens obtained by core needle biopsy or excision surgery within the past 5 years. dMMR-MSI-H status (if the patient's MMR and MSI status has not been pre-determined, testing should be performed at a local laboratory) Part 2: In-house TMB in the G9.2-17(IGG4) + anti-PD1 antibody combo treatment group only Tumor type-related biomarkers

[0309] Treatment period Each treatment cycle was 28 days in duration. For timing of visits, see Table 5 for Part 1, Cohorts 1-6 and Table 6 for Part 1, Cohorts 7 and 8.

[0310] Treatment procedure on day 1 of each cycle (CXD1; ±2 days from the start of cycle 2) The following procedure will be performed on day 1 of each treatment cycle. Study procedures and tests Concomitant medication ●AE ●12-lead ECG (QTcF) Physical Examination ECOG performance status Vital signs Clinical Laboratory Pregnancy testing for WOCBP ●Hematology Serum Chemistry ●TSH, fT4, lipase, amylase, PTH, FSH, LH, free cortisol ●Blood coagulation Urine tests PK / PD assessment ●PD blood sampling ●PK blood sampling ●ADA blood collection Tumor type-related biomarkers Administration of investigational drug • Administer only after all pre-administration evaluations and procedures have been completed. In addition, starting on Day 1 of Cycle 3, the following assessments will be performed every 8 weeks: Tumor imaging evaluation (CT or MRI, with or without contrast; or PET-CT, preferably CT with contrast) In addition, starting on Day 1 of Cycle 4, the following evaluations will be performed every 3 months: ●ECHO / MUGA Cohorts 1-6: Treatment procedures on days 2 and 8 of cycle 1 and cycle 3 (CXD2 ± 1 days and CXD8 ± 1 days) Study procedures and tests Concomitant medication ●AE PK / PD assessment ●PD blood sampling ●PK blood sampling Cohorts 1-6: Treatment procedure on day 15 of each cycle (CXD15 ± 1 days for cycle 1 and ± 2 days for the start of cycle 2)

[0311] The following procedure is performed on day 15 of each treatment cycle. Study procedures and tests Concomitant medication ●AE Physical Examination ECOG performance status Vital signs Clinical Laboratory ●Hematology Serum Chemistry ●Blood coagulation Urine tests PK / PD assessment ● PD blood sampling only on C1D15 and C3D15 ●PK blood sampling for C1D15 and C3D15 only Tumor type-related biomarkers Tumor biopsy on C3D15±7 (Cycle 3 only; may be omitted if deemed too high risk for patient) Administration of investigational drug • Administer only after all pre-administration evaluations and procedures have been completed. Cohorts 7 and 8: Treatment Procedures on Day 3 of Cycle 1 and Cycle 3 (C1D3±1 days and C3D3±1 days) Study procedures and tests Concomitant medication ●AE PK / PD assessment ●PD blood sampling ●PK blood sampling Cohorts 7 and 8: Treatment procedure on day 8 of each cycle (CXD8 ± 1 days) Study procedures and tests Concomitant medication ●AE Physical Examination ECOG performance status Vital signs Clinical Laboratory ●Hematology Serum Chemistry ●Blood coagulation Urine tests PK / PD assessment ●PD blood sampling PK blood sampling only in odd-numbered cycles Administration of investigational drug • Administer only after all pre-administration evaluations and procedures have been completed. Cohorts 7 and 8: Treatment procedures on days 15 and 22 of each cycle (CXD15 ± 1 days in cycle 1 and ± 2 days at the start of cycle 2)

[0312] The following procedures will be performed on days 15 and 22 of each treatment cycle. Study procedures and tests Concomitant medication ●AE Physical Examination ECOG performance status Vital signs Clinical Laboratory ●Hematology Serum Chemistry ●Blood coagulation Urine tests PK / PD assessment ● PD blood sampling only on C1D15 and C3D15 PK blood sampling only in odd-numbered cycles Tumor type-related biomarkers Tumor biopsy on C3D15±7 (Cycle 3 only; may be omitted if deemed too high risk for patient) ●ADA blood collection for C1D15 and C2D15 only Administration of investigational drug • Administer only after all pre-administration evaluations and procedures have been completed.

[0313] Additional treatment after cycle 4 Treatment cycles beyond cycle 4 may be repeated as indicated in the SoA (Tables 5-6). If the patient is experiencing clinical benefit, they may continue treatment even if they have progressed radiologically.

[0314] Procedures for terminating or early discontinuation of treatment The following procedures will be performed 30 days (± 3 days) after the last dose, including patients who are discontinuing treatment early. Study procedures and tests Concomitant medication ●AE Physical Examination ECOG Vital signs • Tumor imaging evaluation: confirmatory scan if study end was >8 weeks since previous scan. Clinical Laboratory Pregnancy testing for WOCBP ●Hematology Serum Chemistry ●TSH, fT4, lipase, amylase, PTH, FSH, LH, free cortisol ●Blood coagulation Urine tests PD evaluation ●PD blood sampling ●ADA blood collection Tumor type-related biomarkers

[0315] IMAR 90-day follow-up All patients receiving combination treatment with an anti-PD1 antibody in Part 2 must visit for safety follow-up on Day 90 ± 7 to evaluate any potential delayed IMAR. Visits include: Study procedures and tests ●AE Physical Examination Vital signs Clinical Laboratory ●Hematology Serum Chemistry ●TSH, fT4, lipase, amylase, PTH, FSH, LH, free cortisol ●Blood coagulation Urine tests

[0316] Long-term follow-up OS will be assessed every 3 months for up to 2 years after patients complete / prematurely discontinue treatment. Tumor imaging assessments will continue if patients are able to discontinue treatment for reasons other than disease progression and not receive additional systemic anti-cancer treatment.

[0317] Survival data and information on new anticancer therapies initiated after disease progression will be collected at least every 3 months. They may be collected more frequently to aid in data cleaning or regulatory submission efforts. Follow-up may be conducted by telephone interview, electronic messaging, or chart review and will be reported on the CRF. During the follow-up period, cause of death, regardless of causality, will be collected and reported within 24 hours of discovery or notification of the event.

[0318] RECIST v1.1 criteria for tumor assessment For screening tumor assessment, tumor lesions / lymph nodes are classified as measurable or non-measurable, and measurable tumor lesions are recorded according to the longest diameter of the measurement surface (excluding pathological lymph nodes, which are measured in their shortest axis). If multiple measurable lesions are present at screening, a total of up to five total lesions (and a maximum of two lesions per organ) representing all involved organs should be identified as target lesions. Target lesions should be selected based on their size (lesions with the longest diameter). The sum of the diameters of all target lesions is calculated and reported as the baseline sum diameter.

[0319] All other lesions (or sites of disease), including pathological lymph nodes, should be identified as non-target lesions and also recorded at screening. No measurements are required and these lesions should be tracked as "present," "absent," or "definite progression."

[0320] Tumor target lesions will be assessed according to RECIST v1.1 guidelines (Eisenhauer et al., 2009) using the following disease response measures:

[0321] Target Lesion Assessment: ● Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes (whether targeted or non-targeted) must have a reduction of less than 10 mm in the short axis. ●Partial response (PR): At least a 30% reduction in the sum of the diameters of the target lesions, based on the baseline sum diameter. ● Progressive Disease: The sum of the diameters of the target lesions increases by at least 20% with reference to the study minimum sum (for the study minimum, this includes the baseline sum). In addition to the relative increase of 20%, the sum must also show an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progression). ● Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, based on the smallest total diameter on study.

[0322] Non-target lesion assessment: CR: All non-target lesions disappear and tumor marker levels normalize. All lymph nodes must be non-pathological in size (short axis <10 mm). • Non-CR / non-progressive disease (non-PD): persistence of one or more non-target lesion(s) and / or maintenance of tumor marker levels above normal limits. ● Progressive disease: clear progression of existing non-target lesions. (Note: the appearance of one or more new lesions is also considered progression).

[0323] A summary is provided in Table 7 below. [Table 12]

[0324] Measurement of disease response at various time points allows for the calculation of: • Disease control rate (DCR), defined as the proportion of patients achieving CR, PR, and SD. ●Objective response rate (ORR), defined as the percentage of patients who experience a predetermined amount of reduction in tumor size (≥30% tumor shrinkage). Progression-free survival (PFS) is defined as the time from start of study drug treatment to disease progression (tumor growth ≥ 30%). Duration of response (DoR), defined as the length of time the tumor continues to respond to treatment without the cancer growing or spreading. Overall survival (OS) is defined as the time from start of study drug treatment to death from any cause.

[0325] Safety assessment Physical Examination The medical and physical examination must be performed by a licensed physician, nurse practitioner, or physician assistant and must include a thorough examination of all body systems. In addition, height (at screening only) and weight will be measured.

[0326] Vital signs Vital signs are measured after 5 minutes of rest in the supine position and include temperature, blood pressure (systolic and diastolic), heart rate, and respiratory rate.

[0327] electro-cardiogram A 12-lead ECG is obtained as outlined in the SoA (see Tables 5-6) using an ECG machine that automatically calculates heart rate and measures heart rate, PR interval, QRS duration, the distance in time in the ECG tracing from the start of the QRS complex to the end of the T wave (QT) interval, and the QTcF interval.

[0328] Clinical safety experimental evaluation Patients will have blood samples drawn (approximately 5 mL at each time point) for routine laboratory tests according to the SoA (Tables 5-6); additional tests may be performed at any time during the study if deemed necessary.

[0329] Clinical laboratory parameters will be analyzed at the facility's local laboratory. Laboratory evaluations completed include hematology and serum chemistry, defined as follows: ● Serum chemistry: Includes glucose, total protein, albumin, electrolytes [sodium, potassium, chloride, magnesium, phosphorus], calcium, bilirubin (total, direct), SGPT (ALT) or SGOT (AST), alkaline phosphatase, gamma glutamyl transferase (gamma GT), lactate dehydrogenase (LDH), creatinine, hemoglobin A1c (HgbA1c) (only if there is a history of type 1 or type 2 diabetes), blood urea nitrogen, and creatine phosphokinase (CPK). TSH, fT4, lipase, amylase, PTH, FSH, LH, and free cortisol at designated visits Fasting blood glucose levels are assessed only when clinically indicated. • Hematology: Includes complete blood count, differential, platelets, and hemoglobin. ● Coagulation: includes prothrombin time (PT) and PTT, activated partial thromboplastin time (APTT) and INR (with tolerated anticoagulants), C-reactive protein (CRP), and troponin. Urinalysis: The patient collects a urine specimen for routine urinalysis, including color, appearance, and specific gravity gauges, protein, leukocyte esterase, glucose, ketones, urobilinogen, nitrites, white blood cell count (WBC), red blood cell count (RBC), and pH, as well as urine culture (if the patient is clinically symptomatic). If clinically significant values ​​do not return to normal / baseline or grade 1 within a reasonable time period, an etiology must be identified. All protocol-required laboratory tests must be performed in accordance with the test manuals and SoAs (Tables 5-6). ● If a non-protocol-specified clinical test result performed in the site's local laboratory requires a change in the participant's management or is deemed clinically important (e.g., an SAE or AE or dose modification), the result must be recorded.

[0330] Pregnancy test Only WOCBP after menstrual period has been confirmed and a high-sensitivity urine or serum pregnancy test has been negative should be included.

[0331] Additional pregnancy testing should be performed according to local need during the treatment period and at end-of-treatment / early discontinuation visits in accordance with the SoA (Tables 5-6).

[0332] A pregnancy test is performed whenever a menstrual cycle is late or if pregnancy is otherwise suspected.

[0333] If the patient has a history of bilateral salpingo-oophorectomy and / or hysterectomy, record these surgical procedures; pregnancy testing is not required for these patients.

[0334] Pharmacokinetic evaluation Where possible, the following serum PK parameters will be calculated for G9.2-17(IGG4): ●AUC 0-336h ●C max ●T max ●t 1 / 2 Serum concentration vs. time profile

[0335] Approximately 5 mL blood samples are collected and processed into serum at each time point specified in the SoA (Tables 5-6).

[0336] PK Schedule for Cohorts 1-6: Day 1 of Cycle 1 and Cycle 3 Before administration ●End of injection (EOI) ●2 hours (±30 minutes) from EOI ● 4 hours (± 30 minutes) from EOI Day 15 of Cycle 1 and Cycle 3 Before administration ●At EOI Days 2 and 8 (non-treatment days) of Cycle 1 and Cycle 3 At any point during the visit Day 1 of Cycle 2 and Cycle 4 Before administration ●At EOI Day 1 of every 2 cycles after Cycle 4 (i.e., C6D1, C8D1, etc.) Before administration ●At EOI

[0337] PK Plan for Cohorts 7 and 8: Day 1 of every odd-numbered cycle (i.e., C1D1, C3D1, etc.) Before administration ●End of injection (EOI) ● 1 hour after EOI (±15 minutes) Day 3 of every odd-numbered cycle (i.e., C1D3, C3D3, etc.) At any point during the visit Days 8, 15, and 22 of every odd-numbered cycle (i.e., C1D8, C3D8, etc.) Before administration ●At EOI Day 1 of every even-numbered cycle (i.e., C2D1, C4D1, etc.) Before administration ●At EOI

[0338] If it is determined that study drug administration has been interrupted, additional PK and safety assessments will be collected upon resumption of administration; additional PK assessments may be performed during the interruption. If the dose of study drug is reduced, additional PK assessments will be collected prior to administration of the reduced dose (within 2 hours prior to dosing) and 2-4 hours after initiation of reduced study drug administration. Additional PK and other hematological assessments may be performed if clinically indicated. Sites unable to accommodate patients more than 2 hours after dosing due to COVID-19 restrictions will give samples at EOI and 2 hours after dosing.

[0339] Instructions for collection and handling of biospecimens will be provided. The actual date and time (24-hour clock time) of each sample will be recorded.

[0340] Samples are used to assess serum concentration levels of total G9.2-17 (IGG4) and free / partially free G9.2-17 (IGG4) by a routine laboratory. Concentrations are measured using a validated assay. At least two 50 μL aliquots of serum are required to measure total G9.2-17 (IGG4) concentrations. At least two 100 μL aliquots of serum are required to measure free and partially free G9.2-17 (IGG4) concentrations as well as a third aliquot of residual serum. Samples collected for analysis of G9.2-17 (IGG4) plasma concentrations may also be used to assess safety or efficacy aspects related to concerns arising during or after the clinical trial.

[0341] No genetic analysis will be performed on these blood samples. Participant confidentiality will be maintained. At visits where blood samples for safety laboratory determination of PD, ADA, and G9.2-17 (IGG4) are taken, one sample of sufficient quantity may be used.

[0342] Genetics Genetics were not evaluated in this study.

[0343] Pharmacodynamic Biomarkers Planned time points for biomarker assessment are provided at the SoA (Tables 5-6); sampling may be reduced to every third cycle after 6 months of treatment.

[0344] Collection of biological specimens for other biomarker studies is also part of this trial. The following specimens are required for biomarker studies and will be collected from all participants in this trial as specified in the SoA. A blood sample to be collected prior to administration of the investigational drug (approximately 15 mL prior to administration) Tumor biopsy (tissue sample)

[0345] Samples will be tested for PD biomarkers (by flow cytometry, ELISA, IHC, or multiplexed phenotyping) using validated assays to assess association with observed clinical response to G9.2-17(IGG4).

[0346] The following biomarkers will be evaluated in this trial: Tumor markers (blood): CA15-3, CA-125, carcinoembryonic antigen (CEA), CA19-9, alpha-fetoprotein, neuron specific enolase (NSE), cytokeratin fragment 21 (CYFRA-21) to be assessed pre- and post-cycle as needed for each tumor type. This may be decreased as needed every 3 cycles after 6 months of treatment, following the same schedule as tumor imaging assessments. PBMC phenotype (blood): e.g., CD3, CD4, CD8, CD45RO, forkhead box protein P3 (FOXP3), CD11B, CD14, CD15, CD16, CD33, CD68, human leukocyte antigen (HLA)DR, CD163, arginase 1, granzyme B, KI67, PD-1, PDL1, pancytokeratin (PAN CK) ● Cytokines (blood): e.g., interferon gamma (IFNγ), IL10, IL12p70, IL13, IL1β, IL2, IL4, IL6, IL8, TNFα, MIP-1b, monocyte chemoattractant protein 1 (MCP-1), MIP-1a, IL17a, IL5, TGFβ Gal-9 in blood and tumor tissues ●PD-L1 (organization) ● Repair Status Mismatch (Organization) Tumor mutation burden (TMB)

[0347] Changes in exploratory biomarkers, if any, will be correlated with safety and response outcomes.

[0348] Samples may be stored for up to 2 years (or according to local regulations) after the last patient's last visit for the trial at selected facilities to allow for further analysis of the effects of G9.2-17(IGG4) on pharmacodynamic biomarkers.

[0349] Immunogenicity assessment Blood samples (approximately 3 mL) will be collected from all participants according to the SoA (Tables 5-6) and processed into serum. In addition, serum samples should also be collected at the end-of-treatment / early discontinuation visit from patients who discontinue the study intervention or withdraw from the study. Cohorts 1-6: Day 1 of Cycle 1-Cycle 4 Before administration Cohorts 1-6: Day 1 of every 2 cycles after Cycle 4 (i.e., C6D1, C8D1, etc.): Before administration Cohorts 7 and 8: Day 1 of each cycle Before administration Cohorts 7 and 8: Day 15 of Cycle 1 and Day 15 of Cycle 2 only Before administration

[0350] At least two 500 μL aliquots of serum each are obtained, and the remaining serum is obtained in a third tube. The samples are shipped to a designated laboratory for analysis using a validated assay. These samples are then tested.

[0351] Serum samples are screened for antibodies that bind to G9.2-17(IgG4)(ADA) and titers of confirmed positive samples are reported. Other analyses may be performed to verify the stability of antibodies to G9.2-17(IgG4) and / or to further characterize the immunogenicity of G9.2-17(IgG4).

[0352] Detection and characterization of antibodies to G9.2-17(IgG4) will be performed using a validated assay. All samples collected for detection of antibodies to the intervention study will be evaluated for G9.2-17(IgG4) serum concentrations to allow interpretation of antibody data. Antibodies may be further characterized and / or evaluated for their ability to neutralize the activity of the investigational intervention. Samples may be stored for up to 2 years (or in accordance with local regulations) after the last patient's last visit for the study in a suitable facility to allow further analysis of the immune response to G9.2-17(IgG4).

[0353] Other evaluations Patient background At screening, patient demographics are collected, including age, sex, race, and ethnicity.

[0354] Medical history Medical history will include oncology history, surgery / transplant history, radiation therapy history, and COVID19 history and testing. • Personal medical history including prior procedures / surgeries (record of any implants in situ or past implants, prior and / or current use of medical devices, concomitant medications (name, indication, dose, route, dose modification with start and end dates if necessary, and reason), pre-existing conditions, and AEs), family history and genetic disorders of risk based on a complete family history to the best of the patient's knowledge). Records of dental treatments performed in the past 12 months • For patients with previously resected pancreatic adenocarcinoma, record whether the primary tumor was located in the head, body, or tail of the pancreas. Bowel habits / typical frequency and consistency ● Record any dietary requirements or preferences (e.g., following a specific dietary regimen: intermittent fasting, keto diet, etc.). - Records of past and current allergies (allergens, severity)

[0355] Prior and concomitant medications Prior and concomitant medications, including vaccines and complementary treatments / supplements, will be documented for each patient at each scheduled visit (Tables 5-6).

[0356] Tumor imaging evaluation Tumor evaluation is performed using CT or MRI with or without contrast; PET-CT studies are performed.

[0357] CT with contrast is the preferred modality (MRI, PET-CT, or other imaging modalities in lieu of or in addition to a CT scan if, at a given site of disease, CT is not feasible or appropriate). Evaluation should include chest / abdomen / pelvis at a minimum and should include other anatomical regions as indicated based on the patient's tumor type and / or medical history. Imaging scans must be de-identified and archived in native format as part of the patient's trial file. The type of scan will be obtained depending on the disease, but the same methodology should be used for the duration of the trial.

[0358] In the trial, evaluations will be performed every 8 weeks ± 7 days according to the SoA (i.e., C3D1, C5D1, C7D1, C9D1, etc.) and at the end of treatment if not evaluated within the past 4-6 weeks. Evaluations may be performed more frequently if clinically indicated. For Part 2 only, a confirmatory scan will be performed after 4 weeks (+7 days) if the scan shows an objective response. After the confirmatory scan, scheduled scans should resume at a frequency of every 8 weeks (± 7 days) from the date of the confirmatory scan.

[0359] Tumor biopsy Pre- and intra-treatment biopsies will be collected. A pre-treatment biopsy will be collected during screening. If a pre-treatment biopsy is not available for reasons outlined in the inclusion criteria and the patient is enrolled in the study, archival tumor tissue specimens from the patient will be collected from the primary tumor and / or metastatic deposits. Excision or core biopsies (FFPE tissue block(s) or fresh tissue in formalin) obtained currently or within 5 years prior to study initiation from the primary tumor lesion or metastatic deposit. If both primary and metastatic tissue are available, preference will be given to the use of metastatic deposit tissue. If information on the treatment(s) received before and after tissue collection is available, this will also be collected.

[0360] Intra-treatment biopsies are scheduled for C3D15±7 days and should be performed only after the cycle 3 tumor imaging scan. If the procedure cannot be performed within the time frame specified in the protocol, alternatives may be permitted but should be discussed with the Investigator / Medical Oversight. It is recognized that a variety of clinical factors may make it difficult to obtain sufficient specimens. The decision not to complete an intra-treatment biopsy should be discussed with Medical Oversight.

[0361] ECHO / MUGA ECHO and / or MUGA will be obtained at the time points indicated in the SoA (Tables 5-6). Assessments will be repeated every 3 months if clinically indicated.

[0362] ECOG ECOG performance status will be assessed at the time points indicated on the SoA (Tables 5-6) using the following grading (Oken et al., 1982): Grade 0: Fully active and able to continue all pre-disease performance without restriction Grade 1: Physically strenuous activity is limited, but the patient is able to walk and perform light or sedentary tasks, e.g., light housework, clerical work. Grade 2: Able to walk and perform all self-care activities, but unable to perform any work activities. Active for approximately 50% or more of waking time. Grade 3: Limited self-care capabilities and confined to bed or chair for more than 50% of waking hours. Grade 4: Completely disabled. Unable to continue any self-care. Completely bed or chair confined. Grade 5: Death

[0363] Adverse Events (AEs), Serious Adverse Events (SAEs), and Other Safety Reports An AE is defined in the ICH guidelines for GCP as "an undesirable medical occurrence in a patient receiving a medicinal product or in a clinical research patient that does not necessarily have a causal relationship to this treatment."

[0364] In this study, the definition of AE is expanded to include any such occurrence (e.g., sign, symptom, or diagnosis) or worsening of an existing medical condition from the time the patient signs informed consent through the time of initiation of investigational drug. Aggravation indicates an increase in the severity, frequency, or duration of symptoms of an existing medical condition (e.g., diabetes, migraine, gout, hypertension, etc.) or an association with a significantly worse outcome.

[0365] Serious Adverse Events An SAE is defined as an AE that: Death is caused • Life threatening (putting the patient at risk of immediate death). • Hospitalization or extension of existing hospitalization is required.

[0366] A hospitalization that meets the definition of "serious" is one that includes at least an overnight stay in a health care facility. Inpatient hospitalization does not include rehabilitation facilities, hospice facilities, skilled nursing facilities, nursing homes, routine emergency room admissions, same-day surgery (as an outpatient / same-day / outpatient procedure), or community hospitalizations (e.g., where the patient has no place to sleep). resulting in persistent or significant impairment / incapacity; or ● Whether the child has a birth abnormality / birth defect A significant medical event that is not fatal, life-threatening, or may require hospitalization may be considered an SAE if, based on appropriate medical judgment, it may endanger the patient and require medical or surgical intervention to prevent one of the outcomes described in this definition. Examples of such medical events include anaphylaxis and allergic bronchospasm that require intensive treatment in the emergency room or at home, blood disorders or convulsions that do not require hospitalization.

[0367] Related For all AEs, sufficient information must be obtained to determine the causality of the AE (e.g., to the study drug or to another illness). The relationship of the AE to the study treatment will be assessed according to the following definitions: • Unrelated: Any event that does not follow a reasonable temporal sequence from administration of the investigational drug and is likely to have been caused by the patient's clinical condition or other treatments administered to the patient. • Low relatedness: Any event that does not follow a reasonable temporal sequence from administration of the investigational drug or that is more likely to have been caused by the patient's clinical condition or other treatments administered to the patient. • Potentially related: any reaction that follows a reasonable temporal sequence from administration of the investigational drug or that follows a known pattern of response to the drug in question, and any reaction that cannot be reasonably explained by known features of the patient's clinical condition or other treatments administered to the patient. • Related: any reaction that follows a reasonable temporal sequence from administration of the investigational drug and follows a known pattern of response to the suspected drug, recurs with rechallenge, and / or improves with cessation or dose reduction of the drug.

[0368] Adverse Event Management AEs will not be recorded prior to the first dose of study drug. AEs that begin after administration of study drug or worsening symptoms related to medical history will be recorded. AEs should be followed until they resolve, return to baseline, or are determined to be stable or chronic. All SAEs will be collected up to 30 days after the last dose of study drug. All study procedure-related SAEs must be collected from the date of the patient's written consent.

[0369] Immune-Mediated Adverse Reactions Immune-mediated adverse reactions (IMARs) have been identified to anti-PD1 antibodies.

[0370] Specific IMARs of interest are: ●Immune-mediated hepatitis ●Immune-mediated nephritis ●Immune-mediated pneumonia ●Immune-mediated pneumonia Immune-mediated colitis and diarrhea Immune-mediated endocrine disorders ●Immune-mediated skin reactions • Other immune-mediated adverse reactions: arthritis, encephalitis, rhabdomyolysis, myositis, myocarditis, pancreatitis, and uveitis.

[0371] The monitoring plan is intended to limit the severity and duration of IMARs occurring during combination development and includes scheduled visits for physical examination, vital signs, safety laboratory assessments including hematology, biochemistry, endocrine function assessments every day (pre-dose) 1 of a new dosing cycle, coagulation status assessments, and urinalysis. The evaluation schedule (Tables 5-6) also includes evaluation of ejection fraction every 3 months and regular ECGs.

[0372] An overview of the management of IMAR caused by G9.2-17(IgG4), alone or in combination with other therapeutic agents, is provided below in Tables 8-9. [Table 13-1] [Table 13-2] [Table 13-3] [Table 13-4] [Table 13-5]

[0373] [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4] [Table 14-5] [Table 14-6] [Table 14-7]

[0374] Dose reduction procedures for adverse event management In the event that dose reductions are used for AE management in Part 2 of the study, two dose reductions of 50% each will be allowed. Dose reductions may continue to be followed and guided if clinical benefit is anticipated.

[0375] Evaluation of Laboratory and Other Abnormalities as AEs and SAEs Abnormal laboratory findings (e.g., clinical chemistry, hematology, and urinalysis) or other abnormal evaluations (e.g., ECG or vital signs) that are judged to be clinically significant will be recorded as AEs and SAEs if they meet the definition of an AE or SAE. Clinically significant abnormal laboratory findings or other abnormal evaluations that are detected during the study or that are present at screening and that have deteriorated significantly after the start of the study will be reported as AEs or SAEs. However, clinically significant abnormal laboratory findings or other abnormal evaluations that are related to the disease being studied or that are present or detected at the start of the study and do not deteriorate will not be reported as AEs or SAEs unless the patient's condition is judged to be more severe than expected.

[0376] Laboratory values ​​that deviate clinically significant from prior measurements may be repeated. If warranted, additional or more frequent testing than specified in the protocol should be performed to provide adequate documentation of and resolution of the AE.

[0377] Duration and frequency of collecting AE and SAE information All AEs and SAEs will be collected at the time points specified in the SoA, from the start of the intervention until the follow-up visit (Tables 5–6).

[0378] Medical events that began before the start of the study intervention and after informed consent was obtained will be recorded as a medical history / current condition and not as an AE.

[0379] All SAEs will be recorded and reported immediately and, under no circumstances, should exceed 24 hours.

[0380] Follow-up of AEs and SAEs After the initial AE / SAE report, each participant should be actively followed up at subsequent visits / contacts. All SAEs will be followed until recovery, stabilization, the event is otherwise described, or the participant is lost to follow-up.

[0381] Statistical considerations The study will be completed when the last patient completes their last visit. The database will be locked for the primary analysis after the last patient experiences a primary endpoint event. Final trial analysis will be performed after study completion.

[0382] Statistical hypotheses The current study will identify the MTD of G9.2-17 (IgG4) (Part 1) by assessing DLTs, followed by evaluating drug activity (alone or in combination) in the three disease types using a Simon two-stage optimal design. The trial hypotheses for Part 2 are detailed below.

[0383] CRC and CCA G9.2-17 (IgG4) single agent treatment group Null hypothesis: ORR3 is ≦5% ●Alternative hypothesis: ORR3 is ≥ 15%

[0384] CRC and CCA G9.2-17 (IgG4) + anti-PD1 antibody combination treatment group Null hypothesis: ORR3 is ≦10% ●Alternative hypothesis: ORR3 is ≥ 25%

[0385] Analysis Set Unless otherwise specified, the intent-to-treat (ITT) population is defined as patients who received at least one dose of study drug. The primary efficacy analysis will be performed on the ITT. Patients will be treated in the ITT.

[0386] The efficacy population is defined as all patients with at least one measurable ORR3 or PFS6 assessment within the ITT population. This population will be used for sensitivity analyses.

[0387] The per-protocol (PP) population is defined as patients who received at least one cycle of G9.2-17 (IGG4) and had no major protocol deviations.

[0388] The safety population (SAF) is defined as all patients who receive at least one dose of study drug. Safety analyses will be performed on the SAF.

[0389] The PK / PD population will be defined as patients receiving at least one cycle of G9.2-17(IGG4).

[0390] Primary endpoint(s) Safety Analysis - Part 1 and Part 2 Unless otherwise specified, all safety analyses will be performed on the SAF.

[0391] Adverse events An investigational emergent adverse event (TEAE) is defined as an event occurring at or after the first dose of an investigational drug. The MedDRA coding dictionary is used to code the AEs. Treatment-related TEAEs, severe or CTCAE grade 3 or grade 4 TEAEs, and TEAEs are summarized by treatment group, overall, by system organ class, and preferred term. These summarize the number of events and the number and percentage of patients with a given event. In addition, the number and percentage of patients with TEAEs are provided by maximum severity. An overview of all TEAEs by system organ class occurring in ≥ 5% of patients in any treatment group and preferred terms is provided.

[0392] The DLT, MTD, and RP2D are summarized.

[0393] Experimental evaluation All clinical laboratory-based data will be presented as a list of all values ​​and any abnormal results judged to be clinically significant (reported as AEs). A numerical summary of all observed findings and changes from baseline screening laboratory assessments, including chemistry, hematology, and urinalysis results, will be provided by visit and treatment group. No inferential comparisons are planned.

[0394] Vital signs Numerical summaries of all observed findings and changes from baseline screening vital signs, including blood pressure, heart rate, respiratory rate, and temperature, will be provided by time point and treatment group. No inferential analyses are planned for vital signs.

[0395] ECG, ECHO / MUGA, and physical examination Physical examination data and changes will be presented as lists. ECG results will be presented as lists and summarized by treatment group and visit based on the incidence of clinically significant abnormalities. No inferential comparisons between treatment groups are planned.

[0396] Primary Efficacy Analysis – Part 2 Disease response will be assessed according to RECIST v1.1 and will be summarized narratively for the ITT, PP, and efficacy populations.

[0397] The primary efficacy endpoints were: ORR3 in CRC and CCA PFS6 for PDAC

[0398] Secondary endpoint(s) Pharmacokinetics, Pharmacodynamics, and Immunogenicity PK, PD, and immunogenicity are summarized narratively for the PK / PD populations in both Part 1 and Part 2.

[0399] Secondary Efficacy Analysis – Part 2 Disease responses (ORR, PFS, DCR, DoR, and OS) will be assessed according to RECIST v1.1 and will be summarized narratively for the ITT, PP, and efficacy populations.

[0400] Exploratory Endpoints Analyses of exploratory endpoints will be detailed in the SAP.

[0401] Other analyses Other collected data not specifically mentioned will be presented in the patient listing.

[0402] Disposition, demographics, baseline characteristics, and medical history Processing information is compiled including the number of patients enrolled, the number who failed screening, the number who were treated, and the number who withdrew for any reason.

[0403] Demographics, baseline characteristics, and medical history will be summarized by treatment group and overall using ITT and PP descriptive statistics.

[0404] Prior and concomitant medications The numbers and percentages of patients taking prior and concomitant medications will be summarized by treatment group and overall for the ITT and PP.

[0405] Example 2: Stability Study of Anti-Galectin-9 Antibodies The candidate IgG4 antibodies underwent stability analysis after storage under several different conditions and at different concentrations. Stability analysis was performed by size exclusion chromatography (SEC) using a TOSOH TSKgel Super SW mAb column. The SEC profiles before and after storage were compared to identify any issues with protein stability (e.g., aggregation or degradation).

[0406] Materials and Methods Sample preparation Anti-galectin-9 antibody was stored at -80°C until use. Prior to analysis, samples were thawed in a room temperature water bath and stored on ice until analysis. Absorbance at 280 nm was measured using a Nanodrop before handling. The instrument was blanked using TBS (20 mM Tris (pH 8.0), 150 mM NaCl). Samples were then transferred to polypropylene microcentrifuge tubes (USA Scientific, 1615-5500) and centrifuged at 16.1 k×g for 30 min at 4°C. Samples were filtered through a 0.22 μm filter (Millipore; SLGV004SL). Absorbance after filtration was measured.

[0407] HPLC analysis Sample conditions tested included: ambient stability (0 hours at room temperature, 8 hours at room temperature), refrigerated stability (0 hours at 4°C, 8 hours at 4°C, 24 hours at 4°C), and freeze / thaw stability (1x freeze) / thaw, 3x freeze / thaw, 5x freeze / thaw). Each condition was run in duplicate at three different concentrations: stock, 10x dilution, and 100x dilution. 100 μL samples were prepared for each condition and stored in polypropylene microcentrifuge tubes. Dilutions were prepared in TBS as needed. Absorbance was read at 280 nm prior to analysis. Room temperature samples were stored on the benchtop for the specified period. 4°C samples were stored on ice or in a 4°C refrigerator for the period indicated in Table 10. Freeze-thaw samples were flash frozen in liquid nitrogen and then thawed in a room temperature water bath. The freezing and thawing process was performed once, three times, or five times, and samples were stored at 4°C until analysis.

[0408] SEC analysis was performed using a TOSOH TSKgel SuperSW mAbHR column on a Shimadzu HPLC with a UV detector at 280 nm. 25 μL of sample was loaded onto the column and run at 0.5 mL / min for 40 min. KBI buffer formulation was used as the mobile phase.

[0409] result Antibody concentrations were determined using UV absorbance measurements before and after filtration, as shown in Table 10. Two 2 mL samples provided by KBI were thawed, one vial used at room temperature and freeze / thaw conditions, and the other at 4° C. Absorbance readings showed nearly complete recovery after filtration. [Table 15]

[0410] Two or three higher molecular weight peaks were observed eluting earlier than the major peak (Figure 2). These peaks constituted approximately 5% of the total sample under each condition assayed (Table 11). No significant differences in protein concentration were observed under all assay conditions. [Table 16]

[0411] In summary, the anti-galectin-9 antibody showed consistent stability after storage under all conditions analyzed, as indicated by no significant changes in the SEC profile. There was no significant loss of protein after filtration, and two to three high molecular weight peaks were identified, accounting for approximately 5% of the total sample. The results suggest that the antibody is stable under all conditions tested, with no aggregate formation or disassembly observed.

[0412] Example 3. Evaluation of Galectin-9 Expression in Tumor Biopsy-Derived Organoid Fractions Tumor organoids can be applied to predict patient outcomes, since using tumor models with similar characteristics to the original tumor may more accurately predict patient drug responses (see, for example, Trends in Biotechnology; 36(4):358-371, April 01, 2018).

[0413] Galectin-9 levels in tumors can serve as a predictor of drug response. Biopsy-derived organoids can be used as a surrogate to assess galectin-9 levels in the original tumor. Therefore, we tested the ability to assess galectin-9 levels in single cells or organoid fractions.

[0414] Biopsies were taken from representative pancreatic adenocarcinomas and colorectal cancers and processed as follows: Surgically resected human tumor specimens were received fresh in DMEM medium on ice and minced into 10 cm dishes. Minced tumors were resuspended in DMEM + 10% FBS containing 100 U / mL collagenase type IV to obtain spheroids. Partially digested samples were pelleted, resuspended in fresh DMEM + 10% FBS, and filtered through both 100 mm and 40 mm filters to generate S1 (>100 mm), S2 (40-100 mm), and S3 (<40 mm) spheroid fractions, which were maintained on ultra-low adherent tissue culture plates.

[0415] The S2 fraction was digested with trypsin for 15 min to generate single cells. In preparation for flow cytometry, cell pellets from S2 and S3 fractions were resuspended and, after Fc receptor block (#422301; BioLegend, San Diego, CA), cell labeling was performed by incubating cells with fluorescently conjugated mAbs against human CD45 (HI30), CD3 (UCHT1), CD11b (M1 / 70), Epcam (9C4) and Gal9 (9M1-3; all Biolegend), or Gal9Fab or Fab isotypes of G9.2-17. Dead cells were excluded from the analysis using Zombie Yellow (BioLegend). Flow cytometry was performed on an Attune NxT flow cytometer (Thermo Scientific). Data were analyzed using FlowJov.10.1 (Treestar, Ashland, OR).

[0416] The results are shown in Figures 3A-3F, 4A-4F, and 5A-5F and show that the levels of galectin-9 detected with Gal9 G9.2-17 Fab in S2 single cell and S3 organoid fractions correlated, thus both S2 single cell and S3 organoids can be used to assess galectin-9 levels in tumor biopsy-derived organoids.

[0417] Example 4. Preparation of patient-derived organotypic tumor spheroids (PDOTs) for cell analysis Biopsy-derived organoids can be a useful measure to evaluate the ability of therapeutic agents to stimulate immune responses. Therefore, the S2 fraction described in the previous Example 3 above, which was used for ex vivo culture, was treated with anti-galectin-9 antibody G9.2-17 and prepared for immune profiling.

[0418] An aliquot of the S2 fraction was pelleted and resuspended in type I rat tail collagen (Corning) at a concentration of 2.5 mg / mL after adding 10x PBS containing phenol red, pH adjusted using NaOH. PANPEHA Whatman paper (Sigma-Aldrich) was used to confirm pH 7.0-7.5. The spheroid-collagen mixture was then injected into the central gel region of a 3-D microfluidic culture device described in: Jenkins et al., Cancer Discov. 2018 Feb;8(2):196-215; Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids, the contents of which are incorporated herein by reference in their entirety. Collagen hydrogels containing patient-derived organotypic tumor spheroids (PDOTS) were hydrated with medium with or without anti-galectin-9 monoclonal antibody G9.2-17 after 30 min at 37 °C. The PDOTS were then incubated at 37 °C for 3 days.

[0419] The cell pellet was resuspended in FACS buffer, initially at 1 × 10 6Cells were stained with Zombie Yellow (BioLegend) to exclude dead cells. After viability staining, cells were incubated with anti-CD16 / CD32 mAb (eBiosciences, San Diego, CA) to block FcγRIII / II, and then antibody stained with 1 μg of fluorescently conjugated extracellular mAb. Intracellular staining for cytokines and transcription factors was performed using the Fixation / Permeabilization Solution Kit (eBiosciences). Useful human flow cytometry antibodies include CD45 (HI30), CD3 (UCHT1), CD4 (A161A1), CD8 (HIT8a), CD44 (BJ18), TNFα (MAb11), IFNγ (4S.B3), and Epcam (9C4) (all Biolegend). Flow cytometry was performed on an LSR-II flow cytometer (BD Biosciences). Data were analyzed using FlowJov.10.1 (Treestar, Ashland, OR).

[0420] Example 5. Evaluation of Galectin-9 Levels in Plasma and Serum of Cancer Patients Plasma and serum Galectin-9 levels were assessed in patient samples and compared to healthy volunteers. Blood (10 ml) was collected from peripheral venous access of 10 healthy controls and 10 inoperable cancer patients. Serum and plasma were extracted from each blood sample. Blood was collected in standard EDTA tubes and analyzed using PicoKine™ ELISA; Catalog Number: EK1113 essentially according to the manufacturer's instructions. The results of the individual values ​​are tabulated in Tables 12 and 13. [Table 17] [Table 18]

[0421] Example 6. Evaluation of Galectin-9 Expression and Localization Using Immunohistochemistry Tumor samples from paraffin-embedded biopsies were used to evaluate the ability to determine galectin-9 expression levels in tumors using immunohistochemical analysis.

[0422] Briefly, slides were deparaffinized (xylene: 2 × 3 min, absolute alcohol: 2 × 3 min, methanol: 1 × 3 min) and rinsed with cold tap water. For antigen retrieval, citrate buffer (pH 6) was preheated to 100 °C in a water bath and slides were incubated in citrate buffer for 5 min. Slides were allowed to cool at room temperature for approximately 10 min and placed in running water. Slides were washed with PBS, a circle was drawn around the sections with a pomp pen, and sections were incubated in blocking buffer (DAKO-Peroxidase Blocking Solution-S2023) for 5 min. Serum-free blocker (Novocastra serum-free protein blocker) was added and then rinsed with PBS. Primary antibody (Sigma, anti-galectin-9 clone 1G3) was used at a dilution of 1:2000 in DAKO-S2022 diluent and sections were incubated overnight at 4 °C. Slides were wa...

Claims

1. A pharmaceutical composition for use in treating a solid tumor in a subject, the pharmaceutical composition comprising an antibody that binds to human galectin-9 (anti-Gal9 antibody), the anti-galectin-9 antibody being: (a) a light chain comprising a light chain (LC) complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:1, a LC complementarity determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO:2, and a light chain variable region (VL) comprising a LC complementarity determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO:3, and (b) a heavy chain comprising a heavy chain (HC) complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 4, a HC complementarity determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 5, and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 6; The pharmaceutical composition, wherein the anti-galectin-9 antibody is administered to the subject once a week at a dose of about 0.2 mg / kg to about 32 mg / kg.

2. The pharmaceutical composition for use according to claim 1, wherein the anti-galectin-9 antibody is administered to the subject once a week at a dose of about 10 mg / kg to about 16 mg / kg.

3. The pharmaceutical composition for use according to claim 2, wherein the anti-galectin-9 antibody is administered to the subject once a week at a dose of 10 mg / kg or 16 mg / kg.

4. 2. The pharmaceutical composition for use according to claim 1, wherein the anti-galectin-9 antibody is administered to the subject at a dose of about 650 mg to about 1120 mg once a week, at a dose of about 650 mg to about 700 mg once a week, or at a dose of about 1040 mg to about 1120 mg once a week.

5. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the solid tumor is a metastatic solid tumor.

6. 5. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the solid tumor is pancreatic ductal adenocarcinoma (PDAC), colorectal carcinoma (CRC), hepatocellular carcinoma (HCC), cholangiocarcinoma (CAA), renal cell carcinoma (RCC), urothelial carcinoma, head and neck cancer, breast cancer, lung cancer, or a gastrointestinal (GI) solid tumor.

7. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein said anti-galectin-9 antibody is administered to said subject by intravenous infusion.

8. The V of the anti-galectin-9 antibody L The pharmaceutical composition for use according to any one of claims 1 to 4, wherein said V H of said anti-galectin-9 antibody comprises the amino acid sequence of SEQ ID NO:8 and said V H of said anti-galectin-9 antibody comprises the amino acid sequence of SEQ ID NO:

7.

9. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein said anti-galectin-9 antibody is a human IgG4 molecule having an altered Fc region compared to the wild-type human IgG4 counterpart.

10. The pharmaceutical composition for use according to claim 9, wherein said anti-galectin-9 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:19 and a light chain comprising the amino acid sequence of SEQ ID NO:

15.

11. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the subject is a human patient not receiving other anti-cancer therapies simultaneously with the treatment comprising the anti-galectin-9 antibody.

12. A pharmaceutical composition for use according to any one of claims 1 to 4, wherein the pharmaceutical composition is for use in combination with an immune checkpoint inhibitor.

13. the immune checkpoint inhibitor is an antibody that binds to PD-1; 13. The pharmaceutical composition for use according to claim 12, wherein the antibody that binds to PD-1 is selected from the group consisting of pembrolizumab, nivolumab, tislelizumab, dostallimab, and cemiplimab.

14. 14. The pharmaceutical composition for use according to claim 13, wherein the subject is a human patient who has not been exposed to any anti-PD-1 or anti-PD-L1 agent in any prior line of therapy and who does not have microsatellite instability (MSI-H) and / or defective mismatch repair (dMMR), or a combination thereof.

15. the subject is a human patient who has undergone one or more prior anti-cancer therapies; The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the one or more prior anti-cancer therapies comprise chemotherapy, immunotherapy, radiation therapy, therapy involving a biological agent, or a combination thereof.

16. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the subject is a human patient having elevated serum or plasma levels of galectin-9 compared to control values.

17. The subject is monitored for the occurrence of side effects. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the dose of said anti-galectin-9 antibody is reduced if side effects are observed.

18. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the subject receives multiple doses of the anti-galectin-9 antibody, with later doses being higher than earlier doses.

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