CN117500522A

CN117500522A - Anti-galectin-9 antibodies and therapeutic uses thereof

Info

Publication number: CN117500522A
Application number: CN202280041556.8A
Authority: CN
Inventors: A·菲利波维奇; E·艾伦科; H·帕登; C·科思
Original assignee: Pure Technology Lyt Co ltd
Current assignee: Pure Technology Lyt Co ltd
Priority date: 2021-04-30
Filing date: 2022-04-29
Publication date: 2024-02-02

Abstract

Disclosed herein are methods of treating 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 gastrointestinal solid tumors) using an anti-galectin-9 antibody, e.g., as monotherapy or as a combination therapy with an immune checkpoint inhibitor.

Description

Anti-galectin-9 antibodies and therapeutic uses thereof

Cross Reference to Related Applications

The present application claims the benefit of U.S. c. ≡119 (e) from U.S. provisional application nos. 63/182,521, 2021, 5, 26, and 63/193,357, 2022, 25, each of which is incorporated herein by reference in its entirety.

Sequence listing

The present application contains a sequence listing that has been electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created at 26, 4, 2022, named 112174-0211-NP009WO1_SEQ. Txt and was 89,119 bytes in size.

Background

The immune system has great potential to recognize and destroy cancer cells, but a complex network that controls tumor immune escape is a widely effective obstacle to immunomodulation (Martinez-Bosch N et al Immune Evasion in Pancreatic Cancer: from Mechanisms to therapeutics (Basel) 2018;10 (1)). Approved Immunooncology (IO) agents can gradually improve survival of many tumor types (e.g., melanoma, lung cancer, kidney cancer, bladder cancer, some colon cancers, etc.), and are rapidly integrated as a standard of care in addition to and in conjunction with surgery, chemotherapy, and radiation therapy. However, there are still significant gaps in the treatment and survival of a variety of other invasive malignancies. For example, the 5 year survival rates for metastatic pancreatic ductal adenocarcinoma (PDAC or PDA), cholangiocarcinoma (CCA), and colorectal cancer (CRC) remain <9%, <5%, and <15%, respectively. These gastrointestinal tumors are very invasive, many patients are at a late stage of disease at the time of their visit, and the effectiveness of approved immunotherapy is not ideal (Rizvi et al, cholangiococcinoma-evolving concepts and therapeutic strategies; nat Rev Clin Oncol.2018;15 (2): 95-111; kalman et al, updates on immunotherapy for colorectal cancer; J Gastrointest Oncol.2018;9 (1): 160-169).

The success of the first generation checkpoint inhibitors (anti-PD-1, anti-PD-L1 and anti-CTLA 4) resulted in new IO clinical trial efficacy and differential outbreaks (hol et al, examining Peripheral and Tumor Cellular Immunome in Patients with Cancer; front immunol.2019; 10:1767). However, while successful, there are a number of unfortunate cases of development failure, and thus, there remains a need for more novel and effective treatments.

Galectin-9 is a tandem repeat lectin consisting of two Carbohydrate Recognition Domains (CRDs) and was first discovered and described in 1997 in patients with Hodgkin Lymphoma (HL) (Tureci et al, J.biol. Chem.1997,272, 6416-6422). Three isoforms exist and may be located either intracellular or extracellular. Elevated levels of galectin-9 have been observed in a variety of cancers, including melanoma, hodgkin lymphoma, hepatocellular carcinoma, pancreatic cancer, gastric cancer, colon cancer, and clear cell renal cell carcinoma (Wdowiak et al, int.j.mol. Sci.2018,19,210). In renal cancer, patients with high galectin-9 expression showed more advanced disease progression with larger tumor sizes (Kawashima et al; BJU int.2014; 113:320-332). In Melanoma, galectin-9 was expressed in 57% of the tumors and significantly increased in the plasma of patients with advanced Melanoma compared to healthy controls (Enning ga et al, melanoma Res.2016, 10 months; 26 (5): 429-441). Many studies have shown the utility of galectin-9 as a prognostic marker and have recently also shown its utility as a potential new drug target (Enning et al 2016; kawashima et al BJU Int 2014;113:320-332; kageshita et al, int J cancer.2002, 20 th day; 99 (6): 809-16, and references therein).

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 that galectin-9 plays a role in supporting immunomodulation of tumors, e.g., by down-regulating Th 1-type responses, th2 polarization, and polarization of macrophages to the M2 phenotype. This work also included studies demonstrating that galectin-9 was involved in direct inactivation of T cells through interaction with T cell immunoglobulin and mucin 3 (TIM-3) receptors (Dardalkon et al, J Immunol.,2010,185,1383-1392; sanchez-Fuyo et al, nat Immunol.,2003,4,1093-1101).

Galectin-9 has also been found to play a role in polarizing T cell differentiation towards tumor suppression phenotypes and promoting tolerogenic macrophage programming and adaptive immunosuppression (Daley et al, nat med.,2017,23,556-567). In a mouse model of Pancreatic Ductal Adenocarcinoma (PDAC), blocking checkpoint interactions between galectin-9 found on innate immune cells in the Tumor Microenvironment (TME) has been shown to increase anti-tumor immune responses in TME and slow tumor progression (Daley et al, nat med.,2017,23,556-567). Galectin-9 was also found to bind to CD206 (a surface marker of M2 type macrophages) resulting in decreased secretion of CVL22 (MDC), a macrophage derived chemokine associated with longer survival and lower risk of recurrence of lung cancer (Enning ga et al J Pathol.2018, month 8; 245 (4): 468-477).

Disclosure of Invention

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 carcinoma, head and neck cancer, breast cancer, lung cancer, or other GI solid tumors involving antibodies capable of binding to human galectin-9, 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 (IgG 4) has a faster clearance rate in human subjects compared to other antibody therapeutics. Thus, a treatment regimen comprising a weekly dosing schedule was developed to ensure a suitable plasma concentration of anti-galectin-9 antibodies, such as therapeutic systemic exposure levels, for achieving a therapeutic effect.

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

In some embodiments, an anti-galectin-9 antibody (e.g., G9.2-17 (IgG 4)) may be administered to a subject at a dose of 0.2mg/kg, 0.63mg/kg, 2mg/kg, 6.3mg/kg, 10mg/kg, or 16mg/kg once every two weeks to once every four weeks. In some examples, the anti-galectin-9 antibody may be administered to the subject once every two weeks. In particular embodiments, an anti-galectin-9 antibody (e.g., G9.2-17 (IgG 4)) is administered to a subject at a dose of 10mg/kg or 16mg/kg once every two weeks to once every four weeks (e.g., once every two weeks).

Alternatively, the anti-Gal-9 antibody, such as G9.2-17 (IgG 4), may be administered to the subject at a dose of about 650mg to about 1120mg once every 2-6 weeks (e.g., once every 2 weeks, once every 3 weeks, or once every 4 weeks). In some examples, the anti-Gal-9 antibody is administered to the subject at a dose of about 650mg to about 700mg once every 2-6 weeks (e.g., once every 2 weeks, once every 3 weeks, or once every 4 weeks). In other examples, the anti-Gal-9 antibody is administered to the subject at a dose of about 1040mg to about 1120mg every 2-6 weeks (e.g., every 2 weeks, every 3 weeks, or every 4 weeks).

In some embodiments, the anti-galectin-9 antibody (e.g., G9.2-17 (IgG 4)) is administered to the subject at a dose of 0.2mg/kg, 0.63mg/kg, 2mg/kg, 6.3mg/kg, 10mg/kg, or 16mg/kg once a week. In particular embodiments, an anti-galectin-9 antibody (e.g., G9.2-17 (IgG 4)) is administered to a subject at a dose of 10mg/kg or 16mg/kg once a week. Alternatively, an anti-Gal-9 antibody disclosed herein, such as G9.2-17 (IgG 4), can be administered to a subject at a dose of about 650mg to about 1120mg once a week. For example, the anti-Gal-9 antibody may be administered to a subject at a dose of 10mg/kg once a week or at a plateau dose of about 650-700mg once a week. Alternatively, the anti-galectin-9 antibody may be administered to the subject at a dose of 16mg/kg once a week or at a plateau dose of about 1040-1120mg once a week.

In some embodiments, an anti-galectin-9 antibody may comprise:

(a) Comprising a light chain variable region (V _L ) A Light Chain (LC) complementarity determining region 1 (CDR 1) comprising the amino acid sequence of SEQ ID NO. 1, a LC complementarity determining region 2 (CDR 2) comprising the amino acid sequence of SEQ ID NO. 2 and a LC complementarity determining region 3 (CDR 3) comprising the amino acid sequence of SEQ ID NO. 3, and

(b) Comprising a heavy chain variable region (V _H ) Comprising a Heavy Chain (HC) complementarity determining region 1 (CDR 1) comprising the amino acid sequence of SEQ ID NO. 4, a HC complementarity determining region 2 (CDR 2) comprising the amino acid sequence of SEQ ID NO. 5 and a HC complementarity determining region 3 (CDR 3) comprising the amino acid sequence of SEQ ID NO. 6.

In some examples, V of the anti-galectin-9 antibody _L Comprising the amino acid sequence of SEQ ID NO. 8. Alternatively or additionally, V of an anti-galectin-9 antibody _H Comprising the amino acid sequence of SEQ ID NO. 7.

In some cases, the anti-galectin-9 antibody is a full length antibody, such as an IgG1 or IgG4 molecule. In some examples, the anti-galectin-9 antibody is a human IgG4 molecule. Such IgG4 molecules may have a modified Fc region relative to wild-type human IgG4 counterparts. In some examples, the modified Fc region comprises the amino acid sequence of SEQ ID NO. 14. In a specific example, an 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 anti-galectin-9 antibodies may be G9.2-17 (IgG 4) as disclosed herein.

In some embodiments, the solid tumor to be treated by any of the methods disclosed herein may be 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 tumor. In some cases, the solid tumor is a metastatic tumor. In some embodiments, the methods comprise administering to a subject having a solid tumor (e.g., PDAC, CRC, HCC or CCA) an effective amount of an antibody that binds human galectin-9 (referred to herein as an anti-Gal 9 antibody or an anti-galectin-9 antibody). In some cases, the subject has one or more of the following features: (i) no resectable cancer; (ii) not infected with SARS-CoV-2; (iii) no active brain or leptomeningeal metastasis; and (iv) has unresectable metastatic cancer, which is adenocarcinoma, optionally squamous cell carcinoma.

In some embodiments, the subject does not receive additional anti-cancer therapies concurrent with the anti-galectin-9 antibody. Alternatively, the method may further comprise administering an immune checkpoint inhibitor to the subject. In some examples, the immune checkpoint inhibitor is an antibody that binds PD-1. Examples include pamglizumab (pembrolizumab), nivolumab (nivolumab), tirelizumab (tislealizumab), doralimab (dostarlimab) or cimetiab Li Shan (cemiplimab). In some cases, the subject is not exposed to any anti-PD-1 or anti-PD-L1 agent, does not receive microsatellite instability (MSI-H) and/or defective mismatch repair (dMMR), or a combination thereof, in any previous treatment line number.

In one example, the antibody that binds PD-1 is nivolumab. In some cases, the nivolumab is administered to the subject at a dose of 240mg once every two weeks. In another example, the antibody that binds PD-1 is tirelizumab. In some cases, tirelimumab is administered intravenously at a dose of about 200mg once every 3 weeks or at a dose of about 400mg every six weeks.

In some embodiments, the anti-galectin-9 antibody is administered to the subject at a dose of about 0.2mg/kg to about 32mg/kg (e.g., about 3mg/kg to about 15mg/kg, or about 2mg/kg to about 16mg/kg, or higher dose levels, or about 0.2mg/kg to about 15mg/kg, or about 0.2mg/kg to about 16mg/kg, or higher dose levels) once every 2-3 weeks. In some embodiments, the anti-galectin-9 antibody is administered to the subject at a dose selected from the group consisting of 0.2mg/kg, 0.63mg/kg, 2mg/kg, 4mg/kg, 6mg/kg, 6.3mg/kg, 8mg/kg, 10mg/kg, 12mg/kg, or 16mg/kg or higher dose levels. In some embodiments, the anti-galectin-9 antibody is administered to the subject at a dose selected from the group consisting of 2mg/kg, 4mg/kg, 8mg/kg, 12mg/kg, or 16mg/kg or higher. In some embodiments, the anti-galectin-9 antibody is administered to the subject at a dose selected from the group consisting of 0.2mg/kg, 0.63mg/kg, 2mg/kg, 4mg/kg, 6mg/kg, 6.3mg/kg, 10mg/kg, or 16mg/kg or higher dose level. In some embodiments, the antibody is administered once every 2 weeks. In some embodiments, the anti-galectin-9 antibody is administered to the subject at a dose selected from the group consisting of 2mg/kg, 4mg/kg, 8mg/kg, 12mg/kg, or 16mg/kg or higher at a dose level once every 2 weeks. In some embodiments, the anti-galectin-9 antibody is administered to the subject at a dose selected from the group consisting of 0.2mg/kg, 0.63mg/kg, 2mg/kg, 4mg/kg, 6mg/kg, 6.3mg/kg, 10mg/kg, or 16mg/kg or higher at a dose level once every 2 weeks. In some embodiments, the anti-galectin-9 antibody is administered once every 2 weeks for one period, once every 2 weeks for two periods, once every 2 weeks for 3 periods, once every 2 weeks for 4 periods, or once every 2 weeks for more than 4 periods. In some embodiments, the duration of treatment is 0-3 months, 3-6 months, 12-24 months, or longer. In some embodiments, the duration of treatment is 12-24 months or longer. In some embodiments, the period extends for a duration of 3 months to 6 months or 6 months to 12 months or 12 months to 24 months or more. In some embodiments, the cycle length is modified, e.g., temporarily or permanently, to a longer duration, e.g., 3 weeks 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 any of the cancers described above. In some embodiments, the method of treatment comprising administration of an anti-galectin-9 antibody does not include any other concurrent anti-cancer therapy.

In some embodiments, the method of treatment using an anti-galectin-9 antibody includes another concurrent anticancer therapy. Thus, in some embodiments, the method of treatment using an anti-galectin-9 antibody further comprises administering to the subject an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an antibody that binds PD-1, such as palbociclib, nivolumab, tirelib, rituximab, or cimetidine Li Shan antibody. In some embodiments, the antibody that binds PD-1 is nivolumab, which is administered to the subject at a dose of 240mg once every two weeks. In some embodiments, the antibody that binds PD-1 is nivolumab, which is administered to the subject at a dose of about 240mg every two weeks or at a dose of about 480mg every 4 weeks. In some embodiments, the antibody that binds PD-1 is palbociclib, which is administered at a dose of 200mg once every 3 weeks. In some embodiments, the antibody that binds PD-1 is a cimrpu Li Shan antibody, which is administered at a dose of about 350mg once every 3 weeks. In some embodiments, the antibody that binds PD-1 is tirelimumab, which is administered at a dose of about 200mg once every 3 weeks or at a dose of about 400mg once every six weeks. In some embodiments, the antibody that binds PD-1 is rituximab, which is administered at a dose of about 500mg once every 3 weeks or at a dose of about 1000mg once every six weeks. In some embodiments, the immune checkpoint inhibitor is administered by intravenous infusion.

In some cases, subject (v) is not exposed to any anti-PD-1 or anti-PD-L1 agent, is not receiving microsatellite instability (MSI-H) and/or defective mismatch repair (dMMR), or a combination thereof, in any previous treatment line number. In some cases, the subject receives microsatellite instability (MSI-H) and/or defective mismatch repair (dMMR) or a combination thereof.

In some examples, the checkpoint inhibitor is administered to the subject on the day the subject receives an anti-galectin-9 antibody. Alternatively, the checkpoint inhibitor and the anti-galectin-9 antibody are administered to the subject for two consecutive days. For example, administration of the checkpoint inhibitor is performed prior to administration of the anti-galectin-9 antibody, and vice versa.

In some embodiments, the subject has undergone one or more prior anti-cancer therapies. In some examples, the one or more previous anti-cancer therapies include chemotherapy, immunotherapy, radiation therapy, therapies involving biological agents, or a combination thereof. In some cases, the subject has progressed on or is resistant to one or more previous therapies by one or more previous anti-cancer therapies.

In some cases, the subject is a human patient having elevated galectin-9 levels relative to a control value. For example, human patients have elevated serum or plasma levels of galectin-9 relative to control values. In some examples, the human patient has cancer cells that express galectin-9. Alternatively, or in addition, the human patient has immune cells expressing galectin-9. In some examples, the cancer cells are in a tumor organoid derived from a human patient. In some embodiments, the control value is based on a value obtained from a healthy human subject.

Any of the methods disclosed herein can further comprise monitoring the subject for the occurrence of an adverse effect. In some examples, the method may further comprise reducing the dose of anti-galectin-9 antibody, the dose of checkpoint inhibitor, or both when adverse effects are observed.

In some embodiments, multiple doses of anti-galectin-9 antibody are administered to a subject, and later doses are higher than earlier doses.

The use of any of the pharmaceutical compositions and anti-galectin-9 antibodies for treating a solid tumor (e.g., those described herein and including metastatic solid tumors) in the manufacture of a medicament for treating a solid tumor, alone or in combination with a checkpoint inhibitor (such as any of the anti-PD-1 antibodies disclosed herein), is also within the scope of the present disclosure.

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will become apparent from the following drawings and detailed description of several embodiments, and the appended claims.

Drawings

The following drawings form a part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which aspects may be better understood by reference to the drawings in conjunction with the detailed description of the specific embodiments presented herein.

Fig. 1 is a schematic diagram depicting an exemplary study protocol. CRM: re-evaluation method; RP2D: recommended phase 2 dose; PK: pharmacokinetics; PD: pharmacodynamics; PDAC: pancreatic ductal adenocarcinoma; CRC: colorectal cancer; CCA: bile duct cancer; TBD: to be determined.

FIG. 2 is a graph showing a representative Size Exclusion Chromatography (SEC) profile of an anti-galectin-9 antibody. The high molecular weight peaks are labeled.

FIGS. 3A-3F include a chart showing the use of anti-galectin-9G9.2-17 Fab fragments and commercially available anti-galectin-9 antibodies (9M 1-3) as measured in S2 and S3 organoid fractions derived from pancreatic adenocarcinoma biopsies (CD 3) ⁺ ) Bar graph of the level of galectin-9 expression in macrophages (CD11b+) and tumor cells (Epcam+). S2 fraction: organoids. S3 fraction: single cells. The corresponding isoforms of the G9.2-17Fab ("Fab isoforms") and "fluorescence minus one" (FMO) 9M1-3 ("Gal 9 FMO") were used as controls for specificity, background staining, and fluorescence exudation from other channels. FIG. 3A shows CD3 as measured in S3 fraction ⁺ Levels of galectin-9 in cells. FIG. 3B shows CD11B as measured in S3 fraction ⁺ Levels of galectin-9 in cells. FIG. 3C shows Epcam as measured in S3 fraction ⁺ Levels of galectin-9 in cells. FIG. 3D shows CD3 as measured in S2 fraction ⁺ Levels of galectin-9 in cells. FIG. 3E shows CD11b as measured in S2 fraction ⁺ Levels of galectin-9 in cells. FIG. 3F shows Epcam as measured in S2 fraction ⁺ Levels of galectin-9 in cells.

FIGS. 4A-4F include illustrations showing the use of anti-galectins9G9.2-17Fab fragment and commercially available anti-galectin-9 antibody (9M 1-3) as measured in S2 and S3 organoid fractions derived from colorectal cancer biopsies (CD 3) ⁺ ) Bar graph of the level of galectin-9 expression in macrophages (CD11b+) and tumor cells (Epcam+). S2 fraction: organoids. S3 fraction: single cells. The corresponding isoforms of G9.2-17Fab ("Fab isoforms") and FMO 9M1-3 ("Gal 9 FMO") were used as controls for specificity, background staining and fluorescence exuded from other channels. FIG. 4A shows CD3 as measured in S3 fraction ⁺ Levels of galectin-9 in cells. FIG. 4B shows CD11B as measured in S3 fraction ⁺ Levels of galectin-9 in cells. FIG. 4C shows Epcam as measured in S3 fraction ⁺ Levels of galectin-9 in cells. FIG. 4D shows CD3 as measured in S2 fraction ⁺ Levels of galectin-9 in cells. FIG. 4E shows CD11b as measured in S2 fraction ⁺ Levels of galectin-9 in cells. FIG. 4F shows Epcam as measured in S2 fraction ⁺ Levels of galectin-9 in cells.

FIGS. 5A-5F include a chart showing the use of anti-galectin-9G9.2-17 Fab fragment and commercially available galectin-9 antibody (9M 1-3) as measured in S2 and S3 organoid fractions derived from a second pancreatic adenocarcinoma biopsy (CD 3) ⁺ ) Bar graph of the level of galectin-9 expression in macrophages (CD11b+) and tumor cells (Epcam+). S2 fraction: organoids. S3 fraction: single cells. The corresponding isoforms of G9.2-17Fab ("Fab isoforms") and FMO 9M1-3 ("Gal 9 FMO") were used as controls for specificity, background staining and fluorescence exuded from other channels. FIG. 5A shows CD3 as measured in S3 fraction ⁺ Levels of galectin-9 in cells. FIG. 5B shows CD11B as measured in S3 fraction ⁺ Levels of galectin-9 in cells. FIG. 5C shows Epcam as measured in S3 fraction ⁺ Levels of galectin-9 in cells. FIG. 5D shows CD3 as measured in S2 fraction ⁺ Levels of galectin-9 in cells. FIG. 5E showsCD11b as measured in S2 fraction ⁺ Levels of galectin-9 in cells. FIG. 5F shows Epcam as measured in S2 fraction ⁺ Levels of galectin-9 in cells.

FIGS. 6A-6C include photographs of immunohistochemical analysis of various tumors using anti-galectin-9 antibody 1G 3. All magnifications were 200X. Figure 6A shows chemotherapy-treated colorectal cancer with heterogeneity intensity scores of 2 and 3 (medium and high) galectin-9 expression. In particular, galectin-9 staining was observed at the cell membrane; in addition, the intra-glandular macrophages are moderately positive and the stromal response in the tumor shows multinucleated macrophage megacells with moderate intensity of galectin-9 expression. Fig. 6B shows liver metastasis of colorectal cancer with high (intensity score 3) galectin-9 expression. Staining was located on the membrane and in the cytoplasm. FIG. 6C shows a bile duct with galectin-9 positive (intensity score of 2) entrapment and a galectin-9 negative cancer.

Fig. 7 includes graphs showing fractions of annexin V and Propidium Iodide (PI) positive cells plotted as a function of the concentration of antibody used. MOLM-13 cells were incubated with different concentrations of G9.2-17 or human IgG4 isotype antibodies and recombinant human galectin-9 for 16 hours. Cells were stained with annexin V and propidium iodide and then analyzed by flow cytometry. Each condition was performed in triplicate. Analysis was performed on FlowJo software.

FIGS. 8A-8B depict graphs showing the results of studies in mice treated with G9.2-17mIgG2a alone or in combination with the alpha PD-1 mAb. Mice with in situ implanted KPC tumors (n=10/group) were treated once a week with commercial αpd-1 (200 μg) mAb or G9.2-17mIg a (200 μg) or a combination of G9.2-17 and αpd-1 or matched isotypes for three weeks. Tumors were removed and weighed (fig. 8A), subsequently treated and stained for flow cytometry (fig. 7B). Each dot represents one mouse; * p <0.05; * P <0.01; * P <0.001; * P <0.0001; by unpaired Student t test. Fig. 8B depicts a bar graph showing flow cytometry at the end of the experiment (day 18) tumor resection from control and treated animals and treatment thereof for intratumoral immune cells and associated activation and immunosuppression markers. Mice tumors were digested prior to flow. Flow cytometry was performed on an Attune NxT flow cytometer (ThermoFisher Scientific, waltham, MA). Data was analyzed using FlowJo v.10.1 (Treestar, ashland, OR).

FIGS. 9A-9B depict graphs showing the results of ADCC assays performed with the IgG1 form of G9.2-17 (FIG. 9A) and the IgG4 form of G9.2-17 (FIG. 9B). As expected for human IgG4mAb, G9.2-17 did not mediate ADCC (fig. 9B). This was tested against the IgG1 human counterparts of G9.2-17, which mediate ADCC and ADCP, as positive controls (fig. 9A).

FIGS. 10A-10B depict graphs showing the effect of 9.2-17 in the B16F10 subcutaneous homology model. Tumors were subcutaneously implanted and treated with G9.2-17 IgG1 mouse mAb, anti-PD-1 antibodies, or a combination of G9.2-17 IgG1 mouse mAb and anti-PD-1 antibodies. Fig. 10A depicts a graph showing the effect on tumor volume. Fig. 10B depicts a graph showing intratumoral CD 8T cell infiltration. The results indicate that the intratumoral presence of effector T cells is enhanced in the combination group.

FIGS. 11A-11B include graphs showing isolated tumor cultures (organoids) derived 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 three days. CD44 (fig. 11A) and tnfα (fig. 11B) expression in cd3+ T cells from PDOTS was assessed.

FIG. 12 includes graphs showing the effect of G2.9-17 on TGF- β1 secretion measurements in an exemplary healthy human donor whole blood. TGF- β1 is released from donor cryopreserved macrophages incubated in the presence of the M2 polarization mixture. IgG4 isotype is the negative control antibody. Data represent mean + SEM of triplicate measurements. Significance was determined by two-way ANOVA using the Dunnett multiple comparison test. * p <0.05

FIG. 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 was released from donor cryopreserved macrophages incubated in the presence of M2 polarization mixtures (IL-4/IL-13 or Gal-9). IgG4 isotype is the negative control antibody. Data represent mean (±sem) of triplicate. Significance was determined by two-way ANOVA using Tukey multiple comparison test. * P <0.05.

Detailed Description

Provided herein are methods of treating 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) using an anti-galectin-9 antibody (e.g., G9.2-17 (IgG 4)). In some embodiments, the cancer is metastatic. In some embodiments, the methods disclosed herein provide specific dosages and/or dosing schedules, e.g., 0.2mg/kg to 16mg/kg of antibody once a week (e.g., 10mg/kg or 16mg/kg once a week). G9.2-17 (IgG 4) was found to have unexpectedly rapid clearance in human subjects compared to conventional antibody therapeutics. Thus, a treatment regimen comprising a weekly dosing schedule was developed to ensure systemic exposure levels of anti-galectin-9 antibodies that achieved therapeutic effects. In some cases, the methods disclosed herein target a particular patient population, e.g., a patient who has undergone prior treatment and who has shown disease progression through prior treatment, or a patient who is resistant (from the head or acquired) to prior treatment.

Galectin-9 (a tandem repeat lectin) 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 tissues and other pathological conditions. In some cases, it was also found to circulate in the Tumor Microenvironment (TME).

Galectin-9 was found to interact with Dectin-1 (an innate immune receptor highly expressed on macrophages in PDAC and on cancer cells) (Daley et al, nat Med.2017;23 (5): 556-6). Regardless of the source of galectin-9, disrupting its interaction with Dectin-1 has been shown to cause the binding of CD4 ⁺ And CD8 ⁺ Cells are reprogrammed as an integral mediator of anti-tumor immunity. Thus, galectin-9 acts as a valuable treatment for blocking the signaling mediated by Dectin-1Therapeutic targets. Thus, in some embodiments, an anti-galectin-9 antibody described herein disrupts the interaction between galectin-9 and Dectin-1.

It was also found that galectin-9 can interact with TIM-3, a type I cell surface glycoprotein, expressed on the surface of leukemic stem cells of all kinds of acute myeloid leukemia, except M3 (acute promyelocytic leukemia), but not in normal human Hematopoietic Stem Cells (HSCs). TIM-3 signaling resulting from galectin-9 ligation 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 secretion of tumor necrosis factor- α (TNF- α), causing monocytes to mature into dendritic cells, resulting in inflammation caused by innate immunity (Kuchroo et al, nat Rev immunol.,2008, 8:577-580). Additional galectin-9/TIM-3 signaling has been found to co-activate NF-. Kappa.B 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 effects, particularly on leukemia and other hematological malignancies. Thus, in some embodiments, an anti-galectin-9 antibody described herein disrupts the interaction between galectin-9 and TIM-3.

Furthermore, galectin-9 was found to interact with CD206 (a mannose receptor, highly expressed on M2 polarized macrophages, thereby promoting tumor survival) (Enning ga et al, J Pathol.2018, month 8; 245 (4): 468-477). Tumor-associated macrophages expressing CD206 are mediators of tumor immunosuppression, angiogenesis, metastasis and recurrence (see, e.g., scoodeller et al, sci Rep.2017, month 11, 7; 7 (1): 14655, and references therein). In particular, M1 (also known as classical activated macrophages) is triggered by Th 1-associated cytokines and bacterial products, expresses high levels of IL-12, and has a tumoricidal effect. In contrast, M2 (a so-called surrogate activated macrophage) is activated by Th 2-associated factors, expresses high levels of anti-inflammatory cytokines such as IL-10, and promotes tumor progression (Biswas and Mantovani; nat immunol.10 month 2010; 11 (10): 889-96). The pro-neoplastic effects of M2 include promoting angiogenesis, promoting invasion and metastasis, and protecting tumor cells from chemotherapy-induced apoptosis (Hu et al, tumour biol.2015, 12; 36 (12): 9119-9126, and references therein). Tumor-associated macrophages are believed to have an M2-like phenotype and have a tumorigenic effect. Galectin-9 has been shown to mediate differentiation of bone marrow cells to the M2 phenotype (Enning et al, melanoma Res.2016, 10; 26 (5): 429-41). Galectin-9 binding to CD206 potentially results in reprogramming TAM to M2 phenotype, similar to what was shown previously for Dectin-1. Without wishing to be bound by theory, blocking the interaction of galectin-9 with CD206 may provide a mechanism by which anti-galectin-9 antibodies (e.g., G9.2-17 antibodies) may be therapeutically beneficial. Thus, in some embodiments, an anti-galectin-9 antibody described herein disrupts the interaction between galectin-9 and CD 206.

Galectin-9 has also been shown to interact with Protein Disulfide Isomerase (PDI) and 4-1BB (Bi S et al Proc Natl Acad Sci U S A.2011;108 (26): 10650-5; madireddi et al J Exp Med.2014;211 (7): 1433-48).

Anti-galectin-9 antibodies can serve as therapeutic agents for treating diseases associated with galectin-9 (e.g., those in which galectin-9 signaling plays a role). Without being bound by theory, anti-galectin-9 antibodies may block galectin-9 mediated signaling pathways. For example, an antibody may interfere with the interaction between galectin-9 and its binding partner (e.g., dectin-1, TIM-3, or CD 206), thereby blocking signaling triggered by galectin-9/ligand interactions. Alternatively, or in addition, anti-galectin-9 antibodies may also exert their therapeutic effect by inducing blocking and/or cytotoxicity (e.g., ADCC, CDC or ADCP against galectin-9 expressing pathological cells). Pathological cells refer to cells that directly or indirectly contribute to the occurrence and/or progression 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 for the subject matter and purposes cited herein.

The anti-galectin-9 antibodies disclosed herein are capable of inhibiting galectin-9 mediated signaling (e.g., a galectin-9/Dectin-1 or galectin-9/Tim-3 mediated signaling pathway) or eliminating pathological cells expressing galectin-9 via, for example, ADCC. Thus, the anti-galectin-9 antibodies described herein can be used to inhibit any galectin-9 signaling and/or eliminate galectin-9 positive pathological cells, thereby being beneficial for treating galectin-9 related diseases.

Anti-galectin-9 antibodies such as G9.2-17 (e.g., G9.2-17 (IgG 4)) were found to be effective in inducing apoptosis against galectin-9 expressing cells. Furthermore, the anti-tumor effect of anti-galectin-9 antibodies such as G9.2-17 was demonstrated in a mouse model alone or in combination with checkpoint inhibitors (e.g., anti-PD-1 antibodies). As reported herein, the efficacy of G9.2-17 was tested in mouse models of PDAC and melanoma as well as in patient-derived organoid tumor models (PDOT). The in situ PDAC KPC mouse model used (LSL-KrasG 12D/+; LSL-Trp53R172H/+; pdx-1-Cre) summarizes many of the characteristics of human disease, including anergy to approved checkpoint inhibitors (Bisht and Feldmann G; animal models for modeling pancreatic cancer and novel Drug discovery; expert Opin Drug discovery.2019; 14 (2): 127-142; weidinhofer et al Animal models of pancreatic cancer and their application in clinical research; gastrointestinal Cancer: targets and Therapy; 6). The B16F10 melanoma mouse model has been a long-term standard for test immunotherapy (Curran et al, PD-1and CTLA-4combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16melanoma tumors;Proc Natl Acad Sci U S A.2010;107 (9): 4275-4280).

PDOT isolated from fresh human tumor samples retains autologous lymphoid and myeloid cell populations, including tumor-infiltrating CD4 and CD 8T lymphocytes that undergo antigen, and is responsive to immunotherapy in short-term ex vivo culture (Jenkins et al Ex Vivo Profiling of PD-1Blockade 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, the expression of galectin-9 on cancer cells was observed in patient-derived organoid assays.

In vivo studies with G9.2-17 mouse IgG1 (G9.2-17 mIgG1 contains exactly the same binding epitope and has the same effector function as G9.2-17 human IgG 4), which antibody has achieved a significant reduction in tumor growth as a single agent in the in situ KPC model where approved checkpoint inhibitors do not work. In the B16F10 model, G9.2-17 significantly exceeded the efficacy against PD-1. In both models, the use of G9.2-17mIgG1 was demonstrated to modulate the intratumoral immune microenvironment and enhance intratumoral CD 8T cell infiltration by up-regulating effector T cell activity and suppressing immunosuppressive signaling.

These results demonstrate that the anti-tumor methods disclosed herein (involving anti-galectin-9 antibodies, optionally in combination with checkpoint inhibitors) will achieve excellent therapeutic efficacy against target solid tumors.

Thus, described herein is the therapeutic use of anti-galectin-9 antibodies as disclosed herein for the treatment of certain cancers.

Antibodies that bind to galectin-9

The present disclosure provides anti-galectin-9 antibodies G9.2-17 and functional variants thereof for use in the methods of treatment disclosed herein.

Antibodies (used interchangeably in plural form) are immunoglobulin molecules capable of specifically binding to a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, 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 (such as Fab, fab ', F (ab') 2, fv), single chains (scFv), mutants thereof, fusion proteins comprising an antibody moiety, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site of a desired specificity, including glycosylated variants of an antibody, amino acid sequence variants of an antibody, and covalently modified antibodies. Antibodies (e.g., anti-galectin-9 antibodies) include any class of antibodies, such as IgD, igE, igG, igA or IgM (or subclass thereof), and the antibodies need not belong to any particular class. Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the antibody heavy chain constant domain. Immunoglobulins fall into five main categories: igA, igD, igE, igG and IgM, wherein several of these classes can be further divided into subclasses (isotypes), e.g. IgG1, igG2, igG3, igG4, igA1 and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

Typical antibody molecules comprise a heavy chain variable region (V _H ) And a light chain variable region (V _L )。V _H And V _L The regions may 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" ("FR")). Each V _H And V _L Typically consisting of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The scope of framework regions and CDRs may be precisely identified using methods known in the art (e.g., by Kabat definition, chothia definition, abM definition, EU definition, "Contact" numbering scheme, "IMGT" numbering scheme, "AHo" numbering scheme, and/or Contact definition, all of which are well known in the art). See, e.g., kabat, e.a. et al (1991) Sequences of Proteins of Immunological Interest, fifth edition, U.S. Pat. No. of Health and Human Services, NIH Publication No.91-3242;chothia et al, (1989) Nature 342:877; chothia, C.et al (1987) J.mol.biol.196:901-917; al-lazikani et Al (1997) J.molecular.biol.273:927-948; edelman et al Proc Natl Acad Sci U S a.1969, month 5; 63 (1) 78-85; and Almagro, J.mol. Recognit.17:132-143 (2004); macCallum et al, J.mol. Biol.262:732-745 (1996); lefranc M P et al, dev Comp Immunol, month 1 2003; 27 (1) 55-77; and honeygger a and plurkthun a, J Mol Biol, 6/8/2001; 309 (3):657-70. See also hgmp.mrc.ac.uk and bioinf.org.uk/abs).

In some embodiments, the anti-galectin-9 antibodies described herein are full length antibodies that contain two heavy chains and two light chains, each comprising a variable domain and a constant domain. Alternatively, the anti-galectin-9 antibody may be an antigen-binding fragment of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of a full-length antibody include (i) Fab fragments, consisting of V _L 、V _H 、C _L And C _H 1 domain; (ii) F (ab') ₂ Fragments, including bivalent fragments of two Fab fragments linked by a disulfide bond at the hinge region; (iii) From V _H And C _H 1 domain-composed Fd fragment; (iv) V by antibody single arm _L And V _H Fv fragments consisting of domains; (v) dAb fragment (Ward et al, (1989) Nature 341:544-546), which is defined by V _H Domain composition; and (vi) an isolated Complementarity Determining Region (CDR) that retains functionality. Furthermore, although the two domains of the Fv fragment V _L And V _H Encoded by separate genes, but can be joined by synthetic linkers using recombinant methods to make them capable of producing a single protein chain, where V _L And V _H The pairing of regions forms monovalent molecules, known as single chain Fv (scFv). See, e.g., bird et al (1988) Science 242:423-426; huston et al (1988) Proc.Natl. Acad.Sci.USA 85:5879-5883.

Any of the antibodies described herein (e.g., an anti-galectin-9 antibody) can be monoclonal or polyclonal. "monoclonal antibody" refers to a homogeneous antibody population, and "polyclonal antibody" refers to a heterogeneous antibody population. These two terms do not limit the source of the antibody or the manner in which it is made.

Reference antibodies G9.2-17 refer to antibodies capable of binding to human galectin-9 and comprise the heavy chain variable region of SEQ ID NO. 7 and the light chain variable region 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 having the same heavy chain Complementarity Determining Regions (CDRs) as the reference antibodies G9.2-17 and/or the same light chain complementarity determining regions as the reference antibodies G9.2-17. With the same V _H And/or V _L Both antibodies of a CDR means that their CDRs are identical when determined by the same method (e.g. Kabat method, chothia method, abM method, contact method or IMGT method as known in the art see e.g. bioinf. Org. Uk/abs /).

The heavy and light chain CDRs of reference antibodies G9.2-17 are provided in table 1 below (determined using the Kabat method):

TABLE 1 heavy and light chain CDRs of G9.2-17

In some examples, an anti-galectin-9 antibody for use in the methods disclosed herein may comprise (according to the Kabat protocol) heavy chain complementarity determining region 1 (CDR 1) as shown in SEQ ID NO. 4, heavy chain complementarity determining region 2 (CDR 2) as shown in SEQ ID NO. 5, and heavy chain complementarity determining region 3 (CDR 3) as shown in SEQ ID NO. 6, and/or may comprise light chain complementarity determining region 1 (CDR 1) as shown in SEQ ID NO. 1, light chain complementarity determining region 2 (CDR 2) as shown in SEQ ID NO. 2, and light chain complementarity determining region 3 (CDR 3) as shown in SEQ ID NO. 3. The anti-galectin-9 antibody (including reference antibody G9.2-17) may be in any form as disclosed herein, such as a full length antibody or Fab. The term "G9.2-17 (IgG 4)" as used herein refers to the G9.2-17 antibody as an IgG4 molecule. Also, the term "G9.2-17 (Fab)" refers to the G9.2-17 antibody as a Fab molecule.

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

Additional galectin-9 antibodies (e.g., binding to CRD1 and/or CRD2 regions of galectin-9) are described in commonly owned, co-pending U.S. patent application 16/173,970 and commonly owned, co-pending international patent applications PCT/US18/58028 and PCT/US2020/024767, the contents of each of which are incorporated herein by reference in their entirety.

In some embodiments, an anti-galectin-9 antibody disclosed herein comprises light chain CDRs that correspond to V of reference antibody G9.2-17 _L CDRs have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments therein) sequence identity compared to either alone or together. Alternatively or additionally, in some embodiments, the anti-galectin-9 antibody comprises a heavy chain CDR that is identical to the corresponding V of the reference antibody G9.2-17 _H CDRs have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments therein) sequence identity compared to either alone or together.

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, as modified in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77,1993. Such algorithms are 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 using the XBLAST program (score=50, word length=3) to obtain amino acid sequences homologous to the protein molecules of the present invention. When gaps exist between the two sequences, gapped BLAST can be used, as described in Altschul et al, nucleic Acids Res.25 (17): 3389-3402, 1997. When using BLAST and Gapped BLAST programs, default parameters for the respective programs (e.g., XBLAST and NBLAST) can be used.

In other embodiments, an anti-galectin-9 antibody described herein comprises V _H The VH comprises HC CDR1, HC CDR2 and HC CDR3 that together contain up to 8 amino acid residue variations (8, 7, 6, 5, 4, 3, 2 or 1 variations, including additions, deletions and/or substitutions) relative to HC CDR1, HC CDR2 and HC CDR3 of reference antibody G9.2-17. Alternatively or additionally, in some embodiments, an anti-galectin-9 antibody described herein comprises V _H The VH comprises LC CDR1, LC CDR2 and LC CDR3 that together contain up to 8 amino acid residue variations (8, 7, 6, 5, 4, 3, 2 or 1 variations, including additions, deletions and/or substitutions) relative to LC CDR1, LC CDR2 and LC CDR3 of reference antibody G9.2-17.

In one example, the amino acid residue variation is a conservative amino acid residue substitution. As used herein, "conservative amino acid substitutions" refer to amino acid substitutions that do not alter the relative charge or size characteristics of the protein in which they are made. Variants may be made according to methods known to those of ordinary skill in the art for altering polypeptide sequences, such as those found in references compiling such methods, for example Molecular Cloning: A Laboratory Manual, J.Sambrook et al, second edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, new York,1989, or Current Protocols in Molecular Biology, F.M. Ausubel et al, john Wiley & Sons, inc., new York. Conservative substitutions of amino acids 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.

In some embodiments, an anti-galectin-9 antibody disclosed herein having heavy chain CDRs disclosed herein contains a polypeptide derived from germline V _H Framework regions of fragment subclasses. Such a line V _H Regions are well known in the art. See, for example, IMGT database (www.imgt.org) or www.vbase2.org/vbstat. Examples include IGHV1 subfamilies (e.g., IGHV1-2, IGHV1-3, IGHV1-8, IGHV1-18, IGHV1-24, IGHV1-45, IGHV1-46, IGHV1-58, and IGHV 1-69), IGHV2 subfamilies (e.g., IGHV2-5, IGHV2-26, and IGHV 2-70), IGHV3 subfamilies (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, IGHV 3-74), IGHV4 subfamilies (e.g., IGHV4-4, IGHV4-28, IGHV4-31, IGHV4-34, IGHV4-4, and IGHV4-6, or IGHV4-6, e.g., and IGHV 1-7.

Alternatively or additionally, in some embodiments, an anti-galectin-9 antibody having light chain CDRs as disclosed herein contains a framework region derived from a germline vκ fragment. Examples include IGKV1 frames (e.g., IGKV1-05, IGKV1-12, IGKV1-27, IGKV1-33, or IGKV 1-39), IGKV2 frames (e.g., IGKV 2-28), IGKV3 frames (e.g., IGKV3-11, IGKV3-15, or IGKV 3-20), and IGKV4 frames (e.g., IGKV 4-1). In other cases, the anti-galectin-9 antibody comprises a light chain variable region containing a framework derived from a germline V lambda fragment. Examples include IGλ1 frames (e.g., IGλV1-36, IGλV1-40, IGλV1-44, IGλV1-47, IGλV 1-51), IGλ2 frames (e.g., IGλV2-8, IGλV2-11, IGλV2-14, IGλV2-18, IGλV 2-23), IGλ3 frames (e.g., IGλV3-1, IGλV3-9, IGλV3-10, IGλV3-12, IGλV3-16, IGλV3-19, IGλV3-21, IGλV3-25, IGλV 3-27), IGλ4 frames (e.g., IGλV4-3, IGλV4-60, IGλV 4-69), λ5 frames (e.g., IGλV5-39, IGλV 5-45), IGλ6 frames (e.g., λV 6-57), IGλ7 frames (e.g., IGλV7-43, λV 7-8, IGλV 7-43), IGλV8 (e.g., IGλV 9-9, or IGλ9-10, e.g., IGλV 8-9).

In some embodiments, the anti-galectin-9 antibody used in the methods disclosed herein may be a heavy chain antibody having the same variable region (V _H ) And/or identical light chain variable regions (V _L ) The antibody of V _H And V _L The region amino acid sequence is provided below:

V _H ：

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSS(SEQ ID NO:7)

V _L ：

DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSTDPITFGQGTKVEIKR(SEQ ID NO:8)

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) to 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) to the light chain variable region of SEQ ID NO: 8.

In some cases, the anti-galectin-9 antibody disclosed herein is a functional variant of reference antibody G9.2-17. Functional variants may be similar in structure to a reference antibody (e.g., comprising a limited number of amino acid residue variations in one or more of the heavy and/or light chain CDRs of G9.2-17 as disclosed herein, or sequence identity to the heavy and/or light chain CDRs of G9.2-17 or VH and/or VL of G9.2-17 as disclosed herein), with substantially similar binding affinity to human galectin-9 (e.g., with a similar KD value).

In some embodiments, an anti-galectin-9 antibody as described herein may bind to and activate galectin-9Inhibition is at least 20% (e.g., 31%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increments therein). Apparent inhibition constant (Ki) ^app Or K _i,app ) Is related to the concentration of inhibitor required to reduce the enzyme activity and is independent of the enzyme concentration. The inhibitory activity of the anti-galectin-9 antibodies described herein can be determined by conventional methods known in the art.

Antibody K _i, ^app The value may be determined by the following procedure: measuring the inhibition of the extent of the reaction (e.g., enzyme activity) by different concentrations of antibody; the change in the pseudo first order rate constant (v) as a function of inhibitor concentration is fitted to the modified Morrison equation (equation 1) to yield an estimate of the apparent Ki value. Ki for competitive inhibitors ^app Can be extracted from K _i, ^app The y-intercept of the linear regression analysis with the substrate concentration plot was obtained.

Wherein A is equal to v _o E, initial rate of enzymatic reaction in the absence of inhibitor (I) (v _o ) Divided by the total enzyme concentration (E). In some embodiments, an anti-galectin-9 antibody described herein has 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, 5pM or less for a target antigen or epitope ^app Values. In some embodiments, the anti-galectin-9 antibody has a lower Ki for the first target (e.g., CRD2 of galectin-9) relative to the second target (e.g., CRD1 of galectin-9) ^app 。Ki ^app May be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000, or 10 ⁵ Multiple times. In some examples, the first egg is a first egg in a first conformation relative to the second antigen (e.g., the same first egg in a second conformationWhite matter or a mimetic thereof; or a second protein), the anti-galectin-9 antibody inhibits the first antigen (e.g., the first protein or mimetic thereof in a first conformation) more greatly. In some embodiments, any of the anti-galectin-9 antibodies is further affinity matured to reduce the Ki of the antibody to the target antigen or epitope thereof ^app 。

In some embodiments, the anti-galectin-9 antibody inhibits Dectin-1 signaling in tumor-infiltrating immune cells such as macrophages, for example. In some embodiments, an anti-galectin-9 antibody inhibits galectin-9 triggered Dectin-1 signaling by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increments therein). Such inhibition activity may be determined by conventional methods of assay such as conventional assays. Alternatively or additionally, the anti-galectin-9 antibody inhibits galectin-9-initiated T cell immunoglobulin mucin-3 (TIM-3) signaling. In some embodiments, an anti-galectin-9 antibody inhibits T cell immunoglobulin mucin-3 (TIM-3) signaling, e.g., in tumor infiltrating immune cells, by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increments therein), e.g., in some embodiments. Such inhibition activity may be determined by conventional methods of assay such as conventional assays.

In some embodiments, the anti-galectin-9 antibody inhibits CD206 signaling in, for example, tumor infiltrating immune cells. In some embodiments, an anti-galectin-9 antibody inhibits CD206 signaling triggered by galectin-9 by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein). Such inhibition activity may be determined by conventional methods of assay such as conventional assays. In some embodiments, the anti-galectin-9 antibody blocks or prevents binding of galectin-9 to CD206 by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increments therein). Such inhibition activity may be determined by conventional methods of assay such as conventional assays.

In some embodiments, the anti-galectin-9 antibody induces cytotoxicity, such as ADCC, in a target cell expressing galectin-9, e.g., wherein the target cell is a cancer cell or an immunosuppressive immune cell. In some embodiments, an anti-galectin-9 antibody induces apoptosis in 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 inhibition activity may be determined by conventional methods of assay such as conventional assays. In some embodiments, any of the anti-galectin-9 antibodies described herein induces cytotoxicity, such as Complement Dependent Cytotoxicity (CDC), against target cells expressing galectin-9.

Antibody-dependent cell-mediated phagocytosis (ADCP) is an important mechanism of action of antibodies through phagocytosis to mediate some or all of their actions. In this case, the antibody mediates uptake of the specific antigen by the antigen presenting cells. ADCP can be mediated by monocytes, macrophages, neutrophils and dendritic cells through fcyriia, fcyri and fcyriiia, with fcyriia on macrophages (CD 32 a) representing the primary pathway.

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

In some embodiments, an anti-galectin-9 antibody described herein induces cytotoxicity, such as Complement Dependent Cytotoxicity (CDC), against a target cell, e.g., a cancer cell or an immunosuppressive immune cell. In some embodiments, an anti-galectin-9 antibody increases CDC against a target cell by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein).

In some embodiments, the anti-galectin-9 antibody induces T cell activation, e.g., in tumor infiltrating T cells, i.e., directly or indirectly inhibits galectin-9 mediated inhibition of T cell activation. In some embodiments, an anti-galectin-9 antibody increases T cell activation by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increments 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, tnfα, ifnγ, and/or PD-1). In some embodiments, the anti-galectin-9 antibody increases 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, an 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 delta therein). In a non-limiting example, an anti-galectin antibody induces ifnγ expression in cd4+ cells. In some embodiments, the anti-galectin-9 antibody increases ifnγ expression in cd4+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any delta therein). In a non-limiting example, an anti-galectin antibody induces tnfα expression in cd4+ cells. In some embodiments, an anti-galectin-9 antibody increases tnfα 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 some embodiments, the anti-galectin-9 antibody increases cd8+ cell activation by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increments therein). In a non-limiting example, an 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 increase therein). In a non-limiting example, an anti-galectin antibody induces ifnγ expression in cd8+ cells. In some embodiments, the anti-galectin-9 antibody increases ifnγ 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, an anti-galectin antibody induces tnfα expression in cd8+ cells. In some embodiments, an anti-galectin-9 antibody increases tnfα 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 some embodiments, an anti-galectin-9 antibody as described herein has suitable binding affinity for a target antigen (e.g., galectin-9) or an epitope thereof. As used herein, "binding affinity" refers to the apparent association constant or K _A 。K _A Is the dissociation constant (K) _D ) Is the inverse of (c). The anti-galectin-9 antibodies described herein may have at least 10 for a target antigen or epitope ^-5 、10 ^-6 、10 ^-7 、10 ^-8 、10 ^-9 、10 ^-10 M or lower binding affinity (K _D ). The increase in binding affinity corresponds to K _D And (3) lowering. Binding affinity (or binding specificity) can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescent assay). Exemplary conditions for assessing binding affinity are in HBS-P buffer (10 mM HEPES pH7.4, 150mM NaCl, 0.005% (v/v)) Surfactant P20).

These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. Under certain conditions, the fractional concentration of bound binding protein ([ binding ]/[ total ]) is generally related to the concentration of total target protein ([ target ]) by the following equation:

binding/total target/Kd target

However, it is not always necessary to accurately determine K _A Because quantitative measurements of affinity are sometimes obtained (e.g., determined using methods such as ELISA or FACS analysis, with K) _A Proportionality, therefore, may be used for comparison, such as determining if the higher affinity is higher, e.g., by a factor of 2), obtaining qualitative measurements of affinity, or obtaining inferences of affinity (e.g., by activity in a functional assay (e.g., in vitro or in vivo assay) are sufficient. In some cases, the in vitro binding assay is indicative of in vivo activity. In other cases, an in vitro binding assay is not necessarily indicative of in vivo activity. In some cases tight binding is beneficial, but in other cases tight binding is not ideal in vivo, and antibodies with lower binding affinity are more ideal.

In some embodiments, the heavy chain of any of the anti-galectin-9 antibodies as 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, such as human, mouse, rat, or rabbit. In a specific example, the heavy chain constant region is from human IgG (gamma heavy chain) of any IgG subfamily as described herein.

In some embodiments, the heavy chain constant regions of antibodies described herein comprise a single domain (e.g., CH1, CH2, or CH 3) or a combination of any single domain of the constant regions (e.g., SEQ ID NOS: 4, 5, 6). In some embodiments, the light chain constant regions of antibodies described herein comprise a single domain of constant region (e.g., CL). Exemplary light and heavy chain sequences are listed below. Exemplary light and heavy chain sequences are listed below. The hIgG1 LALA sequence includes two mutations (L234A and L235A (EU numbering) that inhibit FcgR binding) and a P329G mutation (EU numbering) to eliminate complement C1q binding, thereby eliminating all immune effector functions. The hIgG4 Fab arm crossover mutant sequence includes a mutation that inhibits Fab arm crossover (S228P; EU numbering). IL2 signal sequence (MYRMQLLSCIALSLALVTNS; SEQ ID NO: 9) may be located at the N-terminus of the variable region. It is used in expression vectors, which are cleaved during secretion and therefore not in mature antibody molecules. The heavy chain of the mature protein (post-secretion) starts with "EVQ" and the light chain with "DIM". The amino acid sequences of exemplary heavy chain constant regions are provided below:

hIgG1 heavy chain constant region (SEQ ID NO: 10)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

hIgG1 LALA heavy chain constant region (SEQ ID NO: 12)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

hIgG4 heavy chain constant region (SEQ ID NO: 13)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK*

hIgG4 heavy chain constant region (SEQ ID NO: 20)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

hIgG4 mutant heavy chain constant region (SEQ ID NO: 14)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK*

hIgG4 mutant heavy chain constant region (SEQ ID NO: 21)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

In some cases, the heavy chain constant region in an anti-galectin-9 antibody (e.g., G9.2-17) disclosed herein can remove a C-terminal lysine (K) residue for manufacturing purposes, for example. Corresponding amino acid sequences to those without terminal K residues are provided below:

hIgG1 heavy chain constant region without C-terminal lysine (SEQ ID NO: 24)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

hIgG1 LALA heavy chain constant region without C-terminal lysine (SEQ ID NO: 25)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

hIgG4 heavy chain constant region without C-terminal lysine (SEQ ID NO: 26)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG*

hIgG4 heavy chain constant region without C-terminal lysine (SEQ ID NO: 27)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*

hIgG4 mut heavy chain constant region without C-terminal lysine (SEQ ID NO: 28)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG*

hIgG4 mut heavy chain constant region without C-terminal lysine (SEQ ID NO: 29)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*

In some embodiments, an anti-galectin-9 antibody having any one of the above heavy chain constant regions is paired with a light chain having the following light chain constant regions:

light chain constant region (SEQ ID NO: 11)

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Exemplary full-length anti-galectin-9 antibodies are provided below:

G9.2-17 hIgG1 heavy chain (SEQ ID NO: 16)

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

G9.2-17 hIgG1 heavy chain without C-terminal lysine residue (SEQ ID NO: 30)

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

G9.2-17 hIgG1 LALA heavy chain (SEQ ID NO: 17)

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

G9.2-17 hIgG1 LALA heavy chain without C-terminal lysine residue (SEQ ID NO: 31)

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

G9.2-17 hIgG4 heavy chain (SEQ ID NO: 18)

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK*

G9.2-17 hIgG4 heavy chain without C-terminal lysine residue (SEQ ID NO: 32)

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG*

G9.2-17 hIgG4 heavy chain (SEQ ID NO: 22)

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

G9.2-17 hIgG4 heavy chain without C-terminal lysine residue (SEQ ID NO: 33)

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*

G9.2-17 hIgG4 Fab arm exchange mutant heavy chain (SEQ ID NO: 19)

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK*

G9.2-17 hIgG4 Fab arm exchange mut heavy chain without C-terminal lysine residue (SEQ ID NO: 34)

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG*

G9.2-17 hIgG4 Fab arm exchange mutant heavy chain (SEQ ID NO: 23)

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

G9.2-17 hIgG4 Fab arm exchange mut heavy chain without C-terminal lysine residue (SEQ ID NO: 35)

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*

Any of the heavy chains described above may be paired with a light chain as shown below (SEQ ID NO: 15):

DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSTDPITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

in some embodiments, an 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 increments 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.

In some embodiments, an 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 increments 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.

In some embodiments, the constant region is 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 increments 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.

In some embodiments, the constant region is from human IgG4. In one embodiment, an 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 increments 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.

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 increments 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.

In some embodiments, the IgG is a mutant with minimal Fc receptor binding. In one example, the constant region is 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 increments 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.

In some embodiments, the anti-galectin-9 antibody comprises a modified constant region. In some embodiments, the anti-galectin-9 antibody comprises an immunologically inert modified constant region, e.g., does not trigger complement-mediated lysis, or does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC activity may be assessed using the methods disclosed in U.S. patent No. 5,500,362. In other embodiments, the constant regions are modified as described in Eur.J.Immunol. (1999) 29:2613-2624, PCT application No. PCT/GB99/01441 and/or British patent application No. 9809951.8. In some embodiments, the IgG4 constant region is a mutant with reduced heavy chain exchange. In some embodiments, the constant region is from human IgG4 Fab arm swap mutant S228P.

In one embodiment, the constant region of an 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 increments 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.

In one embodiment, an 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 increments 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.

In some embodiments, the light chain of the anti-galectin-9 antibody has a chain corresponding to SEQ ID NO. 15; and the amino acid sequence of an exemplary heavy chain corresponds to SEQ ID NO. 10 (hIgG 1); 12 (hIgG 1 LALA); 13 (hIgG 4); 20 (hIgG 4); 14 (hIgG 4 mut); and 21 (hIgG 4 mut).

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 NO. 16-19. In some embodiments, the anti-galectin-9 antibody has a light chain comprising SEQ ID NO. 15 and a heavy chain comprising any one of the sequences selected from the group consisting of SEQ ID NO. 16-19, 22 and 23. In some embodiments, the anti-galectin-9 antibody has a light chain consisting essentially of SEQ ID NO. 15 and a heavy chain consisting essentially of any one of the sequences selected from the group consisting of SEQ ID NO. 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 NO. 16-19, 22 and 23. In a 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.

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 increments 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.

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 increments 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.

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 increments 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.

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 increments 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.

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 increments 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.

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 increments 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.

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 increments 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.

In a specific example, an anti-galectin-9 antibody used in the methods of treatment 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 methods of treatment disclosed herein is G9.2-17 IgG4. In some examples, such anti-galectin-9 antibodies do not have a C-terminal lysine residue in their heavy chain.

Preparation of anti-galectin-9 antibodies

Antibodies capable of binding galectin-9 as described herein may be prepared by any method known in the art, including but not limited to recombinant techniques. An example is provided below.

Nucleic acids encoding the heavy and light chains of an anti-galectin-9 antibody as described herein can be cloned into an expression vector, 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 different promoter. Alternatively, the nucleotide sequences encoding the heavy and light chains may 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.

In some examples, the nucleotide sequences encoding the two chains of the antibody are cloned into two vectors, which may be introduced into the same or different cells. When the two chains are expressed in different cells, each of them may be isolated from the host cell in which it is expressed, and the isolated heavy and light chains may be mixed and incubated under suitable conditions that allow the formation of antibodies.

Generally, nucleic acid sequences encoding one or all of the chains of an antibody can be cloned into a suitable expression vector operably linked to a suitable promoter using methods known in the art. For example, the nucleotide sequence and vector may be contacted with a restriction enzyme under suitable conditions to produce complementary ends on each molecule that can be paired with each other and linked together with a ligase. Alternatively, a synthetic nucleic acid linker may be attached to the end of the gene. These synthetic linkers contain nucleic acid sequences corresponding 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.

A variety of promoters may 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 (Rous sarcoma) viral LTR, HIV-LTR, HTLV-1LTR, simian Virus 40 (SV 40) early promoter, E.coli) lac UV5 promoter, and herpes simplex tk virus promoter.

Regulatable promoters may also be used. Such regulatable promoters include those that use a lac repressor from E.coli as a transcription regulator to regulate transcription of mammalian Cell promoters harboring the lac operon [ Brown, M.et al, cell,49:603-612 (1987) ], those that use a 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); sockelt, P.et al, proc.Natl. Acad.Sci.USA,92:6522-6526 (1995) ]. Other systems include FK506 dimer, VP16 or p65 using estradiol, RU486, diphenol Le Zai ketone (diphenol murislerone) or rapamycin. Inducible systems are available from Invitrogen, clontech and Ariad.

A regulatable promoter comprising a repressor with an operator may be used. In one embodiment, the lac repressor from E.coli may act as a transcription regulator to regulate transcription of a mammalian Cell promoter harboring the lac operon (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)), and the tetracycline repressor (tetR) is combined with a transcriptional activator (VP 16) to produce a tetR-mammalian Cell transcriptional activator fusion protein tTa (tetR-VP 16), wherein the minimal promoter harboring tetO is derived from a human cytomegalovirus (hCMV) major immediate early promoter to produce the tetR-tet operon gene system, thereby controlling gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. When the tetracycline operon is correctly located downstream of the TATA element of the CMVIE promoter, the tetracycline repressor (tetR) alone, rather than the tetR-mammalian cell transcription factor fusion derivative, may act as a potent trans regulator to regulate gene expression in mammalian cells (Yao et al, human Gene Therapy,10 (16): 1392-1399 (2003)). A particular advantage of this tetracycline-inducible switch is that it does not require the use of tetracycline repressor-mammalian cell transactivators or repressor fusion proteins (which in some cases may be cytotoxic) (Gossen et al, natl. Acad. Sci. USA,89:5547-5551 (1992); shock et al, proc. Natl. Acad. Sci. USA,92:6522-6526 (1995)) to achieve its regulatory effect.

In addition, the carrier may contain, for example, some or all of the following: selectable marker genes, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences for high level transcription from immediate early genes of human CMV; transcription termination and RNA processing signals for mRNA stability from SV 40; SV40 polyoma viral origin of replication and ColE1 for appropriate episomal replication; an internal ribosome binding site (IRESE); a multifunctional multiple cloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNAs. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.

Examples of polyadenylation signals that may be used to practice the methods described herein include, but are not limited to, human collagen I polyadenylation signals, human collagen II polyadenylation signals, and SV40 polyadenylation signals.

One or more vectors (e.g., expression vectors) comprising nucleic acids encoding any of the antibodies can be introduced into a suitable host cell to produce the antibodies. The host cell may be cultured under suitable conditions for expressing the antibody or any polypeptide chain thereof. Such antibodies or polypeptide chains thereof may be recovered from the cultured cells (e.g., from the cells or culture supernatant) via conventional methods (e.g., affinity purification). If desired, the polypeptide chains of the antibodies may be incubated under suitable conditions for a suitable period of time to allow for the production of the antibodies.

In some embodiments, the methods for making the antibodies described herein involve recombinant expression vectors encoding the heavy and light chains of an anti-galectin-9 antibody as also described herein. The recombinant expression vector may be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods (e.g., calcium phosphate-mediated transfection). Positive transformant host cells can be selected and cultured under suitable conditions that allow expression of the two polypeptide chains forming the antibody, which can be recovered from the cells or culture medium. If necessary, the two chains recovered from the host cell may be incubated under suitable conditions that allow for antibody formation.

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 recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods (e.g., calcium phosphate-mediated transfection). Alternatively, each of the expression vectors may be introduced into a suitable host cell. Positive transformants may be selected and cultured under suitable conditions that allow expression of the polypeptide chain of the antibody. When both expression vectors are introduced into the same host cell, the antibodies produced therein may be recovered from the host cell or medium. If desired, the polypeptide chain may be recovered from the host cell or culture medium and then cultured under suitable conditions that allow for antibody formation. When two expression vectors are introduced into different host cells, each of them may be recovered from the corresponding host cell or from the corresponding medium. The two polypeptide chains may then be incubated under suitable conditions for antibody formation.

Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture the host cells and recover the antibodies from the culture medium. For example, some antibodies can be isolated by affinity chromatography using protein a or protein G coupled matrices.

Any of a nucleic acid encoding the heavy chain, the light chain, or both of an anti-galectin-9 antibody as described herein, a vector (e.g., an expression vector) containing the nucleic acid, and a host cell comprising the vector is within the scope of the disclosure.

The anti-galectin-9 antibodies thus prepared may be characterized using methods known in the art, whereby a reduction, improvement or neutralization of galectin-9 biological activity is detected and/or measured. For example, in some embodiments, ELISA-type assays are suitable for qualitative or quantitative measurement of galectin-9 inhibition of Dectin-1 or TIM-3 signaling.

The biological activity of anti-galectin-9 antibodies can be verified by incubating the candidate antibodies with Dectin-1 and galectin-9 and monitoring any one or more of the following characteristics: (a) Binding between Dectin-1 and galectin-9 and inhibition of signal transduction mediated by said binding; (b) preventing, ameliorating or treating any aspect of a solid tumor; (c) blocking or reducing Dectin-1 activation; (d) Inhibit (reduce) synthesis, production or release of galectin-9. Alternatively, TIM-3 may be used to verify the biological activity of anti-galectin-9 antibodies using the protocol described above. Alternatively, CD206 may be used to verify the biological activity of anti-galectin-9 antibodies using the protocol described above.

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

Other assays to determine the biological activity of anti-galectin-9 antibodies include measuring cd8+ and cd4+ (conventional) T cell activation (in vitro or in vivo assays, e.g., by measuring inflammatory cytokine levels such as ifnγ, tnfα, CD44, ICOS granzyme B, perforin, IL2 (up-regulation); CD26L and IL-10 (down-regulation)); measurement of macrophages re-programming (in vitro or in vivo) from, for example, the M2 to M1 phenotype (e.g., increased mhc ii, decreased CD206, increased TNF-and iNOS). Alternatively, the level of ADCC may be assessed in an in vitro assay as described herein.

Therapeutic method

The present disclosure provides methods of 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 any one of the anti-galectin antibodies (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 (IgG 4)). Such antibodies may be used to treat diseases associated with galectin-9. In some aspects, the present disclosure provides methods of treating cancer. In some embodiments, the present disclosure provides methods for reducing, ameliorating, or eliminating one or more symptoms associated with cancer.

(A) Exemplary target solid tumor

In some embodiments, the present disclosure provides a method of 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 applicable to human patients suffering from pancreatic cancer (e.g., ductal adenocarcinoma (PDAC)). In some cases, PDAC patients may have metastatic cancer. In some examples, the methods disclosed herein are applicable to human patients with colorectal cancer (CRC). In some embodiments, the colorectal cancer is metastatic. In some examples, the methods disclosed herein are applicable to human patients with hepatocellular carcinoma. In some embodiments, the hepatocellular carcinoma is metastatic. In other examples, the methods disclosed herein are applicable to human patients with cholangiocarcinoma. In some embodiments, the cholangiocarcinoma is metastatic.

Pancreatic Ductal Adenocarcinoma (PDAC) is a devastating disease with few long-term survivors (Yadav et al, gastrology, 2013,144,1252-1261). Inflammation is extremely important in the progression of PDAC, as in the absence of concomitant inflammation, oncogenic mutations alone are insufficient to cause tumorigenesis (Guerra et al, cancer Cell,2007,11,291-302). Congenital and adaptive immunity synergistically promote tumor progression in PDACs. In particular, specific innate immune subpopulations within the Tumor Microenvironment (TME) are prone to adaptive immunity Epidemic effector cells are cultured to a tumor-permissive phenotype. Antigen Presenting Cell (APC) populations, including M2 polarized tumor-associated macrophages (TAM) and myeloid Dendritic Cells (DC), induce the production of immunosuppressive Th2 cells that favor 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) have been shown to eliminate anti-tumor CD8 in PDAC ⁺ Cytotoxic T Lymphocytes (CTL) respond and promote metastatic progression (Connolly et al, JLeuk biol.,2010,87,713-725; pylayeva-Gupta et al, cancer Cell,2012,21,836-847; bayne et al, cancer Cell,2012,21,822-835).

Pancreatic cancer remains a difficult disease to treat due to the generally late onset, relatively high resistance to chemotherapy, and lack of effective immune and targeted therapies. About 455,000 new pancreatic cancer cases were reported in 2018 worldwide, 355,000 new cases are expected to occur annually by 2040, and the number of deaths reported annually is almost as large as the new cases. It is expected to be the second leading cause of cancer-related death in the united states by 2030. Despite intervention, with current treatments, patients with metastatic pancreatic cancer have a median life expectancy of less than 1 year, while most patients (up to 80%) are in the advanced/metastatic stage, at which time the disease has failed to cure. Despite advances in the detection and management of pancreatic cancer, five-year survival of metastatic disease remains at 10%. The current standard of care for metastatic pancreatic cancer is primarily chemotherapy, while few patients (less than 10%) with BRCA1/2 mutations and mismatch repair deficient tumors can benefit from PARP inhibitors and potential anti-PD-1 therapies. However, for the vast majority of patients with this disease, currently approved immunotherapy is generally unsuccessful due to the highly immunosuppressive environment.

Colorectal cancer (CRC) (also known as bowel cancer, colon cancer or rectal cancer) is any cancer that affects the colon and rectum. CRC is known to be driven by genetic alterations of tumor cells and is also affected by tumor-host interactions. Recent reports have demonstrated a direct correlation between the density of certain T lymphocyte subpopulations and favorable clinical outcomes in CRC, supporting the major role of T cell mediated immunity in inhibiting tumor progression of CRC.

CRC is one of the largest cancer burden worldwide. Today, it is the fourth most deadly cancer worldwide, with nearly 900,000 deaths each year worldwide. In the united states, 147,950 cases are expected to occur in 2020, with an estimated mortality of 53,200 (Colorectal Cancer Stats). Despite significant advances in standard care therapies, five-year survival rates for metastatic CRC remain around < 20%. CRC mortality is expected to increase by nearly a factor of 20 in the next 20 years. The current standard of care for CRC is a chemotherapy regimen that binds to and/or orders in selected patients with anti-angiogenic therapy and anti-EGF receptor means. In addition, current immunotherapy is effective only for a small fraction of patients with tumor mismatch repair defects and high microsatellite instability (dMMR/MSI-H), albeit with profound and sustained responses (Dekker et al, 2019). Microsatellite stabilized CRC (most CRC patients) results in poor immunotherapy, which indicates that the medical needs for active immunotherapy are significantly unmet. CRC (dMMR/a small portion of MSI-H) benefits from immunotherapy (Huyghe et al, 2019), but the vast majority of patients with proficient mismatch repair or microsatellite stabilized CRC cannot benefit.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. Hepatocellular carcinoma most commonly occurs in people with chronic liver disease, such as cirrhosis caused by hepatitis b or hepatitis c infection. HCC is often associated with cirrhosis and extensive lymphocyte infiltration due to chronic viral infection. Many studies have demonstrated that tumor-infiltrating effector cd8+ T cells and T helper 17 (Th 17) cells are associated with increased survival after surgical removal of tumors. However, tumor infiltrating effector T cells are unable to control tumor growth and metastasis (Pang et al Cancer Immunol Immunother 2009; 58:877-886).

Bile duct cancer (CCA) is a group of cancers that begin in the bile duct. Cholangiocarcinomas are typically classified according to their location relative to the liver. For example, intrahepatic cholangiocarcinoma accounts for less than 10% of all cholangiocarcinoma cases, beginning in the small bile duct within the liver. In another example, portal cholangiocarcinoma (also known as klotskin tumor) accounts for more than half of the cholangiocarcinoma cases, beginning in the portal where the two main bile ducts merge and leave the liver. Others are classified as distal bile duct cancers, beginning in the extrahepatic bile duct.

CCA is an invasive tumor and most patients are already in advanced disease stages at the time of their visit. The incidence of CCA is increasing and effective therapies are urgently needed. Gemcitabine plus cisplatin remains the standard first-line systemic therapy for advanced CCA, although it has many shortcomings because of median survival of less than 1 year. In addition to first line therapy failure, there is little evidence available to guide therapeutic decisions. The U.S. Food and Drug Administration (FDA) recently approved the first targeted therapy for this indication for patients with fibroblast growth factor receptor 2 gene fusions and other rearrangements in tumors. The non-ideal response to immunotherapy in human clinical trials means that most CCAs are immune "cold" tumors with non-T cell infiltrating microenvironments. Indeed, immunotherapy has not produced a response rate of more than 17% so far, and by the date of the present disclosure no immune tumour agent has been approved (Zayac and Almhanna, 2020).

These tumors appear silently, coupled with their high invasiveness and intractability to chemotherapy, leading to alarming mortality, with five-year survival still being only 2% for patients with distant disease (Banales et al, 2020;Bile Duct Cancer Survival,2020).

In some embodiments, methods are provided that increase antitumor activity by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more (e.g., decrease cell proliferation, tumor growth, tumor volume and/or tumor burden or decrease the number of metastatic lesions over time) compared to the level in a pre-treatment or control subject. In some embodiments, the decrease 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 of ameliorating one or more symptoms of cancer by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more are provided. In some embodiments, cancer cells and/or biomarkers in a subject are measured in a biological sample (such as blood, serum, plasma, urine, peritoneal fluid, and/or biopsies from tissue or organs) before, during, and after administration of a pharmaceutical composition. In some embodiments, the methods comprise administering a composition of the invention to reduce the tumor volume, size, burden or burden of the 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 volume, size, burden or burden of the subject prior to treatment. In other embodiments, methods are provided for reducing the rate of cell proliferation or tumor growth 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 prior to treatment. In other embodiments, the methods comprise administering a composition of the invention to reduce the development or number or size of a metastatic lesion 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 prior to treatment.

The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, depending in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" may mean within an acceptable standard deviation. Alternatively, "about" may mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5% and more preferably up to ±1% of a given value. Alternatively, particularly for biological systems or processes, the term may mean within an order of magnitude, preferably within a factor of 2, of the value. Where a particular value is described in the application and claims, unless otherwise indicated, the term "about" is implicit and, in this context, means within an acceptable error range for the particular value.

As used herein, the term "treating" refers to the application or administration of a composition comprising one or more active agents to a subject suffering from, or prone to, a target disease or disorder, a symptom of a disease/disorder, with the aim of curing, healing, alleviating, moderating, altering, remediating, alleviating, ameliorating, or affecting the disorder, the symptom of a disease or disorder, or the propensity to a disease or disorder.

Alleviation of a disease/disorder of interest includes delaying the development or progression of the disease or reducing the severity of the disease or prolonging survival. Cure outcomes are not necessarily required to alleviate the disease or to prolong survival. As used herein, "delay" of progression of a target disease or disorder refers to delaying, impeding, slowing, delaying, stabilizing, and/or slowing the progression of the disease. This delay may be of different lengths of time, depending on the history of the disease and/or the individual being treated. A method of "delaying" or alleviating the progression of a disease or delaying the onset of a disease is a method of reducing the likelihood of one or more symptoms of a disease occurring within a given time frame and/or alleviating the extent of symptoms within a given time frame, as compared to the absence of the method. Such comparisons are typically based on clinical studies using a number of subjects sufficient to yield statistically significant results.

"progression" or "progression" of a disease refers to the initial manifestation and/or subsequent progression of the disease. The progression of the disease can be detected and assessed using standard clinical techniques well known in the art. However, development also refers to progress that may not be detectable. For the purposes of this disclosure, development or progression refers to the biological process of symptoms. "progression" includes occurrence, recurrence and onset. As used herein, a "seizure" or "occurrence" of a disease or disorder of interest includes a first seizure and/or recurrence.

(B) Exemplary patient populations for treatment

Subjects suffering from any of the above cancers may be identified by routine medical examinations (e.g., laboratory tests, organ function tests, genetic tests, interventional procedures (biopsies, surgery), any and all related imaging modalities).

In some embodiments, the subject to be treated by the methods described herein is a human cancer patient who has undergone or experienced an anticancer therapy regimen (e.g., chemotherapy, radiation therapy, tumor treatment fields (TTFields), immunotherapy, biologic therapy, small molecule inhibitors, anti-hormonal therapy, cell-based therapy and/or surgery) delivered systemically and/or locally in any combination or order of summarized therapeutic modalities. In some embodiments, the subject has received a prior immunomodulatory agent or any other anti-neoplastic agent or treatment regimen listed above. Non-limiting examples of such immunomodulators include, but are not limited to, anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-TIGIT, anti-PVRIG, anti-LAG-3, anti-CD 47, anti-CD 40, anti-CSFR 1, anti-CD 73, anti-SIRP, anti-A2 AR, anti-OX 40, anti-CD 137, 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 by treatment. In other embodiments, the subject is resistant to treatment (slave or acquired). In some embodiments, such subjects are demonstrated to have advanced malignancy (e.g., inability to operate or metastasize). Alternatively, or in addition, in some embodiments, the subject has no standard treatment options available or is not in compliance with the conditions of the standard treatment options, which refer to therapies commonly used in the clinical setting to treat the corresponding solid tumor.

Tumor therapeutic electric fields (TTFields) are a type of cancer treatment that uses alternating electric fields of medium frequency (-100-500 kHz) and low intensity (1-3V/cm) to disrupt cell division. In any of the embodiments described herein, the anti-galectin-9 antibody may be administered prior to, concurrently with, or after a tumor treatment electric field (TTFields) regimen, alone or in combination with a checkpoint inhibitor such as an anti-PD-1 antibody.

In some cases, the subject may be a human patient with a refractory disease (e.g., refractory PDAC, refractory CRC, refractory HCC, or refractory cholangiocarcinoma). As used herein, "refractory" refers to a tumor that does not respond to treatment or becomes resistant. In some cases, the subject may be a human patient with a recurrent disease (e.g., recurrent PDAC, recurrent CRC, recurrent HCC, or recurrent cholangiocarcinoma). As used herein, "recurrent" or "recurrence" refers to a tumor that reappears or progresses after a period of treatment improvement (e.g., partial or complete response).

In some embodiments, a human patient to be treated by the methods disclosed herein meets one or more of the inclusion and exclusion criteria disclosed in example 1 below. For example, a human patient may be 18 years old or older; patients with histologically confirmed unresectable metastatic or inoperable cancers (e.g., without standard treatment options), with >Life expectancy of 3 months, with recently archived tumor samples available for biomarker analysis (e.g., archived species for galectin-9 tumor tissue expression levels assessed by IHC); with disease measurable according to RECIST v1.1 with eastern tumor cooperative group (ECOG) expression status 0-1 or Karnofsky score>70; there is no available standard of care choice, with MSI-H (high microsatellite instability) and MSS (microsatellite stability); receiving at least one line number of systemic therapies in an advanced/metastatic setting; has sufficient hematology and end organ function (defined in example 1 below; e.g., neutrophil count. Gtoreq.1X10) ⁹ The platelet count is more than or equal to 100x10 ⁹ L, for HCC in part 1, +.50X10) ⁹ L; hemoglobin not transfused for the previous week is not less than 9.0g/dL, creatinine is not more than 1.5 XULN, AST (SGOT) is not more than 3 XULN (not more than 5 XULN when HCC or liver metastasis is present), ALT (SGPT) is not more than 3 XULN (not more than 5 XULN when HCC or liver metastasis is present), bilirubin is not more than 1.5 XULN (bilirubin of not more than 3.0 XULN for patients known to have Gilbert's disease), albumin is not less than 3.0g/dL, INR and PTT are not more than 1.5 XULN; and/or amylase and lipase are less than or equal to 1.5 XULN); treatment of brain metastases, if any, has been completed (see example 1 below); there was no evidence of active infection and no severe infection in the past month; having at least four (4) weeks or 5 half-lives (whichever is shorter) from the last dose of anti-cancer therapy prior to the first administration of the anti-Gal-9 antibody; continuing bisphosphonate treatment or Desulumab (denosumab) to treat bone metastasis (if The application is as follows). CCR or CCA patients undergoing immediate treatment may have received at least one previous line number of therapy in the desired metastatic environment. In some embodiments, a CCR or CCA patient undergoing immediate treatment may have received at least one previous line number of therapy in a metastatic setting.

Alternatively or additionally, a subject suitable for the treatment disclosed herein may not have one or more of the following: metastatic cancer diagnosed as primary unknown; any bleeding that is not controlled in activity, and any patient with bleeding physique (e.g., active peptic ulcer disease); any other study agent is received within 4 weeks or 5 half-lives of administration of the anti-galectin-9 antibody; radiation therapy is received within 4 weeks of the first dose of anti-galectin-9 antibody, except for a limited range of palliative radiation therapy, such as for the treatment of bone pain or focal pain tumor mass; has mushroom tumor mass; for patients with PDAC, the patient had locally advanced PDAC with a previous gemcitabine-containing regimen less than 6 months from treatment initiation; has an active clinical severe infection > grade 2 NCI-CTCAE version 5.0; with symptomatic or active brain metastasis; have ≡ctcae grade 3 toxicity (see details and exceptions in example 1); there was a history of secondary malignancy (see exceptions in example 1); there is evidence of severe or uncontrolled systemic disease, congestive heart failure; there are severe non-healing wounds, active ulcers or untreated fractures; uncontrolled pleural effusion, pericardial effusion or ascites, requiring repeated drainage procedures; there is spinal cord compression without explicit treatment by surgery and/or radiation. With active or previously treated leptomeningeal disease; suffering from major vascular diseases; with active autoimmune disorders (see exceptions in example 1); a need for systemic immunosuppressive therapy; with tumor-associated pain (> grade 3), unresponsive to extensive analgesic interventions (oral and/or patches); despite the use of bisphosphonates, there is uncontrolled hypercalcemia; there is a history of any immune-related grade 4 adverse events due to previous Checkpoint Inhibitor Therapy (CIT); receiving organ transplantation; and/or subjected to dialysis; for HCC and/or CCA patients, any ablative therapy is performed prior to treatment; hepatic encephalopathy or severe liver adenoma; and/or has a Child-Pugh score of 7 or more. In some cases, a human patient may not have metastatic hepatocellular carcinoma that progressed upon receiving at least one previous line of systemic therapy; sorafenib has been rejected or intolerant; standard therapies have either been deemed ineffective, intolerable or inappropriate, or no effective standard therapies are available.

In some embodiments, a human patient to be treated by the methods disclosed herein may meet one or more of the inclusion and exclusion criteria disclosed in example 1 below. For example, a human patient may be older than 18 years and have a histologically confirmed unresectable metastatic cancer (e.g., adenocarcinoma and squamous cell carcinoma). The patient may have a disease that is measurable according to RECIST v.1.1. In some cases, a human patient may have a recently archived tumor sample (e.g., obtained within 5 years) that can be used for biomarker analysis (e.g., galectin-9 tumor tissue expression, which can be assessed by IHC). In some cases, the human patient is a PDAC patient that has received at least one line of systemic therapy in a metastatic cancer setting. In some cases, the human patient is a metastatic PDAC patient who has received or has not received systemic therapy prior to receiving an anti-galectin-9 containing regimen. Such patients may not have received a gemcitabine-containing regimen or have not been treated with a gemcitabine-containing regimen for at least 6 months in the previous disease stage setting. The patient may have an eastern tumor cooperative group (ECOG) performance status of 0-1 and/or Karnofsky score >70. The patient may also have sufficient hematologic and end organ function, e.g., neutrophil count ≡1X10 ⁹ Platelet count 100x10 ⁹ L, for HCC in part 1, +.50X10) ⁹ L; hemoglobin not transfused for the previous week is not less than 9.0g/dL, creatinine is not more than 1.5 XULN, AST (SGOT) is not more than 3 XULN (not more than 5 XULN when HCC or liver metastasis is present), ALT (SGPT) is not more than 3 XULN (not more than 5 XULN when HCC or liver metastasis is present), bilirubin is not more than 1.5 XULN (bilirubin of not more than 3.0 XULN for patients known to have Gilbert's disease), albumin is not less than 3.0g/dL, INR and PTT are not more than 1.5 XULN; and/or amylase and lipase are less than or equal to 1.5 XULN. At the position ofIn some cases, human patients show no evidence of active infection or infection requiring parenteral antibiotics, and no severe infection within 4 weeks before treatment begins. Pancreatic, biliary or intestinal fistulae are allowed, provided that they are controlled with an appropriate uninfected and unobstructed drainage tube.

Alternatively or additionally, a human patient undergoing any of the treatments disclosed herein may not have the following: (i) With primary, non-inventive metastatic cancer, (ii) with clinically significant, actively uncontrolled bleeding, any bleeding constitutions (e.g., active peptic ulcer); (iii) Radiotherapy within 4 weeks of treatment at the first dose, (iv) with a mushroom tumor mass; (v) Since previous cancer therapies have toxicity (except for alopecia and vitiligo) of ≡ctcae grade 3; (v) There is a second history of malignancy, (vi) evidence of serious or uncontrolled systemic disease, congestive heart failure, grade 2 of the New York Heart Association (NYHA), or Myocardial Infarction (MI) occurring within 6 months, (vii) severe non-healing wounds, active ulcers, or untreated fractures; (viii) Uncontrolled pleural effusion, pericardial effusion or ascites, requiring repeated drainage procedures; (ix) Has a history of severe allergic, anaphylactic or other hypersensitivity reactions to chimeric or humanized antibodies or fusion proteins; (x) Significant vascular disease (e.g., aortic aneurysm requiring surgical repair or recent arterial thrombosis) within 6 months after treatment, a history of pulmonary embolism, stroke or transient ischemic attacks within the first 3 months of treatment, and/or a history of abdominal fistulae or gastrointestinal perforation within 6 months of treatment; (xi) With active autoimmune disorders (type I diabetes, hypothyroidism requiring only hormone replacement, vitiligo, psoriasis or alopecia excluded); (xii) in need of systemic immunosuppressive therapy; (xii) Tumor-associated pain (> grade 3), unresponsive to extensive analgesic interventions (oral and/or patches); (xiii) Uncontrolled hypercalcemia despite the use of bisphosphonates; (xiv) receiving an organ transplant.

In some cases, the subject is a human patient having elevated levels of galectin-9 as compared to control levels. The level of galectin-9 may be a plasma or serum level of galectin-9 in a human patient. In other examples, the level of galectin-9 is a level of galectin-9 in a cancer cell within a tumor. In other examples, the level of galectin-9 is a level of galectin-9 of an immune cell within a tumor. In other examples, the level of galectin-9 may be a level of galectin-9 on a cell surface, e.g., a level of galectin-9 on a cancer cell. In one example, the level of galectin-9 may be that of galectin-9 expressed on cancer cells (e.g., on the surface of cancer cells) or galectin-9 expressed in immune cells, as measured in patient-Derived Organotypic Tumor Spheroids (PDOTs) that may be prepared, for example, by the methods disclosed in the examples below. Control levels may refer to the level of galectin-9 in a matched sample of a subject of the same species (e.g., human) without a solid tumor. In some examples, the control level represents a level of galectin-9 in a healthy subject. In some embodiments, the control level may be a baseline level prior to treatment.

To identify such subjects, a suitable biological sample may be obtained from a subject suspected of having a solid tumor, and the biological sample may be analyzed using conventional methods (e.g., ELISA or FACS) to determine the level of galectin-9 (e.g., free, cell surface expression, or total) contained therein. In some embodiments, for example, organoid cultures are prepared as described herein and used to assess galectin-9 levels in a subject. Single cells obtained from certain fractions obtained as part of the organoid preparation process are also useful for assessing galectin-9 levels in a subject. In some cases, the assay for measuring the level of galectin-9 in free form or expressed on the cell surface involves the use of antibodies that specifically bind to galectin-9 (e.g., specifically bind to human galectin-9). Any anti-galectin-9 antibody known in the art can be tested for suitability in any of the assays described above and then used in such assays in a conventional manner. In some embodiments, the antibodies described herein (e.g., G9.2-17 antibodies) can be used, such as in an assay. In some embodiments, antibodies are described in U.S. patent No. 10,344,091 and WO2019/084553, the respective relevant disclosures of each of which are incorporated by reference for the purposes and subject matter cited herein. In some examples, the anti-galectin-9 antibody is a Fab molecule. Assays for determining galectin-9 levels as disclosed herein are also within the scope of the present disclosure.

(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 inhibit galectin-9 (and/or Dectin-1 or TIM-3 or CD 206) by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) in vivo in an immunosuppressive immune cell in a tumor. In other embodiments, an antibody described herein (e.g., G9.2-17) is administered in an amount effective to reduce the level of activity of galectin-9 (and/or Dectin-1 or TIM-3 or CD 206) in immunosuppressive immune cells in a tumor by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) compared to the level in a pre-treatment or 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 by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) compared to the level in the pre-treatment or control subject.

Depending on the type of disease or the site of the disease to be treated, the pharmaceutical composition may be administered to the subject using conventional methods known to those of ordinary skill in the medical arts. In some embodiments, the anti-galectin-9 antibody may be administered to the subject by intravenous infusion.

The injectable composition may contain various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol and polyols (glycerol, propylene glycol, liquid polyethylene glycols, etc.). For intravenous infusion, the water-soluble antibody may be administered by instillation, thereby infusing a pharmaceutical formulation containing the antibody and physiologically acceptable excipients. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, ringer's solution, or other suitable excipients. Intramuscular formulations of antibodies (e.g., sterile formulations in the form of suitable soluble salts) can be dissolved in a pharmaceutical excipient (such as water for injection, 0.9% saline, or 5% dextrose solution) and administered.

An effective amount of a pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of treatment, either systemically or locally via a suitable route. In some embodiments, the anti-galectin-9 antibody is administered by intravenous administration (e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebroventricular, subcutaneous, intraarterial, intraarticular, intrasynovial, intrathecal, intratumoral, subcutaneous, oral, inhalation, or topical route). 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 intraperitoneally to a subject.

As used herein, "effective amount" refers to the amount of each active agent required to impart a therapeutic effect to a subject, either alone or in combination with one or more other active agents. In some embodiments, the therapeutic effect is a reduction in galectin-9 activity and/or amount/expression, a reduction in Dectin-1 signaling, a reduction in TIM-3 signaling, a reduction in CD206 signaling, or an increase in anti-tumor immune response in a tumor microenvironment. Non-limiting examples of increasing anti-tumor responses include increasing the level of activation of effector T cells or switching TAMs from the M2 phenotype to the M1 phenotype. In some cases, the anti-tumor response comprises increasing an ADCC response. It will be apparent to those skilled in the art that determining whether an amount of antibody achieves a therapeutic effect. As will be appreciated by those of skill in the art, the effective amount will vary depending upon the particular condition being treated, the severity of the condition, the individual patient parameters (including age, physical condition, body type, sex, and weight), the duration of the treatment, the nature of concurrent therapy (if any), the particular route of administration, and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with only routine experimentation. It is generally preferred to use the maximum dose of the individual components or combinations thereof, i.e. the highest safe dose according to sound medical judgment.

Empirical considerations (such as half-life) often help determine the dosage. For example, antibodies that are 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 to protect the antibody from attack by the host immune system. The frequency of administration may be determined and adjusted during the course of treatment and is generally, but not necessarily, based on the treatment and/or inhibition and/or amelioration and/or delay of the target disease/disorder. Alternatively, a sustained continuous release formulation of the antibody may be suitable. Various formulations and devices for achieving sustained release are known in the art.

In one example, the dosage of an antibody as described herein is empirically determined in an individual who has been administered one or more antibody administrations. The subject is administered increasing doses of the antagonist. To assess the efficacy of an antagonist, an index of disease/condition may be followed.

(D) Anti-galectin-9 antibody treatment

In some embodiments, the anti-galectin-9 antibodies described herein are used to treat a target cancer disclosed herein, i.e., not receiving additional anti-cancer therapy concurrent with the therapy with the anti-galectin-9 antibodies. In some cases, an anti-galectin-9 antibody such as G9.2-17 (IgG 4) disclosed herein can be used in monotherapy (i.e., the anti-galectin-9 antibody as the sole active agent). In other cases, anti-galectin-9 antibodies disclosed herein, such as G9.2-17 (IgG 4), for example, can be used in combination therapies (e.g., in combination with PD-1 inhibitors such as those disclosed herein).

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 or an effective amount of a pharmaceutical composition comprising an anti-galectin-9 antibody or antigen binding fragment thereof as described herein. Any of the anti-galectin-9 antibodies disclosed herein (e.g., antibodies G9.2-17 (IgG 4) (e.g., having the heavy chain of SEQ ID NO:19 and the light chain of SEQ ID NO: 15)) can be used in the methods disclosed herein.

In some embodiments, the antibody is administered, for example, once every two to six weeks via intravenous infusion. In some examples, the antibody may be administered every 2-4 weeks (e.g., every 2 weeks). In other examples, the antibody may be administered weekly. In some embodiments, an anti-galectin-9 antibody disclosed herein (e.g., G9.2-17 IgG4) is administered intravenously over an infusion period of 30 minutes to 6 hours. In some examples, intravenous infusion of 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 long infusion period (e.g., about 2-6 hours, e.g., about 2-4 hours or about 4-6 hours). In specific examples, exemplary anti-galectin-9 antibodies may be infused intravenously over a period of about 3 hours, about 4 hours, about 5 hours, or about 6 hours.

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

In some embodiments, the anti-galectin-9 antibody is administered once every 2 weeks. In some embodiments, the anti-galectin-9 antibody is administered once every 2 weeks for one period, once every 2 weeks for two periods, once every 2 weeks for 3 periods, once every 2 weeks for 4 periods, or once every 2 weeks for more than 4 periods. In some embodiments, the anti-galectin-9 antibody is administered once every 2 weeks for 4 cycles. In some embodiments, the anti-galectin-9 antibody is administered once every 4 or 6 weeks. In some embodiments, the duration of treatment is 12-24 months or longer. In some embodiments, the period extends for a duration of 3 months to 6 months or 6 months to 12 months or 12 months to 24 months or more. In some embodiments, the cycle length is modified, e.g., temporarily or permanently, to a longer duration, e.g., 3 weeks or 4 weeks. In some embodiments, the use further comprises administering to the subject an immune checkpoint inhibitor as described herein, e.g., an anti-PD-1 antibody, e.g., according to a regimen described herein. In some embodiments, the interval or period is one week. In some embodiments, the interval or period is 2 weeks. In a specific embodiment, the interval or period is 2 weeks. In a specific embodiment, the interval or period is 3 weeks. In a specific embodiment, the interval or period is 4 weeks.

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 one cycle every 2 weeks, two cycles every 2 weeks, three cycles every 2 weeks, four cycles every 2 weeks, or more than four cycles every 2 weeks. In some embodiments, the treatment is for 1 to 3 months once every 2 weeks, 3 to 6 months once every 2 weeks, 6 to 12 months once every 2 weeks, or 12 to 24 months or more once every 2 weeks. In some embodiments, the antibody is administered via intravenous infusion.

In some embodiments, the regimen or dosing schedule is one period every 3 weeks or 4 weeks, two periods every 3 weeks or 4 weeks, three periods every 3 weeks or 4 weeks, four periods every 3 weeks or 4 weeks, or more than four periods every 3 weeks or 4 weeks. In some embodiments, the treatment is for 1 to 3 months once every 3 weeks or 4 weeks, 3 to 6 months once every 3 weeks or 4 weeks, 6 to 12 months once every 3 weeks or 4 weeks, or 12 to 24 months or longer once every 3 weeks or 4 weeks. In some embodiments, the treatment is for 1 to 3 months once every 3 weeks or 4 weeks, 3 to 6 months once every 6 weeks, 6 to 12 months once every 3 weeks or 4 weeks, or 12 to 24 months or more once every 3 weeks or 4 weeks. In some embodiments, the treatment is for more than 24 months when clinically indicated. In some embodiments, the antibody is administered via intravenous infusion.

In other embodiments, the anti-galectin-9 antibody, such as G9.2-17 (IgG 4), may be administered to a human patient at an appropriate dose (e.g., a dose disclosed herein) once a week. For example, 2.0mg/kg of G9.2-17 (IgG 4) may be administered to a human patient once a week. For example, 6.3mg/kg of G9.2-17 (IgG 4) may be administered once a week to a human patient. In another example, 10mg/kg of G9.2-17 (IgG 4) may be administered to a human patient once a week. Alternatively, 12mg/kg of G9.2-17 (IgG 4) may be administered to a human patient once a week. In yet another example, 16mg/kg of G9.2-17 (IgG 4) may be administered once a week to a human patient.

In some cases, the anti-galectin-9 antibody may be administered to a 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 from 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 exceed 24 months.

In some cases, an anti-Gal-9 antibody disclosed herein, such as G9.2-17 (IgG 4), may be administered to a subject at a steady dose (e.g., about 650mg to about 1120 mg) once per week to once every 4 weeks. In some examples, the anti-Gal-9 antibody is administered to the subject at about 650mg to about 700mg once a week. In some examples, the anti-Gal-9 antibody is administered to the subject from about 650mg to about 700mg once every two weeks. In some examples, the anti-Gal-9 antibody is administered to the subject at about 1040mg to about 1120mg once a week. In some examples, the anti-Gal-9 antibody is administered to the subject from about 1040mg to about 1120mg once every two weeks.

In some embodiments, the dose is adjusted according to the patient's response to treatment. In some embodiments, the dose varies between treatment intervals. In some embodiments, treatment may be temporarily stopped. In some embodiments, treatment may be temporarily stopped. In some embodiments, anti-galectin-9 therapy is temporarily discontinued. In some embodiments, checkpoint inhibitor therapy used in combination with an anti-galectin-9 antibody is temporarily discontinued. In some implementations, both are temporarily stopped.

Alternatively, a human patient may begin with a low dose (e.g., 0.2mg/kg, 0.63mg/kg, or 2 mg/kg) of an anti-galectin-9 antibody disclosed herein, such as G9.2-17 (IgG 4). When no adverse effect is observed, the dosage of the antibody can be increased to, for example, 6.3mg/kg, 10mg/kg or 16mg/kg.

Whereas the tumorigenic effects of galectin-9 are mediated by interactions with immune cells (e.g., interactions with lymphoid cells via TIM-3, CD44 and 41BB, and interactions with macrophages via dectin-1 and CD 206), and whereas galectin-9 is expressed in a large number of tumors, the use of galectin-9 binding antibodies, for example, to target galectin-9 to inhibit interactions with its receptors, provides a treatment that can be applied to a number of different tumor types.

(E) Combination therapy

In some embodiments, any of the anti-galectin-9 antibodies described herein (e.g., G9.2-17 antibodies, such as G9.2-17 (IgG 4) as disclosed herein) can be used in any of the methods described herein in combination with a second therapeutic agent (e.g., a checkpoint inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody). Non-limiting examples of checkpoint inhibitors and administration regimens are provided elsewhere.

Thus, the methods of treatment disclosed herein can further comprise administering to the subject an inhibitor of a checkpoint molecule, such as PD-1. Examples of PD-1 inhibitors include anti-PD-1 antibodies, such as palbociclib, nivolumab, tirelib mab, rituximab, and cimetidine Li Shan antibodies. Such checkpoint inhibitors may be administered simultaneously or sequentially (in any order) with an 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 dewaruzumab, avistuzumab, and acter Li Zhushan antibodies. 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 the group consisting of CD40, GITR, LAG-3, OX40, TIGIT, and TIM-3.

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

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

In some embodiments, antibodies that bind PD-1 are used in conjunction with the anti-galectin-9 antibodies disclosed herein (e.g., G9.2-17 (IgG 4)). In some cases, a smooth dose may be used to administer the anti-PD-1 antibody. In some embodiments, the antibody that binds PD-1 is nivolumab, which can be administered to a subject at a dose of about 240mg every two weeks or about 480mg every 4 weeks. In some embodiments, the antibody that binds PD-1 is palbociclib, which may be administered at a dose of about 200mg once every 3 weeks. In some embodiments, the PD-1 binding antibody is a cimiput Li Shan antibody, which can be administered intravenously at a dose of about 350mg once every 3 weeks. In some embodiments, the antibody that binds PD-1 is tirelimumab, which can be administered intravenously at a dose of about 200mg once every 3 weeks or at a dose of about 400mg once every 6 weeks. In some embodiments, the antibody that binds PD-1 is rituximab, which may be administered intravenously at a dose of about 500mg every three weeks or intravenously at a dose of about 1000mg every six weeks.

In some embodiments, an antibody that binds PD-L1 (anti-PD-L1 antibody) is used in conjunction with an anti-galectin-9 antibody disclosed herein (e.g., G9.2-17 (IgG 4)). In some cases, a smooth dose of antibody that binds PD-L1 is used to administer. In some examples, the anti-PD-L1 antibody is atilizumab, which may be administered intravenously at a dose of 1200mg once every 3 weeks. In some examples, the anti-PD-L1 antibody is avermectin, which may be administered intravenously at a dose of 10mg/kg every 2 weeks. In some embodiments, the anti-PD-L1 antibody is a rivarox You Shan antibody, which can be administered intravenously at a dose of 1500mg every 4 weeks.

In specific examples, any of the methods disclosed herein comprise (i) administering 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) once every two weeks at a dose of about 0.2 to about 32mg/kg (e.g., about 3mg/kg or about 15 mg/kg) to a human patient having a target solid tumor as disclosed herein (e.g., pancreatic ductal adenocarcinoma (PDAC or PDAC), CRC, HCC, CCA, RCC, urothelial cancer, head and neck cancer, breast cancer, lung cancer, or other GI solid tumor); and (ii) administering an effective amount of an anti-PD-1 antibody (e.g., nal Wu Shankang, pamil mab, tirelimumab or cimapril Li Shan antibody, ritalimumab, divali You Shan antibody, avilamab or atelizumab) to the human patient.

In other specific examples, any of the methods disclosed herein comprise (i) administering 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) once per week at a dose of about 0.2 to about 32mg/kg (e.g., about 10mg/kg or about 16 mg/kg) to a human patient having a target solid tumor as disclosed herein (e.g., pancreatic ductal adenocarcinoma (PDAC or PDAC), CRC, HCC, CCA, RCC, urothelial cancer, head and neck cancer, breast cancer, lung cancer, or other GI solid tumor); and (ii) administering an effective amount of an anti-PD-1 or anti-PD-L1 antibody (e.g., nal Wu Shankang, pamil mab, tirelimumab or cimaprzumab Li Shan, rituximab, divali You Shan, avilamentamab or atelizumab) to the human patient.

Without being bound by theory, it is believed that the anti-galectin-9 antibodies may reprogram immune responses against tumor cells by inhibiting Dectin-1, for example, via inhibiting activity of γδ T cells infiltrating into the tumor microenvironment and/or by enhancing immune surveillance against tumor cells, for example, by activating cd4+ and/or cd8+ T cells. Thus, it is expected that the combined use of an anti-galectin-9 antibody and an immunomodulator (such as those described herein) will significantly enhance anti-tumor efficacy.

In some embodiments, methods are provided wherein an anti-galectin-9 antibody is administered concurrently 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 topically. In some embodiments, the checkpoint inhibitor is administered by intravenous administration (e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebroventricular, subcutaneous, intraarterial, intraarticular, intravesical, intrasynovial, intrathecal, intratumoral, or subcutaneous route). In one embodiment, the checkpoint inhibitor is administered to the subject by intravenous infusion.

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 (IgG 4), may be administered on the same day. In some examples, the checkpoint inhibitor may be administered to the subject prior to administration of the anti-galectin-9 antibody. In other cases, administration of the checkpoint inhibitor (e.g., anti-PD-1 antibody) and administration of the anti-galectin-9 antibody are performed two consecutive days. A checkpoint inhibitor (e.g., an anti-PD-1 antibody) may be administered to a subject on a first day of administration, and an anti-galectin-9 antibody may be administered to the subject on a subsequent day.

In other cases, a checkpoint inhibitor (such as any of the anti-PD-1 antibodies disclosed herein) may be administered about 1-7 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).

In some examples, an anti-galectin-9 antibody may be administered to a subject prior to administration of a checkpoint inhibitor (e.g., an anti-PD-1 antibody). In other cases, administration of the anti-galectin-9 antibody and administration of the checkpoint inhibitor (e.g., anti-PD-1 antibody) are performed for two consecutive days. An anti-galectin-9 antibody may be administered to a subject on a first day of administration, and a checkpoint inhibitor (e.g., an anti-PD-1 antibody) may be administered to the subject on a subsequent day.

In other cases, an anti-galectin-9 antibody (such as G9.2-17) disclosed herein may be administered about 1-7 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).

In any of the method embodiments described herein, the anti-galectin-9 antibody (alone or in combination with the anti-PD-1 antibody) may be administered once every 2 weeks for one cycle, once every 2 weeks for two cycles, once every 2 weeks for three cycles, once every 2 weeks for four cycles, or once every 2 weeks for more than four cycles. In some embodiments, the treatment is 1 to 3 months, 3 to 6 months, 6 to 12 months, 12 to 24 months, or longer. In some embodiments, the treatment is for 1 to 3 months once every 2 weeks, 3 to 6 months once every 2 weeks, 6 to 12 months once every 2 weeks, or 12 to 24 months or more once every 2 weeks.

Alternatively or additionally, anti-galectin-9 antibodies can be used in combination with a regimen comprising UGN-102, UGN-201, or UGN-302. In one embodiment, UGN-102, UGN-201, or UGN-302 is formulated in a hydrogel (e.g., a hydrogel based on inverse thermal gelation technology). In some examples, the anti-galectin-9 antibody can be administered prior to UGN-102, UGN-201, or UGN-302. In some examples, the anti-galectin-9 antibody can be administered concurrently with UGN-102, UGN-201, or UGN-302. In some examples, the anti-galectin-9 antibody can be administered subsequent to UGN-102, UGN-201, or UGN-302.

(F) Monitoring treatment response

Responses to treatment (e.g., treatment of solid tumors as described herein) may be assessed according to RECIST or RECIST 1.1 criteria and/or irRC, irRECIST, iRECIST, imRECISTPDAC, as described in example 1 and the following documents: 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 these documents are incorporated herein by reference in their entirety.

In some embodiments, methods are provided for improving and/or controlling overall response/tumor burden/tumor size (e.g., at about 2, 3, 6, or 12 months or at a later time) e.g., as compared to baseline levels obtained prior to the onset of a G9.2-17 IgG4 treatment regimen, comprising administering an anti-galectin-9 antibody described herein. In some embodiments, the method is used to improve and/or control overall response/tumor burden/tumor size at about 2 months. In some embodiments, when an 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 can improve or control the overall response/tumor burden/tumor size (e.g., at about 2, 3, 6, or 12 months or at a later time), e.g., compared to a baseline level obtained prior to the initiation of the treatment. In some embodiments, methods of producing a complete response, partial response, or stable disease (e.g., as measured at about 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated point in time) are provided, comprising administering an anti-galectin-9 antibody described herein. Such a response may be temporary or permanent for a period of time.

In some embodiments, the method increases the likelihood of complete response, partial response, or stable disease (e.g., as measured at about 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated point in time), e.g., as compared to baseline levels obtained prior to the onset of the G9.2-17 IgG4 treatment regimen. Such a response may be temporary or permanent for a period of time. In some embodiments, the treatment may result in a progressive disease that is reduced or attenuated, e.g., as compared to baseline levels obtained prior to the onset of the G9.2-17 IgG4 treatment regimen (e.g., as measured at about 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated point in time). This attenuation may be temporary or permanent. In any of these embodiments, an anti-galectin-9 antibody may be administered in combination with a checkpoint inhibitor (e.g., an anti-PD-1 antibody).

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

In any of the methods described herein, partial response, stable disease, complete response, partial response, stable disease, progressive disease, disease progression (e.g., as measured at about 2 months, 3 months, 6 months, or 12 months, or at a later time or at any other clinically indicated point in time) can be assessed according to irC criteria, RECIST1.1, irec or im RECIST criteria, or other criteria known in the art (see, e.g., borcom et al, annals of Oncology 30:385-396,2019' irc: hos et al, j. Immunother.30 (1): 1-15).

Partial response is a decrease in response to treatment of tumor size or in vivo cancer range (i.e., tumor burden) compared to baseline levels prior to initiation of treatment. For example, according to RECIST response criteria, the partial response is defined as a reduction in the sum of diameters of target lesions by at least 30% with reference to the baseline total diameter. Progressive disease is a growing, diffuse or worsening disease. For example, according to RECIST response criteria, progressive diseases include diseases in which a sum of target lesion diameters is observed to increase by at least 20% and the sum must also exhibit an absolute increase of at least 5 mm. Furthermore, the appearance of one or more new lesions is also considered to be progressive. Tumors that were neither reduced nor increased in extent or severity compared to baseline levels prior to initiation of treatment were considered stable diseases. For example, according to RECIST response criteria, with reference to the minimum overall diameter at study, stable disease occurs when neither sufficient shrinkage nor sufficient increase satisfies the conditions of partial response, nor the conditions of progressive disease.

In some embodiments, the present disclosure provides methods for reducing or maintaining tumor size in a subject (including a human subject) permanently or for a minimum period of time relative to a baseline tumor size prior to initiation of treatment in the subject (e.g., as measured at about 2 months, 3 months, 6 months, or 12 months, or at a later time or at any other clinically indicated point in time), 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 according to specific measurement protocols (e.g., as described in Eisenhower et al, referenced above) in combination with various software tools. Thus, in some embodiments, tumor size is measured in regularly scheduled re-staging scans (e.g., CT with/without contrast agent, MRI with/without contrast agent, PET-CT (diagnostic CT), and/or X-ray, ultrasound, and/or other related imaging modalities). In some embodiments, reducing tumor size, maintaining tumor size refers to the size of the target lesion. In some embodiments, tumor size reduction, maintenance of tumor size refers to the size of a non-target lesion. According to RECIST 1.1, when there is more than one measurable lesion at baseline, all lesions representing up to a total of five lesions for all affected organs (and up to two lesions per organ) should be identified as target lesions. All other lesions (or disease sites) including pathological lymph nodes should be identified as non-target lesions.

In some embodiments, the disclosure provides methods for increasing the likelihood of reducing or maintaining tumor burden (e.g., as measured at about 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated point in time), comprising administering to a subject a therapeutically effective amount of an anti-galectin-9 antibody as disclosed herein, alone or in combination with a checkpoint inhibitor (e.g., an anti-PD-1 antibody). In some embodiments, treatment may result in a greater likelihood of reducing tumor burden or maintaining tumor burden (e.g., as measured at about 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated point in time). As used herein, tumor burden refers to the number of cancers, the size or volume of the tumor in the subject, taking into account 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).

In some embodiments, the methods described herein increase the time to disease progression or progression free survival (e.g., as measured at about 2 months, 3 months, 6 months, or 12 months after initiation of treatment, or at a later time, or at any other clinically indicated point in time). The progression-free survival may be permanent or a progression-free survival over a period of time. In some embodiments, the method provides a greater likelihood of progression-free survival (a permanent progression-free survival or a progression-free survival over a period of time (e.g., 3, 6, or 12 months or measured, for example, at about 2 months, 3 months, 6 months, or 12 months after initiation of treatment, at a later time, or at any other clinically indicated point in time)). 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 due to any cause. In some embodiments, the methods achieve a longer survival or greater likelihood of survival, e.g., at a time, e.g., at 6 or 12 months.

The response to treatment (e.g., treatment of a solid tumor as described herein) can be assessed according to the iRECIST criteria, such as Seymour et al, iRECIST: guidelines for response criteria for use in trials; the Lancet, volume 18, month 3 2017 (The contents of which are incorporated herein by reference in their entirety). iRECIST was developed to use the modified RECIST1.1 standard, particularly in cancer immunotherapy trials, to ensure consistent design and data collection, and can be used as a guideline for standard methods of solid tumor measurement, as well as definition of objective changes in tumor size for use in trials using immunotherapy. iRECIST is based on RECIST 1.1. Responses assigned using irec have the prefix "i" (i.e., immunity), e.g., an "immune" complete response (iCR) or partial response (iPR) and an unproven progressive disease (iCPD) or an proven progressive disease (iCPD) or stable disease (iSD) to distinguish them from responses assigned using RECIST1.1, all of which are defined in Seymour et al RECIST 1.1. In some embodiments, the criteria may be compared to a baseline level prior to initiation of treatment. In any of these embodiments, an anti-galectin-9 antibody may be administered alone or in combination with a checkpoint inhibitor (e.g., an anti-PD-1 antibody, such as those disclosed herein).

Thus, in some embodiments, the present disclosure provides methods for Improving Overall Response (iOR) or achieving an "immune" complete response (iCR), partial response (iPR), or stable disease (iSD) as compared to a baseline level of disease prior to initiation of treatment (e.g., as measured at about 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated point in time). The decrease in the "immune" response (e.g., iCR, iPR, or iSD) may be temporary or permanent over a period of time. In some embodiments, the treatment may increase the likelihood of a complete response (iCR), partial response (iPR), or stable disease (iSD) (e.g., as measured at about 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated point in time). For example, in some embodiments, the present disclosure provides methods for attenuating disease progression or attenuating progressive disease (e.g., attenuating unproven progressive disease (ibpd) or attenuating proven progressive disease (iCPD)) (e.g., as measured at about 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated point in time) comprising administering to a subject a therapeutically effective amount of an anti-galectin-9 antibody as disclosed herein. Any of these above-mentioned irec criteria can be compared to baseline levels prior to initiation of treatment. In any of these methods, an anti-galectin-9 antibody may be administered alone or in combination with a checkpoint inhibitor (e.g., an anti-PD-1 antibody).

The decrease in iCPD or iCPD may be temporary or permanent for a period of time. In some embodiments, the treatment may result in a greater likelihood of overall reduction of the unproven progressive disease (ibpd) or the confirmed progressive disease (iCPD) (e.g., as measured at about 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated point in time). In some embodiments, the present disclosure provides methods for reducing the number of new lesions in a subject (e.g., as measured at about 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated point in time) according to the irec criteria, the methods comprising administering to the subject a therapeutically effective amount of an anti-galectin-9 antibody as disclosed herein. The reduction in the number of lesions may be relative to a baseline level prior to initiation of treatment, and the reduction may be temporary, or permanent, over a period of time. In any of these embodiments, an anti-galectin-9 antibody may be administered in combination with a checkpoint inhibitor (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody).

Additional criteria may be used to measure the therapeutic response. For example, tumor burden can be measured according to irRC criteria (Hoos et al, 2007). In irRC, the tumor burden is measured by combining an "index" lesion with a new lesion, i.e. the new lesion is considered as a change in tumor burden. In irRC, the immune-related complete response (irCR) is the disappearance of all lesions, measured or not, and no new lesions; immune-related partial response (irPR) is a 50% decrease in tumor burden relative to baseline as defined by irRC; and immune related progressive disease (irPD) is a 25% increase in tumor burden relative to the lowest level recorded. Everything else is considered to be immune-related stable disease (irSD).

Immune-related RECIST (irRECIST) is based on one-dimensional measurements of RECIST, and specific immune-related criteria are further redefined in irectist. Recently, a new standard, namely immune-modified RECIST (imRECIST), was evaluated based on the atilizumab data in NSCLC, requiring confirmation of disease progression at least 4 weeks after initial assessment (Hodi et al, JCO 2018;36 (9): 850-858). For a comparison of RECIST 1.1., irRC, irRECIST, iRECIST and imRECIST see, e.g., fig. 4 in borcom et al Annals of Oncology 30:385-396,2019; nishino et al, clin Cancer Res2013;19 (14) 3936-3943, the contents of which are incorporated herein by reference in their entirety. Any of these criteria are suitable for determining the response rate of any of the methods described herein.

The occurrence of an adverse effect (e.g., a severe adverse effect) in a subject treated with any of the anti-galectin-9 antibodies disclosed herein (e.g., G9.2-17) alone or in combination with a checkpoint inhibitor as disclosed herein (e.g., an anti-PD-1 or anti-PD-L1 antibody) can be monitored. Exemplary adverse effects to be monitored are provided in example 1 below. If the occurrence of adverse effects is observed, the treatment conditions of the subject may be altered. For example, the dosage of anti-galectin-9 antibody may be reduced and/or the dosing interval may be prolonged. The suitability and extent of the reduction can be assessed by a qualified clinician. In one embodiment, a 30% or 50% reduction level of the previous dose level is implemented. In one specific example, a reduction level of at least 30% (to dose level 1, the level at which the first dose was reduced) according to the clinician's assessment is performed. If desired, a further 30% dose reduction of dose level-1 (dose level-2, level at the second dose reduction) is performed. In another example, another dose reduction of 50% of dose level-1 (dose level-2) is performed. In some embodiments, administration of one or more doses reduces the previous dose level by about 10% to about 80%. In some embodiments, one or more doses are administered to reduce the previous dose level by 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%. In some embodiments, one or more dose reductions are performed by 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, or 70% to 80% of the previous dose level. In some embodiments, administration of one or more doses reduces the previous dose level by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%. In some embodiments, administration of one or more doses reduces the previous dose level by 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%. Alternatively, or in addition, the dose of the checkpoint inhibitor may be reduced and/or the dosing interval of the checkpoint inhibitor may be prolonged. In some cases (e.g., where life threatening adverse effects occur), the treatment may be terminated.

(G) Modulating immune responses

The response to treatment may also be characterized by one or more of the following: immunophenotype in blood and tumor, cytokine profile (serum), soluble galectin-9 level in blood (serum or plasma), galectin-9 tumor tissue expression level and expression pattern obtained by immunohistochemistry (tumor, stroma, immune cells), tumor Mutational Burden (TMB), PD-L1 expression (e.g. by immunohistochemistry), mismatch repair status or disease related tumor markers (e.g. as measured at about 3 months, 6 months or 12 months or at a later time or at any other clinically indicated time point). Examples of such tumor markers include, but are not limited to, CA15-3, CA-125, CEA, CA19-9, alpha-fetoprotein. These parameters can be compared to baseline levels prior to 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).

In any of the methods disclosed herein, the subject may be examined for one or more of the following characteristics prior to, during, and/or after treatment: (a) One or more tumor markers in a blood sample from a subject, optionally wherein the one or more tumor markers comprise CA15-3, CA-125, CEA, CA19-9, and/or alpha-fetoprotein, and any other tumor type specific tumor markers; (b) cytokine profile; and (c) galectin-9 serum/plasma levels, d) peripheral blood mononuclear cell immunophenotyping, e) tumor tissue biopsy/resection specimen multiplex immunophenotyping, f) tumor tissue biopsy/resection specimen galectin-9 expression levels and patterns, g) any other immune scoring test such as: PD-L1 immunohistochemistry, tumor Mutational Burden (TMB), tumor microsatellite instability status, and groups such as: in NanoString Gene expression system, 18-gene marker and PanCancer IO 360 ^TM Assay (NanoString Technologies) et al>HalioDx, immunoSeq-Adaptive Biotechnologies, TIS. Other suitable biomarkers specific for a target tumor, such as PDAC, may also be used. In one non-limiting example, PD-L1 (SP 263) (Roche, ventana) may be used to detect PD-L1 in cancer tissue using immunohistochemistry.

In some embodiments, described herein are methods for altering the levels of immune cells and immune cell markers (e.g., immune activation) in blood or in tumors comprising administering an anti-Gal-9 antibody alone or in combination with a checkpoint inhibitor (e.g., an anti-PD-1 antibody). Such changes may be measured in patient blood and tissue samples using methods known in the art, such as multiple flow cytometry and multiple immunohistochemistry. For example, a set of phenotypic and functional PBMC immunomarkers may be evaluated at baseline before the start of treatment and at different time points during treatment. Table 2 lists non-limiting examples of markers that can be used in these assessment methods. Flow Cytometry (FC) is a rapid and informative first-choice technique for analyzing cellular phenotypes and functions, and has been prominent in immunophenotyping. It allows the characterization of many cell subsets, including rare subsets, in complex mixtures (such as blood) and represents a rapid method of acquiring large amounts of data. The FC has the advantages of high speed, high sensitivity and strong specificity. Standardized antibody sets and procedures can be used to analyze and classify immune cell subtypes. Multiplex IHC is a powerful research tool that provides objective quantitative data describing tumor immune background in terms of immune subset number and location, and allows multiple markers to be assessed on a single tissue section. Computer algorithms can be used to quantify IHC-based biomarker content in whole slice images of patient biopsies, combining chromogenic IHC methods and staining with digital pathology methods.

Thus, in some embodiments, described herein are methods for modulating an immune response (e.g., modulating an immune activation marker, such as those in table 2) comprising administering an anti-gal 9 antibody alone or in combination with a checkpoint inhibitor therapy. In some embodiments, the adjusting comprises one or more of: (1) an increase in more CD8 cells in the plasma or tumor tissue, (2) a decrease in T regulatory cells (tregs) in the plasma or tumor tissue, (3) an increase in M1 macrophages in the plasma or tumor tissue, and (4) a decrease in MDSCs in the plasma or tumor tissue, and (5) a decrease in M2 macrophages in the plasma or tumor tissue (e.g., as measured at about 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated time point). In some embodiments, the marker assessed using the techniques described above or known in the art is selected from the group consisting of CD4, CD8, CD14, CD11b/c and CD25. These parameters can be compared to baseline levels prior to initiation of treatment.

TABLE 2 PBMC typing markers

In some embodiments, described herein are methods for modulating pro-inflammatory and anti-inflammatory cytokines comprising administering anti-gal 9 alone or in combination with checkpoint inhibitor therapy. In some embodiments, methods are provided for one or more of the following: (1) increasing the level of ifnγ in plasma or tumor tissue; (2) increasing the level of tnfα in plasma or tumor tissue; (3) Lowering the level of IL-10 in plasma or tumor tissue (e.g., as measured at about 3 months, 6 months, or 12 months, or at a later time or at any other clinically indicated point in time). These parameters can be compared to baseline levels prior to initiation of treatment.

In some embodiments, cytokine levels or immune cell levels can be assessed between 1 tumor biopsy prior to administration and repeated biopsies taken at a viable time. In some embodiments, cytokine levels or immune cell levels can be assessed between 2 duplicate biopsies. In some embodiments, methods are provided for modulating one or more of the soluble galectin-9 level in blood (serum or plasma) or galectin-9 tumor tissue expression level and expression pattern obtained by immunohistochemistry (tumor, stroma, immune cells) (e.g., as measured at about 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated time point). In some embodiments, the method reduces the level of soluble galectin-9 or galectin-9 tumor tissue expression level in blood (serum or plasma) or expression pattern obtained by immunohistochemistry (tumor, stroma, immune cells) (e.g., as measured at about 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated time point). Galectin-9 levels can be compared to baseline levels prior to initiation of treatment. In some embodiments, galectin-9 levels can be compared to untreated control groups or healthy subjects. In any of these embodiments, an 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, methods for modulating PD-L1 expression (e.g., as assessed by immunohistochemistry) are provided, 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 methods modulate (increase or decrease) one or more tumor markers associated with the disease (e.g., as measured at about 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated point in time). Examples of such tumor markers include, but are not limited to, CA15-3, CA-125, CEA, CA19-9, alpha-fetoprotein. These parameters can be compared to baseline levels prior to initiation of treatment. In any of these embodiments, an 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, the present disclosure provides methods 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 a change or modification, and includes up-and down-regulation. For example, "modulating an immune response" means changing or altering the state of one or more immune response parameters. Exemplary parameters of a T cell mediated immune response include the level of T cells (e.g., an increase or decrease in effector T cells) and the level of T cell activation (e.g., an increase or decrease in the production of certain cytokines). Exemplary parameters of a B cell mediated immune response include an increase in B cell levels, B cell activation, and B cell mediated antibody production.

When the immune response is modulated, some parameters of the immune response may decrease, while others may increase. For example, in some cases, modulating the immune response causes an increase (or up-regulation) in one or more immune response parameters and a decrease (or down-regulation) in one or more other immune response parameters, and the result is an overall increase in the immune response, e.g., an overall increase in the inflammatory immune response. In another example, modulating the immune response causes an increase (or up-regulation) in one or more immune response parameters and a decrease (or down-regulation) in one or more other immune response parameters, and the result is an overall decrease in the immune response, e.g., an overall decrease in inflammatory response. In some embodiments, the increase in the overall immune response, i.e., the increase in the overall inflammatory immune response, is determined by a decrease in tumor weight, tumor size, or tumor burden, or any RECIST or irec criteria described herein. In some embodiments, the increase in the overall immune response is determined by an increase in the level of one or more pro-inflammatory cytokines (e.g., including two or more, three or more, etc.) or a majority of pro-inflammatory cytokines (one or more, two or more, etc. or a majority of anti-inflammatory and/or immunosuppressive cytokines and/or one or more of the most potent anti-inflammatory or immunosuppressive cytokines decrease or remain constant). In some embodiments, the increase in the overall immune response is determined by an increased level of one or more of the most potent pro-inflammatory cytokines (one or more anti-inflammatory and/or immunosuppressive cytokines including one or more of the most potent cytokines decrease or remain unchanged). In some embodiments, the increase in the overall immune response is determined by a reduced level of one or more including a majority of immunosuppression and/or anti-inflammatory cytokines (the level of one or more or most of pro-inflammatory cytokines including, for example, the most potent pro-inflammatory cytokines increases or remains unchanged). In some embodiments, the increase in the overall immune response is determined by the increased level of one or more of the most potent anti-inflammatory and/or immunosuppressive cytokines (one or more or most pro-inflammatory cytokines including, for example, the most potent pro-inflammatory cytokines are increased or remain unchanged). In some embodiments, the increase in the overall immune response is determined by a combination of any of the above. Furthermore, an increase (or up-regulation) of one type of immune response parameter may result in a corresponding decrease (or down-regulation) of another type of immune response parameter. For example, an increase in the production of certain pro-inflammatory cytokines may result in the down-regulation of certain anti-inflammatory and/or immunosuppressive cytokines, and vice versa.

In some embodiments, the present disclosure provides methods for modulating an immune response in a subject (including a human subject) (e.g., as measured at about 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated point in time) comprising administering to the subject a therapeutically effective amount of an anti-galectin-9 antibody as disclosed herein. In some embodiments, the present disclosure provides methods for modulating the levels of immune cells and immune cell markers (including but not limited to those described herein in table 2) in the blood or in tumors of a subject (including a human subject), e.g., compared to baseline levels prior to the initiation of treatment (e.g., compared to baseline levels obtained prior to the initiation of an anti-Gal 9 antibody treatment regimen), comprising administering to the subject a therapeutically effective amount of an anti-galectin-9 antibody as disclosed herein. In some embodiments, the overall result of modulation is up-regulation of pro-inflammatory immune cells and/or down-regulation of immunosuppressive immune cells. In some embodiments, the present disclosure provides methods for modulating the level of immune cells, wherein the modulating comprises one or more of: (1) increasing CD8 cells in plasma or tumor tissue, (2) reducing Treg in plasma or tumor tissue, (3) increasing M1 macrophages in plasma or tumor tissue and (4) reducing MDSCs in plasma or tumor tissue, and (5) reducing M2 macrophages in plasma or tumor tissue, and wherein the method comprises administering to a subject a therapeutically effective amount of an anti-galectin-9 antibody as disclosed herein. In some embodiments, markers that assess the level 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 the levels of pro-inflammatory and immunosuppressive cytokines in the blood or in tumors of a subject (including a human subject) (e.g., as measured at about 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated point in time), e.g., compared to a baseline level prior to initiation of treatment, the methods comprising administering to the subject a therapeutically effective amount of an anti-galectin-9 antibody as disclosed herein. In some embodiments, the overall result of modulation is up-regulation of pro-inflammatory cytokines and/or down-regulation of immunosuppressive cytokines. In some embodiments, the present disclosure provides methods for modulating the level of a cytokine cell, wherein modulating encompasses one or more of the following: (1) increasing the level of ifnγ in plasma or tumor tissue; (2) increasing the level of tnfα in plasma or tumor tissue; (3) decrease the level of IL-10 in plasma or tumor tissue.

In some embodiments, the present disclosure provides methods for altering one or more of the soluble galectin-9 level or galectin-9 tumor tissue expression level in blood (serum or plasma) and expression pattern obtained by immunohistochemistry (tumor, matrix, immune cells) (e.g., as measured at 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated time point) comprising administering to a subject a therapeutically effective amount of an anti-galectin-9 antibody as disclosed herein. In some embodiments of the method, one or more of the soluble galectin-9 level or galectin-9 tumor tissue expression level in blood (serum or plasma) and the expression pattern obtained by immunohistochemistry (tumor, stroma, immune cells) remains unchanged. In some embodiments, the methods provided herein reduce one or more of the soluble galectin-9 level or galectin-9 tumor tissue expression level in blood (serum or plasma) and expression pattern obtained by immunohistochemistry (tumor, matrix, immune cells) (e.g., as measured at 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated time point). Galectin-9 levels can be compared to baseline levels prior to initiation of treatment. In some embodiments, galectin-9 levels may be compared to healthy subjects. In some embodiments, the treatment results in an alteration in PD-L1 expression (e.g., by immunohistochemistry). A dosage level of 16mg/kg or higher, a dosage level of 16mg/kg or higher.

In some embodiments, the disclosure provides methods for altering PD-L1 expression (e.g., as assessed by immunohistochemistry) (e.g., as measured at 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated point in time), comprising administering to a subject a therapeutically effective amount of an anti-galectin-9 antibody as disclosed herein. In some embodiments of the methods, PD-L1 expression (e.g., as assessed by immunohistochemistry) remains unchanged. PD-L1 levels can be compared to baseline levels prior to initiation of treatment. In some embodiments, the methods provided herein reduce PD-L1 expression (e.g., as assessed by immunohistochemistry). The PD-L1 level may be measured using conventional methods known in the art. In one non-limiting example, PD-L1 (SP 263) (Roche, ventana) may be used to detect PD-L1 in cancer tissue using immunohistochemistry.

In some embodiments, the present disclosure provides methods for altering (increasing or decreasing) one or more tumor markers associated with a disease (e.g., as measured at 2 weeks, 4 weeks, 1 month, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated point in time), comprising administering to a subject a therapeutically effective amount of an anti-galectin-9 antibody as disclosed herein. In some embodiments of the methods, one or more tumor markers associated with the disease (increase or decrease) 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 tumor markers can be compared to baseline levels prior to initiation of treatment. In some embodiments, the methods provided herein reduce the occurrence of one or more tumor markers associated with a disease.

In some embodiments, the present disclosure provides methods for altering (increasing or decreasing) one or more biomarkers associated with a disease (e.g., as measured at 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated point in time), the methods comprising administering to a subject a therapeutically effective amount of an anti-galectin-9 antibody as disclosed herein. Conventional methods may be used to measure the level of a biomarker in clinical tissue from a patient, such as multiple immunofluorescence (mIF) techniques, as described in the examples herein. One exemplary set of biomarkers may 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.

In any of these methods described herein for modulating an immune response, a cytokine, a biomarker such as galectin-9 or PD-L1 levels, or a tumor marker, any of the anti-galectin-9 antibodies described herein may be used. In some embodiments, the antibody comprises light chain complementarity determining region 1 (CDR 1) as shown in SEQ ID NO. 1, light chain complementarity determining region 2 (CDR 2) as shown in SEQ ID NO. 2, and light chain complementarity determining region 3 (CDR 3) as shown in SEQ ID NO. 3, and/or comprises heavy chain complementarity determining region 1 (CDR 1) as shown in SEQ ID NO. 4, heavy chain complementarity determining region 2 (CDR 2) as shown in SEQ ID NO. 5, and heavy chain complementarity determining region 3 (CDR 3) as shown 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.

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

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 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 solid tumor is a metastatic tumor.

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., as measured at about 1 month, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated point in time) comprising administering to the subject a therapeutically effective amount of an anti-galectin-9 antibody as disclosed herein. Improved quality of life and symptom control can be compared to a baseline prior to initiation of treatment. In some embodiments, improvement may be measured on an ECOG scale.

Kit for treating galectin-9-related diseases

The present disclosure also provides kits for treating or alleviating diseases associated with galectin-9 (e.g., galectin-9 associated with a cell surface glycoprotein (e.g., dectin-1, TIM3, CD206, etc.) or pathological cells expressing galectin-9 (e.g., cancer cells). Examples include solid tumors, such as PDAC, CRC, HCC, cholangiocarcinomas, and other GI solid tumors, as well as other solid tumors described herein. Such kits can include one or more containers comprising an anti-galectin-9 antibody (e.g., any of those described herein), and a second therapeutic agent (e.g., a checkpoint inhibitor, such as an anti-PD-1 antibody as disclosed herein) also as described herein, optionally to be used with the anti-galectin-9 antibody.

In some embodiments, the kit may include instructions for use according to any of the methods described herein. Included instructions may include descriptions of administration of an anti-galectin-9 antibody and optionally a second therapeutic agent to treat, delay onset, or reduce a target disease such as those described herein. In some embodiments, the kit further comprises a description of selecting an individual suitable for treatment based on identifying whether the individual has the target disease (e.g., applying a diagnostic method as described herein). In other embodiments, the instructions comprise a description of administering the antibody to an individual at risk of developing the target disease.

Instructions relating to the use of anti-galectin-9 antibodies typically include information about the dosage, dosing regimen, and route of administration of the intended treatment. The container may be a unit dose, a bulk package (e.g., a multi-dose package), or a subunit dose. The instructions provided in the kits of the invention are typically written instructions on a label or package insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disc) are also acceptable.

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

The kits of the invention are in a suitable package. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packages (e.g., sealed mylar or plastic bags), and the like. Packages for use in combination with specific devices, such as inhalers, nasal administration devices (e.g. nebulizers) or infusion devices (such as micropumps), are also envisaged. In some embodiments, the kit has a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). In some embodiments, the container also has a sterile access port (e.g., the container is an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-galectin-9 antibody as described herein.

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

General technique

The practice of the present invention employs, 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 well explained in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al, 1989) Cold Spring Harbor Press; oligonucleotide Synthesis (m.j. Gait edit, 1984); methods in Molecular Biology, humana Press; cell Biology A Laboratory Notebook (J.E.Cellis editions, 1998) Academic Press; animal Cell Culture (r.i. freshney edit, 1987); introduction to Cell and Tissue Culture (J.P.Mather and P.E.Roberts, 1998) Plenum Press; cell and Tissue Culture: laboratory Procedures (A.Doyle, J.B.Griffiths and D.G.Newell et al, 1993-8) J.Wiley and Sons; methods in Enzymology (Academic Press, inc.); handbook of Experimental Immunology (d.m. weir and c.c. blackwell editions); gene Transfer Vectors for Mammalian Cells (J.M.Miller and M.P.Calos. Editions, 1987); current Protocols in Molecular Biology (F.M. Ausubel et al, 1987); PCR: the Polymerase Chain Reaction (Mullis et al, 1994); current Protocols in Immunology (J.E. Coligan et al, editions, 1991); short Protocols in Molecular Biology (Wiley and Sons, 1999); immunobiology (c.a. janeway and p.convers, 1997); antibodies (P.Finch, 1997); antibodies a practical approach (D.Catty. Eds., IRL Press, 1988-1989); monoclonal antibodies: a practical approach (P.shepherd and C.dean editions, oxford University Press, 2000); using anti-ibodies a laboratory manual (E.Harlow and D.Lane (Cold Spring Harbor Laboratory Press, 1999); the Antibodies (M.Zanetti and J.D.Capra editions Harwood Academic Publishers, 1995).

Without further elaboration, it is believed that one skilled in the art can, based on the preceding description, utilize the present invention to its fullest extent. Accordingly, the following specific embodiments are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. For the purposes or subject matter recited herein, all publications recited herein are incorporated by reference.

Examples

Example 1: 1/2-phase open-label, multicenter study of safety, pharmacokinetics and anti-tumor Activity of anti-galectin-9 monoclonal antibodies alone and in combination with anti-PD-1 antibodies in patients with metastatic solid tumors

Galectin-9 is a molecule that is overexpressed by many solid tumors, including those in pancreatic, colorectal, and hepatocellular carcinoma. Furthermore, galectin-9 is expressed on tumor-associated macrophages as well as on intratumoral immunosuppressive γδ T cells, thereby acting as an effective mediator of cancer-associated immunosuppression. As described herein, monoclonal antibodies (e.g., G9.2-17, igg 4) have been developed that target galectin-9. The data indicate that G9.2-17 stagnates pancreatic tumor growth by 50% in the in situ KPC model and prolongs survival of KPC animals by more than one time. Without wishing to be bound by theory, it is believed that anti-galectin-9 antibodies reverse the M2 phenotype Transition to M1 phenotype, thereby promoting intratumoral CD8 ⁺ T cell activation. In addition, antibodies G9.2-17 (IgG 4) (having the heavy chain of SEQ ID NO:19 and the light chain of SEQ ID NO: 15) have been found to act synergistically with anti-PD-1.

G9.2-17 (IgG 4) is a fully human IgG4 monoclonal antibody (mAb) targeting the galectin-9 (gal-9) protein. Gal-9 acts as an immunosuppressant, conferring tumor cell immune privileges and disabling immune-mediated cancer attack by modulating the sensitivity of macrophages, T cells, myeloid-derived suppressor cells, and cancer cells to cytotoxic T cell-induced death. Based on available data, blocking gal-9 by G9.2-17 (IgG 4) would interfere with the immunosuppressive function of gal-9, resulting in effective immune activation and tumor growth inhibition in multiple preclinical models.

Gal-9 can be overexpressed and/or secreted in many solid tumor types, including pancreatic adenocarcinoma, cholangiocarcinoma (CCA), colorectal cancer (CRC), breast cancer, bladder cancer, ovarian cancer, non-small and small cell lung cancer, nasopharyngeal cancer, malignant melanoma, ovarian cancer, etc., and high levels of tissue and/or circulating Gal-9 are associated with invasive tumor characteristics and poor survival outcomes.

Thus, the target indication for G9.2-17 (IgG 4) is recurrent or refractory metastatic solid tumor, where G9.2-17 (IgG 4) can be studied both as a single agent and in combination with a checkpoint inhibitor (e.g., a programmed cell death 1[ pd 1] antibody, such as na Wu Shankang, palbociclizumab, cimiput Li Shan antibody, rituximab, or tirelimumab).

Dose escalation (part 1) was performed in all solid tumor types in order to establish the safety and tolerability profile of G9.2-17 (IgG 4), evaluate their immunogenic potential, establish Pharmacokinetic (PK) and Pharmacodynamic (PD) profiles, and reach recommended phase 2 doses (RP 2D). This may be the Maximum Tolerated Dose (MTD). The extension group is calculated (part 2) in the following terms: first line metastatic Pancreatic Ductal Adenocarcinoma (PDAC), as well as CRC and CCA, can be combined with both single agents and anti-PD 1 antibodies.

No other therapies targeting gal-9 are currently approved or in clinical trials for any indication.

In the non-clinical studies conducted so far, no significant toxicity was observed at doses about 500 times higher than those expected for human administration. Furthermore, G9.2-17 (IgG 4) has been shown to be highly specific for gal-9 and has been shown to be effective in a variety of animal models of cancer. The target patient population into the group was at advanced disease and failed in standard-of-care treatment prior to the study into the group. G9.2-17 (IgG 4), alone or in combination with a checkpoint inhibitor such as an anti-PD-1 antibody (e.g., nano Wu Shankang, palbociclizumab, tirelimumab, multi-tarolimumab, or cimetidine Li Shan antibody), is expected to be beneficial in the treatment of malignant tumors, such as malignant solid tumors.

Target and endpoint

Part 1: dose escalation

Part 2: group extension

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Study design

This is an open-label, non-control, multicenter phase 1/2 study with up-dosing phase (part 1) and group expansion phase (part 2) in patients with recurrent/refractory metastatic solid tumors. This study was conducted in up to 20 study centers in the united states. The duration of the study was estimated to be 12-24 months. Survival follow-up lasted for up to 2 years. The study protocol is presented in figure 1.

The study included monotherapy with G9.2-17 (IgG 4) and a combination of G9.2-17 and an anti-PD-1 antibody such as nivolumab. The dosage of G9.2-17 may be in the range of about 3mg/kg to 15mg/kg once every two weeks. In alternative embodiments, the dosage of G9.2-17 may be in the range of about 0.2mg/kg to 16mg/kg or higher dosage levels once every two weeks. Antibodies were administered by intravenous infusion.

Duration of treatment and treatment period

Duration of treatment

Study drug administration was continued until disease progression, unacceptable toxicity occurred, or study was withdrawn. Patients who discontinued study medication prior to disease progression and were not treated with other systemic anti-cancer therapies will receive study follow-up until disease progression.

Treatment period

The study consisted of the following phases in part 1 and part 2:

screening period: up to 4 weeks (day-28 to day-1) before the first dose

Treatment period: the 28 day treatment cycle treatment later period as presented in the evaluation schedule (SoA; tables 5-6 below): 30 days after the last treatment (treatment end visit/early termination visit)

IMAR follow-up period: 90 days after the last treatment (G9.2-17 (IgG 4) +anti-PD-1 antibody group)

Follow-up period: patients who have discontinued treatment for reasons other than disease progression and have not received additional systemic anti-cancer treatment are subjected to long-term follow-up for up to 2 years (visit once every 3 months)

Part 1: dose escalation stage

Dose discovery studies were performed using the Continuous Reassessment Method (CRM) (O' Quigley et al, 1990) to establish DLT and RP2D. Two to six patients per treatment group 1-6 were assigned G9.2-17 (IGG 4) to receive higher IV injections every two 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 group receive the corresponding study dose for that group. They received study medication at one of 8 dose levels until disease progressed, unacceptable toxicity occurred, or the study was exited for other reasons. Patients who have been withdrawn for reasons other than toxicity or tolerability problems during the first treatment cycle are replaced only.

For groups 1-6, two patients were dosed at a time according to the CRM design. Dose escalation is based on analysis of patient safety data, focusing on the occurrence of DLT at previous dose levels and other relevant safety and dosing data in previous cohorts. The dose escalation may occur after at least 28 days (1 cycle). Skipping of dose levels is not allowed.

After group 6 was completed according to the CRM design, weekly (QW) G9.2-17 (IGG 4) dosing regimens were evaluated, provided RP2D had not been reached in the CRM design. Groups 7 and 8 were not evaluated with the CRM design. Once DLT was not identified, only patients were allowed to enter group 7.

For groups 7 and 8, four patients were dosed once per group. Four patients at each dose level in groups 7 and 8 were assigned G9.2-17 (IGG 4) that received higher IV injections weekly (QW) on days 1, 8, 15, and 22 of each 28 day cycle. Starting from the first four patients in group 7, dose escalation to the next group only occurs without identification of DLT. If a single DLT is recorded in group 7, no other patients are dosed within the group and group 8 is not activated.

Part 1, groups 1-8, were included in approximately 36 patients. A total of 6 dose levels were evaluated in the CRM design:

Dose escalation cohort 1=0.2 mg/kg Q2W

Dose escalation cohort 2=0.63 mg/kg Q2W

Dose escalation cohort 3=2 mg/kg Q2W

Dose escalation cohort 4=6.3 mg/kg Q2W

Dose escalation cohort 5=10 mg/kg Q2W

Dose escalation cohort 6=16 mg/kg Q2W

Consider RP2D, which includes another 2 dose levels:

dose escalation cohort 7=10 mg/kg QW

Dose escalation cohort 8=16 mg/kg QW

Patients treated in early groups prior to identification of RP2D were allowed to dose escalation up to the highest dose level cleared. After a complete cycle, dose escalation may occur after at least 28 days (1 cycle). The dose escalation may not occur in the middle of the cycle. Patients may continue to dose escalation to the highest approved dose level until they discontinue due to toxicity or disease progression or for other reasons (e.g., patient selection discontinues the study).

The dose escalation is based on DLT production in patients treated at previous dose levels. For each dose group, a priori DLT probabilities were specified based on GLP-compliant toxicity studies and preclinical models. For a prescribed target DLT rate and total number of dose levels, a skeleton of the power model d ∈p (a) is generated according to the methods of Lee and Cheung using a priori MTD at the median dose level adjusted by PK/PD data and a spacing metric of δ=0.05 (Lee and Cheung, 2011). The prior distribution of parameter "a" has a normal distribution with zero mean, with minimal prior variance information. If the lower limit of the Agrest and Coull two Confidence Intervals (CI) for the lowest study dose level exceeds the target DLT rate, the trial is stopped for safety considerations (Agrest and Coull, 1998). RP2D is the MTD dose derived from part 1.

If DLT occurs in any patient during the first 28 days of treatment, administration of the study drug to that patient is permanently discontinued.

For patients experiencing toxicity outside the DLT window, including IMAR, dose reduction is only allowed if clinical benefit is expected and clinical benefit can continue to be obtained with lower doses of G9.2-17 (IgG 4). The dose of G9.2-17 (IgG 4) was initially reduced by 50%, and possibly further reduced by 50%, as defined by the dose modification guidelines provided in table 3. No further dose reduction is allowed.

Table 3 recommended dose modification (AE outside DLT Window and outside IMAR) for G9.2-17 (IgG 4)

Part 1 completion

Part 1 is complete when up to six patients have received doses identified as RP 2D. RP2D is based in part on continuous re-assessment method (CRM) study design, PK and PD data parameters, additional safety and efficacy data, and any other factors to be considered.

Backfill group

The purpose of the backfill group was to assess the safety, tolerability and biological effects of G9.2-17 (IGG 4) in patients with tumors positive for gal-9. The gal-9 state of the RP2D group is determined retrospectively. If fewer than 6 gal-9 positive tumor patients were treated with RP2D, patients designated for backfill groups required prospective assessment of gal-9 tumor status by IHC. Additional patients with up to 6 additional tumors positive for gal-9 may be enrolled in the backfill group at RP2D dose levels.

Part 2: group extension phase

The second part of the protocol was designed with a Simon two-stage optimization and included approximately 223 patients. Groups of extended PDACs, CRCs, and CCA and/or potentially other solid tumor types are planned, based on tumor-specific considerations implemented for the extended group and clinical trial endpoints. The rationale behind this approach is to ensure the feasibility of recruitment, as well as capture the clinical needs of a particular indication.

CRC and CCA patients received one of two treatments (4 treatment groups total):

LYT 200 as a single agent

G9.2-17 (IGG 4) +anti-PD-1 antibodies as combination therapy.

anti-PD-1 antibodies should be administered before G9.2-17 (IgG 4). If the same day of administration cannot be completed for any reason, the anti-PD-1 antibody should be administered on the first day and G9.2-17 (IgG 4) on the second day.

In some cases, the present study may investigate the use of anti-galectin-9 antibody G9.2-17 (IgG 4) alone (single agent group of the present study) or in combination with nivolumab (e.g., administered once every two weeks at a steady dose of 240 mg).

CRC and CCA patients

Treatment of a single agent group or a combined agent group of CRC and CCA patients may be performed in parallel.

G9.2-17 (IgG 4) monotherapy

The initial dose of G9.2-17 (IgG 4) in monotherapy was RP2D identified in section 1. For the CRC and CCA monotherapy groups, an optimized two-stage design (stages I and II) was used to examine the null hypothesis for ORR 3.ltoreq.5% with the alternative hypothesis for ORR 3.gtoreq.15% within the single agent group.

After testing 23 patients for study drug in stage I, the trial group was terminated if 1 patient responded. If the trial proceeds to phase II of Simon optimization design, approximately 33 patients are additionally treated in each of the single agent groups. If the total number of response patients is less than or equal to 5, the study medication in the group is rejected. If ≡6 patients have confirmed ORR3, part 3 extension cohort of the group is activated and described in protocol revision.

Dose reduction is only allowed if clinical benefit is expected and clinical benefit can continue to be expected with lower doses of G9.2-17 (IgG 4). The dose of G9.2-17 (IgG 4) was initially reduced by 50%, and possibly further reduced by 50%, as defined by the dose modification guidelines provided in this protocol. No further dose reduction is allowed.

G9.2-17 (IgG 4) +anti-PD-1 antibody combination therapy

The dose of G9.2-17 (IGG 4) in combination therapy with an anti-PD-1 antibody (e.g., nivolumab or palbociclizumab) is RP2D-1, which is the dose immediately preceding the RP2D dose identified in section 1. An optimized two-stage design may also be used to verify zero hypotheses of 10% or less for ORR3 and alternative hypotheses of 25% or more for ORR 3.

To ensure patient safety, a safe break-in was performed, with the first 8 patients dosed. Only when less than or equal to 2 patients develop DLT (below 25% Target Toxicity Level (TTL)) will the group continue into the group. If 3 or more patients develop DLT, the combination group is terminated for the type of cancer being treated. In this combination treatment abrasion group, only patients who were withdrawn for reasons other than toxicity or tolerability problems during the first treatment cycle were replaced. If DLT occurs in any of the 8 safely-running-in patients during the first 28 days of treatment, administration of the study drug to that patient is permanently discontinued.

For patients experiencing toxicity outside the DLT window, dose reduction is allowed when clinical benefit is expected and clinical benefit can continue to be obtained with lower doses of G9.2-17 (IgG 4). The dose of G9.2-17 (IgG 4) was initially reduced by 50%, and possibly further reduced by 50%, as defined by the dose modification guidelines provided in this protocol. No further dose reduction is allowed. Dose modification of anti-PD-1 antibodies was allowed.

If IMAR onset/recurrence cannot be managed by reducing the dose of either agent, both study medications must be discontinued.

After the 18 patients were tested in combination in the first stage, if 2 patients were responding, the corresponding test group was terminated. If the trial continued to phase II of Simon optimization design, approximately 25 additional patients were treated in each of the combination groups. If the total number of responding patients is less than or equal to 7, combinations within the group are rejected. If ≡8 patients have confirmed ORR 3, the expanded cohort of the group is activated and described in protocol revision.

PDAC patient

Part 2 cohort of metastatic PDAC patients required combination therapy of G9.2-17 (IgG 4) in a linear metastatic setting.

The dose of G9.2-17 (IGG 4) is the RP2D-1 dose, which is the dose level immediately preceding the RP2D dose identified in section 1. To ensure patient safety, a safe break-in was performed, wherein the first 8 patients were dosed and the group was continued only if less than or equal to 2 patients developed DLT (below 25% Target Toxicity Level (TTL)). If 3 or more patients develop DLT, the combination treatment group is terminated. In this combination treatment abrasion group, only patients who were withdrawn for reasons other than toxicity or tolerability problems during the first treatment cycle were replaced. If DLT occurs in any of the 8 safely-running-in patients during the first 28 days of treatment, administration of the study drug to that patient is permanently discontinued.

For patients experiencing toxicity outside the DLT window, dose reduction is allowed when clinical benefit is expected and clinical benefit can continue to be obtained with lower doses of G9.2-17 (IgG 4). The dose of G9.2-17 (IgG 4) was initially reduced by 50% and possibly by a further 50%. No further dose reduction is allowed.

The primary efficacy endpoint was patient PFS6.

Part 2 completion

The completion of part 2 depends on the patient ORR 3 of the CRC and CCA patients and the PFS6 of the PDAC.

Part 3: expansion of

If a promising efficacy signal attributable to a tumor type is identified in one or more of the test groups, an expanded group is initiated to confirm the findings as described above. The sample size for each extension group is determined based on the point estimate determined in section 2 and a predetermined level of accuracy of 95% CI around ORR/OS and PFS. Prior to initiating section 3, a protocol revision is submitted including detailed information about the expanded population, treatment protocol, and statistical analysis plan.

Dose limiting toxicity criteria

Dose limiting toxicity assessed in this trial was defined as clinically significant hematological and/or non-hematological AE or abnormal laboratory values that were assessed as not associated with metastatic tumor disease progression, concomitant disease or concomitant medication, and possibly associated with or correlated with study drug, and occurred during the first cycle of the study (28 days). The administration of study drug to any patient experiencing DLT in part 1 or part 2 during the first 28 days of treatment was permanently discontinued.

DLT is toxicity meeting any of the following criteria:

any death not explicitly due to underlying disease or external cause

Indication of potential drug-induced liver injury (hyrule cases) as follows:

upper Limit of Normal (ULN) for ALT or AST >3x, confirmed by repeated testing after 24 hours, and

total Bilirubin (TBL) >2x uln after 24 hours was confirmed by repeated testing

No other explanation is given for elevated o TBL and/or AT, such as viral hepatitis (type a, type b or type c), alcoholic or autoimmune hepatitis, existing or acute liver disease, gallbladder obstruction or biliary tract disease, gilbert syndrome, disease progression or other medications that can cause the observed effects.

All grade 4 non-hematologic and hematologic toxicities of any duration

All grade 3 non-hematologic and hematologic toxicities. The exceptions are as follows:

grade 3 nausea, vomiting and diarrhea, no hospitalization or total parenteral nutrition support is required, and can be managed to grade 2 or less within 48 hours by supportive care.

The grade 3 electrolyte abnormality was corrected to grade 2 or less within 24 hours.

Grade 3 electrolyte abnormalities, lasting <24-72 hours, were clinically uncomplicated and spontaneously regressed or responded to routine medical intervention.

The class 3 amylase or lipase is not less than the above, and is irrelevant to symptoms or clinical manifestations of pancreatitis.

End of study definition

The end of the study in part 1 of the study was defined as the point in time at which RP2D had been identified and all patients had been treated with G9.2-17 (IGG 4) until disease progression was confirmed.

The end of study part 2 was defined for each of the three tumor types after completion of Simon two-stage optimization design, and all patients in the group had been treated with G9.2-17 (IGG 4) (alone or in combination) until disease progression was confirmed.

In parts 1 and 2, if patients discontinue treatment for reasons other than disease progression and they do not receive additional systemic anti-cancer therapy, they will receive up to 2 years of OS follow-up after the last dose of G9.2-17 (IgG 4).

The end of the study is defined as the date of the last visit by the last patient.

Test stopping rule

Part 1

If the lower limit of the two items of CI, agrest and Coull, at the lowest study dose level exceeds the target DLT rate, the trial is stopped for safety reasons (Agrest and Coull, 1998).

Part 2

After testing study drugs for 23 patients in phase I of Simon optimization design for CRC and CCA monotherapy groups, if 1 or less patients respond, the corresponding test group is stopped. If the trial proceeds to stage II of Simon's optimization design, the trial group is stopped if the total number of responsive patients in the group is less than or equal to 5.

Similarly, simon optimization also guided the test to stop for the G9.2-17 (IgG 4) +anti-PD-1 antibody combination in CRC and CCA. After testing the combination for 18 patients in phase I, if 2 patients were responding, the corresponding test group was stopped. If the trial proceeds to stage II, the trial group is stopped if the total number of responsive patients in the group is less than or equal to 7.

To ensure patient safety for both combination treatment groups, a safety break-in was performed, with 8 patients in front. For each cancer type (e.g., CCA, CRC, and/or PDAC), the group was continued only if less than or equal to 2 patients developed DLT (below 25% Target Toxicity Level (TTL)). If 3 or more patients with a given cancer type develop DLT in the combination treatment group, the cancer type is terminated into the group.

Study population

Inclusion criteria

Participants were eligible for inclusion in the study only if all of the following criteria were applicable:

part 1 and part 2

1. Written informed consent (patients with normal spirit, able to understand and willing to sign informed consent)

2. Male or non-pregnant female with age equal to or greater than 18 years old

3. Histologically confirmed unresectable metastatic cancers (allowing for adenocarcinoma and squamous cell carcinoma). Patients with resectable disease are excluded.

4. Can follow the research scheme

5. Life expectancy >3 months

6. Eastern tumor cooperative group (ECOG) expression status 0-1

7. Coronavirus SARS-CoV-2 (COVID-19) negative patient

8. Patients can and are willing to receive pre-treatment and mid-treatment/post-treatment biopsies. Planned biopsies should not expose the patient to a significantly increased risk of complications. All efforts are made to biopsy the same lesion in repeated biopsies.

9. Disease measurable according to the solid tumor Response Evaluation Criteria (RECIST) v 1.1. Note that lesions intended for biopsy should not be target lesions.

10. Sufficient hematology and end organ function is defined by the following laboratory results obtained prior to the first dose of study drug treatment:

a. neutrophil count is greater than or equal to 1x10 ⁹ /L

b. Platelet count is greater than or equal to 100x10 ⁹ L; for hepatocellular carcinoma (HCC) in part 1, > 50X10 ⁹ /L

c. Hemoglobin not transfused in the previous week is more than or equal to 9.0g/dL

d. Creatinine is less than or equal to 1.5x Upper Limit of Normal (ULN)

e. Aspartate aminotransferase AST (SGOT). Ltoreq.3XULN (5 XULN when HCC or liver metastasis is present)

f. Alanine aminotransferase (ALT [ SGPT ]) 3x ULN (5 x ULN when HCC or liver metastasis is present)

g. Bilirubin is less than or equal to 1.5 ULN (patients known to have Gilbert's disease may have bilirubin less than or equal to 3.0 ULN)

h. Albumin not less than 3.0g/dL

i. International Normalized Ratio (INR) and Partial Thromboplastin Time (PTT) less than or equal to 1.5 XULN

j. Amylase and lipase are less than or equal to 1.5 XULN

11. There was no evidence of active infection or infection requiring parenteral antibiotics, and no severe infection within 4 weeks prior to study initiation

12. Women with fertility must be negative for pregnancy tests within 72 hours before treatment begins. For women with fertility: consent was given to maintain abstinence (avoiding idiosyncratic exchange) or to use contraceptive methods with a annual failure rate of <1% during the treatment period and for at least 180 days after the last study treatment.

o if the woman is post-infancy, does not reach postmenopausal status (continuous amenorrhea ≡12 months and no clear cause other than menopause) and does not receive surgical sterilization (ovariectomy and/or uterus), the woman has fertility.

Examples of contraceptive methods with a failure rate of <1% per year include bilateral tubal ligation, male sterilization, hormonal contraceptives to inhibit ovulation, hormonal-releasing intrauterine devices and intrauterine copper devices. The reliability of sexual abstinence should be assessed according to the duration of the clinical trial, the patient's preference and the usual lifestyle. Periodic abstinence (e.g., calendar, ovulation, symptomic-thermal) or post-ovulation methods) and in vitro ejaculation (withdrawal) are unacceptable contraceptive methods. Fertility men must employ effective contraceptive methods during the study unless there is a record of infertility.

13. From the last dose of anti-cancer therapy prior to the first administration of G9.2-17 (IgG 4), there is four (4) weeks or 5 half-lives (whichever is shorter)

14. Allowing continued use of bisphosphonate treatment (e.g., zoledronic acid) or Deshumab to treat bone metastases that have been stabilized for at least 6 months prior to C1D1

15. Allowing biliary or gastric outlet obstruction provided that effective drainage is achieved by endoscopic, surgical or interventional means

16. Allowing pancreatic, biliary or intestinal fistulae, provided that they are controlled with appropriate uninfected and unobstructed drainage tubes (if any are in place, it is necessary to confirm patency before the study begins)

In addition, for part 1 only:

17. patient:

a. has received at least one prior line number of systemic therapies for treating metastatic disease, or

b. With tumor types for which no standard care selection is available.

In addition, for part 2 only:

pdac extension group: 1-line metastatic patients who were first receiving a gemcitabine-containing regimen or who were previously untreated with a gemcitabine-containing regimen in a neoadjuvant or adjuvant/locally advanced setting for at least 3 months

CRC and CCA extended group-patients who have received at least one previous line number of therapy in a metastatic environment

Exclusion criteria

Participants were excluded from the study if any of the following criteria were applicable:

1. patients who are reluctant or unable to follow the regimen

2. Patients diagnosed with primary unknown metastatic cancer

3. Previously or now illicit drug addiction (medical and recreational cannabis/Cannabidiol (CBD)/Tetrahydrocannabinol (THC) is not considered "illicit")

4. Clinically significant, uncontrolled bleeding, and any patient with bleeding physique (e.g., active peptic ulcer disease). Allowing prophylactic or therapeutic use of anticoagulants.

5. Pregnant and/or lactating women

6. Any other study agent or any other clinical trial involving another study agent for treatment of solid tumors was received 4 weeks prior to study cycle 1, day 1, or within 5 half-lives of the administered drugs (whichever was shorter), or other study therapy or major surgery was received within 4 weeks of the consent date, or surgery was planned within 4 weeks of the start of the envisaged study (including dental surgery).

7. Radiation therapy was received within 4 weeks of the first dose of study drug, except for limited field palliative radiation therapy, such as used to treat bone pain or focal pain tumor mass, and without compromising the measurable lesions required for response assessment (RECIST v 1.1).

8. Patient with mushroom tumor mass

9. Patients with locally advanced PDAC without distant organ metastatic deposition

10. Grade 4 immune-mediated toxicity was generated with previous checkpoint inhibitors. Grade 2 or grade 3 pneumonia or any other grade 3 checkpoint inhibitor-associated toxicity leading to discontinuation of immunotherapy treatment. Low levels (< 3 levels) of toxicity are allowed, such as neuropathy, manageable electrolyte abnormalities and lymphopenia, hair loss and vitiligo resulting from previous treatments.

11. With a history of a second malignancy, those previously treated healthily for more than five years without recurrence or with a low likelihood of recurrence (e.g., non-melanoma skin cancer, cervical cancer in situ, early (or localized) prostate cancer, or superficial bladder cancer)

12. Active brain or pia metastasis. Brain transfer patients are eligible, provided they show clinically and radiologically Stable Disease (SD) at least 4 weeks after definitive therapy, and no steroid (> 10 mg/day of prednisone or equivalent) is used at least 4 weeks before the first dose of study drug.

13. There is evidence of severe or uncontrolled systemic disease, congestive heart failure > New York Heart Association (NYHA) class 2, myocardial Infarction (MI) within 6 months, or laboratory findings that the patient is not suitable for participation in the trial

14. Any medical condition that is considered to seriously jeopardize the patient's safety or that affects the interpretation of the G9.2-17 (IgG 4) toxicity assessment

15. Severe non-healing wounds, active ulcers or untreated fractures

16. Uncontrolled pleural effusion, pericardial effusion or ascites, require repeated drainage procedures. For the purposes of this study, "recurrence" was defined as ≡3 drainage times over the first 30 days.

17. Has a history of severe allergic, allergic or other hypersensitivity reactions to chimeric or humanized antibodies or fusion proteins

18. Significant vascular disease (e.g., aortic aneurysm requiring surgical repair or recent arterial thrombosis) occurs within 6 months of cycle 1 day 1

19. A history of pulmonary embolism, stroke or transient ischemic attack within 3 months prior to cycle 1, day 1

20. History of abdominal fistulae or gastrointestinal perforation 6 months prior to cycle 1 day 1

21. Active autoimmune disorders (type I/II diabetes mellitus, hypothyroidism requiring only hormone replacement, vitiligo, psoriasis or alopecia areata excluded)

22. Systemic immunosuppression therapies are needed including, but not limited to, cyclophosphamide, azathioprine, methotrexate, thalidomide, and anti-TNF agents. Patients who have received or are receiving acute, low doses of systemic immunosuppressant drugs (e.g.,. Ltoreq.10 mg/day of prednisone or equivalent) may be included in the group. Replacement therapy (e.g., thyroxine, insulin, physiological corticosteroid replacement therapy for adrenal or pituitary insufficiency [ e.g.,. Ltoreq.10 mg/day prednisone equivalent ]) is not considered a form of systemic treatment. The use of inhaled corticosteroids and mineralocorticoids (e.g., fludrocortisone), topical steroids, intranasal steroids, intra-articular and ophthalmic steroids is allowed.

23. Severe tumor-associated pain (grade

24. Hypercalcemia (according to CTCAE v 5.0,3 grade), although bisphosphonates are used

25. Any other disease, metabolic dysfunction, physical examination findings or clinical laboratory findings, such that a reasonable suspicion of diseases or conditions that prohibit the use of research drugs or possibly affect the interpretation of the results or put the patient at high risk of treatment complications

26. Receiving organ transplants

27. Patients undergoing dialysis

28. For patients enrolled into the anti-PD-1 antibody combination group, they were not exposed to any anti-PD-1 or anti-PD-L1 agent in any previous line of therapy. In addition, patients diagnosed with dMMR/MSI-H were excluded.

29. For part 1, patients with metastatic castration-resistant prostate cancer are allowed to continue hormone androgen deprivation therapy.

30. Any ablative therapy (radiofrequency ablation or percutaneous ethanol injection) was performed on HCC <6 weeks prior to entry into the trial

31. Hepatic encephalopathy or severe liver adenoma

child-Pugh score of 7 or more

In addition, for part 2 only:

33. hypersensitivity to the active substances or any excipients listed in the composition of the PD-1 antibody

In addition, only for part 2 combination (G9.2-17 (IGG 4) +anti-PD-1 antibody) group:

34. live vaccine was received within 28 days after initiation of treatment. Allowing the use of inactivated vaccines (i.e., influenza and Covid-19).

Study of drugs and other interventions

Research interventions are defined as any research agent, market product, placebo or medical instrument intended to be administered/used to study participants according to a research regimen.

Pharmaceutical agents for administration in combination with G9.2-17 IgG4

Nawu monoclonal antibody

Nawu monoclonal antibodyIs a blocking antibody of programmed death receptor 1 (PD-1) and is suitable for treating various tumor types. Nivolumab may 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.

Nivolumab may be administered by intravenous infusion at 240mg every 2 weeks over a 28 day period over 30 minutes (unless otherwise indicated). According to the FDA label, nivolumab administration was not contraindicated.

The nivolumab AE is presented in the following table according to its frequency of occurrence.

Na Wu Shankang adverse events (non-IMAR related) reported by frequency

The source is as follows: OPDIVO package insert section 6.1

Adverse events of na Wu Shankang considered IMAR report

Study of intervention administration

All patients received G9.2-17 (IgG 4). G9.2-17 (IgG 4) was administered weekly or every 2 weeks via IV infusion until disease progression, unacceptable toxicity occurred, or consent was withdrawn.

In section 1, patients received G9.2-17 (IgG 4) alone, with doses increasing sequentially starting at 0.2 mg/kg.

In section 2, the patient receives either G9.2-17 (IgG 4) of RP2D as a single agent (as determined in section 1) or G9.2-17 (IgG 4) RP2D-1 in combination with an anti-PD-1 antibody as follows:

CRC or CCA patient

In CRC, G9.2-17 (IgG 4)

O in CCA, G9.2-17 (IgG 4)

G9.2-17 (IgG 4) +anti-PD-1 antibody in CRC

G9.2-17 (IgG 4) +anti-PD-1 antibody in CCA

Other solid tumor types (based on data from section 1)

Omicron G9.2-17 (IgG 4), as a single agent and/or in combination with checkpoint inhibitors or chemotherapy, based on each tumor type determination

A summary description of each study intervention is known with reference to table 4.

Patients experiencing DLT in part 1 did not resume treatment. Patients experiencing DLT in part 2 discontinue treatment. If they are experiencing clinical benefit, their treatment may be resumed with the same or reduced dose of G9.2-17 (IgG 4).

TABLE 4 summary features of study interventions

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IMP: study drug, IV: intravenous, mAb: monoclonal antibodies, RP2D: suggested phase 2 dose; RP2D-1: dose level in group immediately preceding RP2D

Preparation of G9.2-17 (IgG 4)

The manufacture and packaging of research pharmaceutical (IMP) G9.2-17 (IgG 4) meets applicable current good manufacturing practice (Good Manufacturing Practice, cGMP) and the product meets standards applicable to the human body.

The G9.2-17 (IgG 4) drug was diluted to the target dose prior to administration. All dilutions should be made in a controlled and sterile environment (patient doses prepared and delivered via about 60 minutes IV infusion).

G9.2-17 (IgG 4) is a sterile liquid and is stored at 2℃to 8℃and protected from light.

Dose decrementing

If the patient is experiencing clinical benefit and regimen efficacy assessment criteria from G9.2-17 (IgG 4) and the patient is experiencing adverse reactions not attributable to G9.2-17 (IgG 4), treatment with G9.2-17 (IgG 4) alone may continue.

G9.2-17 (IgG 4) may continue if:

the clinical status of the patient does not deteriorate rapidly; and

suspending the combination agent due to AE of the combination agent alone.

Albumin-bound paclitaxel is not recommended in patients with total bilirubin >5x ULN or AST >10x ULN. In addition, the use of albumin-bound paclitaxel is not recommended in metastatic pancreatic adenocarcinoma patients with moderate to severe liver injury (total bilirubin >1.5x ULN and AST +.10x ULN). For patients with moderate or severe liver injury, the initial dose should be reduced.

Dose modification for specific AEs associated with nivolumab administration

The following provides a suggestion of modification of nivolumab based on a particular AE. For hypothyroidism or hyperthyroidism, no dose modification is suggested for nivolumab.

Recommended dose modifications of nivolumab for AEs other than IMAR are provided below:

* Toxicity was graded according to NCI CTCAE V.

a) Recovery treatment when adverse reactions improved to grade 0 or grade 1, source: OPDIVO Highlights

of Prescribing Information, 4 th 2019.

Dose modification for IMAR

If IMAR occurs, see guidelines provided herein for dose management of G9.2-17 IgG4 and/or nivolumab.

Discontinuation of research intervention

In rare cases, it may be necessary for the patient to permanently discontinue the study intervention. If study intervention is permanently stopped for reasons other than disease progression and the patient is not treated with other anti-cancer therapies, the patient is continuously assessed for disease progression for up to 2 years. See SoA for data to be collected at the time of discontinuation of study intervention and follow-up and any further evaluation that needs to be completed.

Researchers must take all the effort to keep patients on receiving study treatment until one of the causes of study treatment termination (disease progression, development of toxicity associated with study drugs, withdrawal of consent) is met. If the patient has radiological progress but no clear clinical progress and no replacement therapy has been initiated, the patient may continue to receive study treatment. However, if the patient has clear clinical progress without radiological progress, the study treatment should be stopped and the patient is advised of the available treatment options.

The patient may discontinue before disease progression for any of the following reasons:

DLT occurs according to the definition in section 3.4.4.

AE occurs/recurs outside the DLT window, requiring discontinuation of study treatment

IMAR occurrence/recurrence, need to discontinue study treatment

PureTech Health, LLC termination study

Preventing further administration of the treatment or complications or medical conditions that may compromise patient safety if the study treatment is continued

Pregnancy

Use of non-regimen anti-cancer therapies

Patients may also discontinue before disease progression for any of the following reasons:

patient partial significant deviation regimen (including lack of compliance)

The cause of discontinuation of study treatment should be recorded in the Case Report Form (CRF). If the patient discontinued study treatment due to toxicity, a "dose limiting toxicity" or "adverse event" was recorded as the primary cause of withdrawal. If the patient ceases the study at any time earlier due to AE or SAE, the patient must be followed until they resolve to grade 2 or lower unless improvement is unlikely due to underlying disease.

Concomitant therapy

Any drugs or vaccines (including over-the-counter or prescription drugs, recreational drugs, vitamins and/or herbal supplements) that the participants are receiving at the time of enrollment or during the study must be recorded along with the following information:

Cause of use

Date of application, including start and end date

Dose information, including dose and frequency

Granting medication

The following concomitant medications are allowed:

any standard of care pre-operative medication of patients receiving the combination treatment regimen.

Continuing treatment with bisphosphonate therapy (e.g., zoledronic acid) or Deshumab to treat bone metastasis that has been stabilized for at least 6 months prior to treatment (C1D 1)

Use of inhaled corticosteroids and mineralocorticoids (e.g., fludrocortisone), topical steroids, intranasal steroids, intra-articular and ophthalmic steroids

Prophylactic or therapeutic use of anticoagulants

Vaccines that allow for vaccination with covd-19, common influenza, and/or other common clinically desirable indications (e.g., tetanus, pneumococci, HBV, etc.) prior to or during the study period. The time and type of vaccination must be recorded.

Illicit drugs

The following drugs were not allowed to be used in this study:

for any indication, other study agents than G9.2-17 (IGG 4) were administered simultaneously.

Systemic immunosuppressive therapy including, but not limited to, cyclophosphamide, azathioprine, methotrexate, thalidomide, and anti-TNF agents. However, patients are allowed to take acute, low dose systemic immunosuppressant medications (e.g., 10mg day's prednisone or equivalent).

Replacement therapy (e.g., thyroxine, insulin, physiological corticosteroid replacement therapy for adrenal or pituitary insufficiency [ e.g.,. Ltoreq.10 mg/day of prednisone equivalent ]) is not considered a form of systemic treatment.

Supportive care

Patients should receive full supportive care including blood transfusions and blood products, as well as treatment with antibiotics, antiemetics, antidiarrheals and analgesics, and other care deemed appropriate and in compliance with institutional guidelines during the study

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Study evaluation and procedure

A signed written ICF approved by the Institutional Review Board (IRB) must be obtained from a potential patient before he/she can participate in any particular study procedure, including a particular study screening procedure.

Once all screening procedures are completed and the patient is determined to meet all qualification criteria, the patient may enter the study.

The study procedures of part 1 groups 1-6 and their corresponding schedules are summarized in SoA (Table 8). The study procedures of part 1 groups 7 and 8 and their corresponding schedules are summarized in SoA (table 9) and do not allow protocol exemption or exemption.

Compliance with all study requirements (including those specified in SoA) is critical and essential for study performance.

When an emergency safety issue occurs or is appreciated, a discussion should be immediately made to determine if intervention is required or to abort the study.

All screening evaluations must be completed and reviewed to confirm compliance of potential participants with all qualification criteria. A screening log is maintained to record details of all participants being screened and to confirm qualifications or record reasons for screening failure (if applicable).

Procedures performed as part of the participants routine clinical management (e.g., blood count) and obtained prior to signing the ICF can be used for screening or baseline purposes provided that the procedures meet the criteria specified by the protocol and are performed within the timeframe defined in the SoA.

Assessment by visit

SoA (tables 5-6) provides a list of evaluations performed during the screening period (up to 28 days), treatment period (presented as a 28 day cycle), end of treatment/premature termination period, IMAR follow-up and long term follow-up period. If a medical indication is present, an optional visit is allowed during each treatment cycle during which any study evaluation may be performed.

During the popularity of covd-19, many governments require citizens to maintain social distance and suggest more vulnerable groups to self-quarantine. These types of limitations may affect the ability to conduct this clinical study as originally planned. Planned study center visits may be adjusted so that the study may continue safely during the pandemic. Possible modifications may include:

Visit and/or study program delay

Replacement with a phone/video call

Replacement with home visit

Visit to an alternative clinical site

Performing the visit by a healthcare provider outside the research team

Visit and/or study procedure is completely cancelled.

Screening period (between day-28 and day-1)

The following procedure must be performed within 4 weeks after the initiation of treatment:

study procedure and examination

Written informed consent

Inclusion and exclusion criteria to verify patient eligibility

Patient demographics

Medical history

Previous and concomitant medications

ECHO/Multi-Gate acquisition scan (MUGA)

12 lead ECG (QT interval corrected using the Fridericia formula [ QTcF ])

Physical examination-neurological examination in patients with stable and pre-treated brain metastases

ECOG performance status

Vital signs

Tumor imaging assessment (computed tomography [ CT ] or Magnetic Resonance Imaging (MRI) with or without contrast agent; or Positron Emission Tomography (PET) -CT; CT with contrast agent is preferred)

Clinical laboratory

Pregnancy test, suitable for women with fertility (WOCBP)

Hematology (Hematology)

Serum chemistry

Thyroid Stimulating Hormone (TSH), free T4 or thyroxine (fT 4), serum lipase, amylase, parathyroid hormone (PTH), follicle Stimulating Hormone (FSH), luteinizing Hormone (LH), free cortisol

Coagulation of blood

Urine analysis

Pharmacodynamics and pharmacokinetics

Tumor biopsy

If a biopsy is considered to be at risk to the patient, this procedure may be omitted.

If biopsies are not available, the research center should make all efforts to obtain archived tumor tissue samples that are available as formalin-fixed paraffin-embedded (FFPE) blocks. Acceptable archived samples include those obtained through core needle biopsy or resection procedures over the past five years.

dMMR-MSI-H status (if the MMR and MSI status of the patient were not previously determined, then a check must be made at the local laboratory)

TMB in tissue against part 2G 9.2-17 (IGG 4) +anti-PD 1 antibody combination group only

Tumor type-related biomarkers

Treatment period

Each treatment cycle had a duration of 28 days. The visit times for groups 1-6 of part 1 are seen in table 5, and the visit times for groups 7 and 8 of part 1 are seen in table 6.

Treatment procedure on day 1 of each cycle (CXD 1; day 2 from cycle 2)

The following procedure was performed on day 1 of each treatment cycle.

Study procedure and examination

Concomitant medication

·AE

12 lead ECG (QTcF)

Physical examination

ECOG performance status

Vital signs

Clinical laboratory

WOCBP pregnancy test

Hematology (Hematology)

Serum chemistry

TSH, fT4, lipase, amylase, PTH, FSH, LH, free cortisol

Coagulation of blood

Urine analysis

PK/PD assessment

PD blood sampling

PK blood sampling

ADA blood sampling

Tumor type-related biomarkers

Study drug administration

Administration was only after all pre-dose evaluations and procedures were completed.

In addition, from day 1 of cycle 3, the following evaluations were performed every 8 weeks:

tumor imaging assessment (CT or MRI with or without contrast agent; or PET-CT; CT with contrast agent being preferred)

Furthermore, starting on day 1 of cycle 4, the following evaluations were performed every 3 months:

·ECHO/MUGA

groups 1-6: treatment course on days 2 and 8 (CXD 2+ -1 and CXD 8+ -1) of cycle 1 and 3 Sequence of steps

Study procedure and examination

Concomitant medication

·AE

PK/PD assessment

PD blood sampling

PK blood sampling

Groups 1-6: every weekDay 15 of the phase (cycle 1 is CXD 15.+ -. 1 day and starting from cycle 2 is.+ -. 2) Day) treatment program

The following procedure was performed on day 15 of each treatment cycle.

Study procedure and examination

Concomitant medication

·AE

Physical examination

ECOG performance status

Vital signs

Clinical laboratory

Hematology (Hematology)

Serum chemistry

Coagulation of blood

Urine analysis

PK/PD assessment

PD blood sampling at C1D15 and C3D15 only

PK blood sampling at C1D15 and C3D15 only

Tumor type-related biomarkers

Tumor biopsies were taken on C3D 15.+ -. 7 days (only on cycle 3; if the risk was considered too great for the patient, it could be cancelled)

Study drug administration

Groups 7 and 8: treatment program on cycle 1 and 3 day 3 (C1D 3.+ -. 1 day and C3D 3.+ -. 1 day)

Study procedure and examination

Concomitant medication

·AE

PK/PD assessment

PD blood sampling

PK blood sampling

Groups 7 and 8: treatment program on day 8 of each cycle (CXD 8.+ -. 1 day)

Study procedure and examination

Concomitant medication

·AE

Physical examination

ECOG performance status

Vital signs

Clinical laboratory

Hematology (Hematology)

Serum chemistry

Coagulation of blood

Urine analysis

PK/PD assessment

PD blood sampling

PK blood sampling only in odd cycles

Study drug administration

Groups 7 and 8: day 15 and day 22 of each cycle (cycle 1 is CXD 15.+ -. 1 day and starts from cycle 2) Beginning with ±2 days) of treatment procedure

The following procedure was performed on days 15 and 22 of each treatment cycle.

Study procedure and examination

Concomitant medication

·AE

Physical examination

ECOG performance status

Vital signs

Clinical laboratory

Hematology (Hematology)

Serum chemistry

Coagulation of blood

Urine analysis

PK/PD assessment

PD blood sampling at C1D15 and C3D15 only

PK blood sampling only in odd cycles

Tumor type-related biomarkers

ADA blood sampling at C1D15 and C2D15 only

Study drug administration

Additional treatment after cycle 4

The treatment cycle following cycle 4 may be repeated as indicated in SoA (tables 5-6). If the patient is experiencing clinical benefit, the patient can continue treatment even with radiological progress.

Treatment end or early termination procedure

The following procedure, including patients who discontinued treatment prematurely, was performed 30 days (±3 days) after the last dose.

Study procedure and examination

Concomitant medication

·AE

Physical examination

·ECOG

Vital signs

Tumor imaging assessment: if the study ended >8 weeks after the previous scan, a confirmatory scan was performed.

Clinical laboratory

WOCBP pregnancy test

Hematology (Hematology)

Serum chemistry

TSH, fT4, lipase, amylase, PTH, FSH, LH, free cortisol

Coagulation of blood

Urine analysis

PD assessment

PD blood sampling

ADA blood sampling

Tumor type-related biomarkers

IMAR 90 day follow-up

All patients receiving the combination treatment with anti-PD 1 antibody in part 2 had to return to a safe follow-up on day 90±7 in order to evaluate any possible delayed IMAR. The visit includes the following:

study procedure and examination

·AE

Physical examination

Vital signs

Clinical laboratory

Hematology (Hematology)

Serum chemistry

TSH, fT4, lipase, amylase, PTH, FSH, LH, free cortisol

Coagulation of blood

Urine analysis

Long-term follow-up

OS was assessed every 3 months for up to 2 years after the end/premature termination of patient treatment. For patients who have discontinued treatment for reasons other than disease progression and who have not received additional systemic anti-cancer treatment, tumor imaging assessment should be continued as much as possible.

Survival data is collected at least once every 3 months and information about any new anti-cancer therapies that begin after disease progression. Survival data may be collected more frequently to support data cleansing or regulatory submission work. The follow-up may be through telephone interviews, electronic messages or chart reviews and reported on the CRF. During the follow-up period, the event is collected and reported within 24 hours after the death (regardless of causality) is discovered or notified.

RECIST v1.1 criteria for tumor assessment

In screening tumor evaluation, tumor lesions/lymph nodes were classified as measurable or non-measurable, with measurable tumor lesions (except pathological lymph nodes, which were measured on the shortest axis) recorded according to the longest diameter in the measurement plane. When there is more than one measurable lesion at the time of screening, all lesions representing up to a total of five lesions (and up to two lesions per organ) of all affected organs should be identified as target lesions. The target lesion (the lesion with the longest diameter) should be selected based on its size. The sum of the diameters of all target lesions was calculated and reported as the baseline total diameter.

All other lesions (or disease sites) including pathological lymph nodes should be identified as non-target lesions and should also be recorded at the time of screening. No measurement is required and these lesions should be tracked as "present", "absent" or "clear progression".

According to RECIST v1.1 guidelines (Eisenhauer et al 2009), tumor target lesions were evaluated using the following disease response metrics:

evaluation of target lesions:

complete Response (CR): all target lesions disappeared. The minor axis of any pathological lymph node (whether targeted or non-targeted) must be reduced to <10mm.

Partial Response (PR): the sum of the diameters of the target lesions is reduced by at least 30% with reference to the baseline total diameter.

Progressive disease: the sum of diameters of target lesions increases by at least 20% with reference to the minimum sum at study (which includes the baseline sum if at study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (note: the appearance of one or more new lesions is also considered to be progression).

Stable Disease (SD): taking the minimum overall diameter at the time of investigation as a reference, neither sufficient shrinkage nor sufficient increase satisfies the conditions of PR.

Evaluation of non-target lesions:

CR: all non-lesions disappeared and tumor marker levels were normalized. All lymph node sizes must be non-pathological (short axis <10 mm).

non-CR/non-progressive disease (non-PD): the persistence of one or more non-target lesions and/or tumor marker levels are maintained above normal limits.

Progressive disease: clear progress in existing non-target lesions. (note: the appearance of one or more new lesions is also considered to be progression).

The summary is provided in table 7 below.

TABLE 7 evaluation of overall time point response for measurable disease patients at baseline

Target lesions	Non-target lesions	New lesions	Overall response
				CR	CR	Whether or not	CR
CR	non-CR/non-PD	Whether or not	PR
				CR	NE	Whether or not	PR
PR	non-PD or NE	Whether or not	PR
				SD	non-PD or NE	Whether or not	SD
Not all are evaluated	non-PD	Whether or not	NE
				Progressive disease	(Any)	Yes or no	Progressive disease
(Any)	Progressive disease	Yes or no	Progressive disease
				(Any)	(Any)	Is that	Progressive disease

CR: full response, non-PD: non-progressive disease, PR: partial response, SD: stabilizing disease, NE: not being able to evaluate

* When the target lesions show SD/PR but not some subset of target lesions are not evaluable, care must be taken to decide whether to refer to the overall response at that time point as SD/PR or NE. This is based on whether non-evaluable lesions (if they show growth) can elicit an overall response of progressive disease in the context of other lesion responses seen. If the non-evaluable non-target lesions account for a significant proportion of the overall disease burden, then the appropriate time point response is NE.

The disease response measures at different time points allow the following to be calculated:

disease Control Rate (DCR), defined as the percentage of patients who achieved CR, PR and SD.

Objective Response Rate (ORR), defined as the proportion of patients whose tumor size decreased by a predetermined amount (tumor shrinkage ≡30%).

Progression Free Survival (PFS), defined as the time from the onset of study drug treatment to disease progression (tumor growth. Gtoreq.30%).

Response duration (DoR), defined as the length of time that a tumor continues to respond to treatment without cancer growth or spread.

Total survival (OS), defined as the time from the start of study drug treatment to death due to any cause.

Security assessment

Physical examination

Medical science and physical examination must be performed by qualified physicians, nurse practitioner or physician assistants, and should include thorough examination of all body systems. In addition, height (only at screening) and weight were measured.

Vital signs

Vital signs including body temperature, blood pressure (systolic and diastolic), heart rate and respiration rate were measured in the supine position after 5 minutes of rest.

Electrocardiogram

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

Clinical safety laboratory assessment

Blood samples from patients were collected according to SoA (tables 5-6) for routine clinical laboratory testing (approximately 5mL per time point); additional tests may be performed at any time during the study, if necessary.

Clinical laboratory parameters were analyzed at the local laboratory of the study center. The laboratory assessment completed included hematology and serum chemistry and was defined as follows:

Serum chemistry: including glucose, total protein, albumin, electrolytes [ sodium, potassium, chlorine, magnesium, phosphorus ], calcium, bilirubin (total, direct), SGPT (ALT) or SGOT (AST), alkaline phosphatase, gamma glutamyl transferase (gamma GT), lactate Dehydrogenase (LDH), creatinine, hemoglobin A1c (HgbA 1 c) (only in the presence of a history of type 1 or type 2 diabetes), blood urea nitrogen, creatine Phosphokinase (CPK)

O TSH, fT4, lipase, amylase, PTH, FSH, LH, free cortisol additionally at the indicated visit

The omicron evaluates fasting glycemia only in the presence of clinical indications

Hematology: comprises differential whole blood count, platelets, and hemoglobin

Coagulation: including Prothrombin Time (PT) and PTT, activated Partial Thromboplastin Time (APTT) and INR (if anticoagulants are allowed to be used), C-reactive protein (CRP) and troponin

Urine analysis: a urine sample from the patient is collected for routine urine analysis. Urine analysis includes color, appearance and specific gravity paper, proteins, leukocyte esterases, glucose, ketones, urobilinogens, nitrites, white blood cell count (WBC), red blood cell count (RBC) and pH, and urine culture if the patient has clinical symptoms.

If the clinically significant value does not revert to normal/baseline or grade 1 within a reasonable period of time, the etiology should be determined.

Laboratory tests required for all protocols must be performed according to the laboratory manual and SoA (tables 5-6).

If laboratory values of non-protocol specified laboratory tests performed at the local laboratory of the institution require changes managed by the participants or are considered clinically significant (e.g., SAE or AE or dose modification), the results must be recorded.

Pregnancy test

WOCBP should be included only after menstrual periods are confirmed and the hypersensitive urine or serum pregnancy test is negative.

Additional pregnancy tests should be performed during the treatment period and at the end of treatment/early termination of the visit according to SoA (tables 5-6) and according to local requirements.

Pregnancy tests are performed whenever the menstrual cycle is missed or pregnancy is suspected.

If the patient had a history of bilateral tubal ovariectomy and/or hysterectomy, these surgeries are recorded; these patients do not need to be subjected to pregnancy tests.

Pharmacokinetic assessment

The following serum PK parameters for G9.2-17 (IGG 4) were calculated, if possible:

·AUC _0-336h

·C _max

·T _max

·t _1/2

serum concentration versus time curve

Approximately 5mL of blood samples were collected and processed into serum at each time point as specified in SoA (tables 5-6).

PK schedule for groups 1-6:

cycle 1 and 3 day 1

Pre-administration of drug

End of infusion (EOI)

2 hours after EOI (+ -30 minutes)

4 hours after EOI (+ -30 minutes)

Cycle 1 and 3 day 15

Pre-administration of drug

At EOI time

Cycle 1 and 3 days 2 and 8 (day of non-administration)

Any point in time during the visit

Cycle 2 and cycle 4 day 1

Pre-administration of drug

At EOI time

Every 2 cycles and 1 day after the 4 th cycle (i.e. C6D1, C8D1, etc.)

Pre-administration of drug

At EOI time

PK schedule for groups 7 and 8:

day 1 of each odd numbered cycle (i.e., C1D1, C3D1, etc.)

Pre-administration of drug

End of infusion (EOI)

1 hour after EOI (+ -15 minutes)

Day 3 of each odd numbered cycle (i.e., C1D3, C3D3, etc.)

Any point in time during the visit

Odd numbered periods of days 8, 15 and 22 (i.e., C1D8, C3D8, etc.)

Pre-administration of drug

At EOI time

Even numbered period day 1 (i.e., C2D1, C4D1, etc.)

Pre-administration of drug

At EOI time

If it is determined that the dose of study drug is to be discontinued, additional PK and safety assessments are collected after resumption of dosing; additional PK evaluations were performed during the interruptible period. If the dose of study drug is reduced, additional PK assessments are collected before administration of the reduced dose (within 2 hours prior to dosing) and 2 to 4 hours after starting the reduced dose of study drug. Additional PK and other blood assessments may be performed if clinical indications exist. Because of the restriction of covd-19, it is not possible to leave the center of the patient above 2 hours post-dose to provide samples only at EOI and 2 hours post-dose.

Instructions for collecting and processing biological samples are provided. The actual date and time of each sample was recorded (24 hours Zhong Shijian).

The designated laboratory uses samples to evaluate serum concentration levels of total G9.2-17 (IGG 4) and free/partially free G9.2-17 (IGG 4). The concentration was determined using a validated assay. A minimum of two 50. Mu.L serum aliquots were required to determine total G9.2-17 (IGG 4) concentration. A minimum of two 100 μl serum aliquots were required to determine the free and partial free G9.2-17 (IGG 4) concentrations, with the remaining serum in the third aliquot. Samples collected for analysis of G9.2-17 (IGG 4) plasma concentrations may also be used to assess safety or efficacy aspects associated with problems occurring during or after the study.

These blood samples were not subjected to genetic analysis. Participant confidentiality is maintained. In the visit to obtain PD, ADA, safety laboratory blood samples for determining G9.2-17 (IGG 4), a sample of sufficient volume may be used.

Genetics of

The study did not evaluate genetics.

Pharmacodynamic biomarkers

The planned time points for biomarker assessment are provided in SoA (tables 5-6); after 6 months of treatment, sampling can be reduced to once every 3 cycles.

Collecting biological samples for other biomarker studies is also part of this study. The following samples were required for biomarker studies and were collected from all participants in the study as specified in SoA:

blood samples collected prior to study drug administration (about 15mL prior to administration)

Tumor biopsy (tissue sample)

PD biomarkers (by flow cytometry, ELISA, IHC, or multiplex phenotyping) of samples were tested to evaluate their association with observed clinical responses to G9.2-17 (IGG 4) using validated assays.

The study assessed the following biomarkers:

tumor markers (blood): CA15-3, CA-125, carcinoembryonic antigen (CEA), CA19-9, alpha-fetoprotein, neuronal Specific Enolase (NSE), cytokeratin fragment-21 (CYFRA-21) were evaluated as tumor type as needed prior to each periodic dose administration. After 6 months of treatment, the time was optionally reduced to once every 3 cycles on the same schedule as the tumor imaging evaluation.

PBMC phenotype (blood): for example, CD3, CD4, CD8, CD45RO, fork frame protein P3 (FOXP 3), CD11B, CD, CD15, CD16, CD33, CD68, human Leukocyte Antigen (HLA) DR, CD163, arginase 1, granzyme B, KI, PD-1, PD L1, ubiquitin (PANCK)

Cytokine (blood): for example, interferon gamma (IFNgamma), IL 10, IL 12p70, IL 13, IL 1 beta, IL 2, IL 4, IL 6, IL 8, TNF alpha, MIP-1b, monocyte chemotactic protein 1 (MCP-1), MIP-1a, IL 17a, IL 5, TGF beta

Gal-9 in blood and tumor tissue

PD-L1 (organization)

Mismatch repair status (tissue)

Tumor Mutation Burden (TMB)

Exploratory biomarker changes (if any) are related to safety and response outcomes.

After the last visit of the last patient of the study, the samples can be stored in the selected facility for up to 2 years (or according to local regulations) to be able to further analyze the effect of G9.2-17 (IGG 4) on the pharmacodynamic biomarkers.

Immunogenicity assessment

Blood samples (approximately 3 mL) were collected from all participants according to SoA (tables 5-6) and processed into serum. In addition, serum samples should also be collected from patients who either discontinue study intervention or withdraw from the study at the end of treatment/early termination of visit.

Groups 1-6: 1 st cycle to 4 th cycle, 1 st day

Pre-administration of drug

Groups 1-6: every 2 cycles and 1 day after 4 th cycle (i.e., C6D1, C8D1, etc.):

pre-administration of drug

Groups 7 and 8: day 1 of each cycle

Pre-administration of drug

Groups 7 and 8: day 15 of cycle 1 only and day 15 of cycle 2

Pre-administration of drug

A minimum of two serum aliquots of 500 μl each were obtained, with the remaining serum in the third tube. The samples were transported to a laboratory designated for analysis using a validated assay. These samples were tested.

Serum samples were screened for antibodies (ADA) that bind to G9.2-17 (IgG 4) and the confirmation of titers of positive samples was reported. Other assays may be performed to verify the stability of antibodies G9.2-17 (IgG 4) and/or to further characterize the immunogenicity of G9.2-17 (IgG 4).

Detection and characterization of antibodies to G9.2-17 (IgG 4) was performed using validated assay methods. All samples collected for detection of antibodies to study intervention were evaluated for G9.2-17 (IgG 4) serum concentration to enable interpretation of antibody data. Antibodies can be further characterized and/or evaluated for their ability to neutralize the activity of the intervention. After the last visit of the last patient of the study, the samples can be stored in a suitable facility for up to 2 years (or according to local regulations) to be able to further analyze the immune response to G9.2-17 (IgG 4).

Other evaluation

Demographic profile

At screening, patient demographics were collected. These data include age, gender, race, and ethnicity.

Medical history

Medical history includes tumor history, surgery/transplant history, radiotherapy history, and covd 19 history and detection.

Personal medical history including previous treatment/surgery, record of any in situ or past implants, previous and/or currently used medical devices, concomitant medication (name, indication, dose, route, date of start and end, dose modification (if any) and cause), preexisting symptoms and AEs), risky genetic diseases based on family history and the patient's most known complete family history

Record of any dental treatment performed within 12 months of expiration

For patients who have previously resected pancreatic adenocarcinoma, record whether the primary tumor is located in the head, body or tail of the pancreas.

Defecation habit/typical frequency and viscosity

Record any dietary requirements or preferences (e.g., carrying out a particular dietary regimen: intermittent fasting, ketogenic diet, etc.)

Record past and present allergic conditions (allergen, severity)

Past and concomitant use of drugs

The past and concomitant medications, including vaccines and supplemental treatments/supplements, for each patient were recorded at each scheduled visit (tables 5-6).

Tumor imaging assessment

Tumor assessment using CT or MRI with or without contrast agent; PET-CT was performed.

CT with contrast agent is the preferred modality (if CT is not feasible or appropriate given the disease location, MRI, PET-CT or other imaging modality is used instead of or in addition to CT scanning). The assessment should include at least chest/abdomen/pelvis and should include other anatomical regions shown based on the patient's tumor type and/or medical history. The imaging scan must be de-identified and archived in raw format as part of the patient study file. Although the type of scan obtained is suitable for the disease, the same method should be used for the duration of the study.

In the study, the evaluation was performed according to SoA (i.e., C3D1, C5D1, C7D1, C9D1, etc.) once every 8 weeks.+ -. 7 days, and at the end of the treatment if no evaluation was performed within the past 4-6 weeks. The evaluation may be performed more frequently if there is a clinical indication. For part 2 only, if an objective response is observed in the scan, a confirmation scan is performed after 4 weeks (+7 days). After the confirmatory scan, the scheduled scan will resume at a frequency of every 8 weeks (+ -7 days) starting from the day of confirmatory scan.

Tumor biopsy

Pre-treatment and in-treatment biopsies were collected. Pre-treatment biopsies were collected during screening. If a pre-treatment biopsy is not available for reasons outlined in inclusion criteria and the patient is admitted to the group study, an archived tumor tissue specimen of the patient is collected from the primary tumor and/or metastatic deposit. Resections or core biopsies (FFPE tissue blocks or fresh tissue in formalin) are obtained currently or within 5 years of the beginning of the study from primary tumor lesions or metastatic deposits. If both primary and metastatic tissue is available, metastatic deposit tissue is preferentially used. If treatment information received before and after tissue acquisition is available, this information is also collected.

In-treatment biopsies are scheduled at C3D15±7 days and should be performed only after the tumor imaging scan of cycle 3. In the event that surgery cannot be performed within the time frame prescribed by the protocol, an alternative may be allowed, but must be discussed with the study master/medical supervisor. It is believed that a variety of clinical factors may lead to difficulty in obtaining a sufficient sample. The decision to not complete the biopsy in treatment should be discussed with the medical inspector.

ECHO/MUGA

ECHO and/or MUGA were obtained at the time points indicated in SoA (tables 5-6). If clinical indications are present, the assessment will be repeated every 3 months.

ECOG

ECOG performance status was assessed using the following ratings (Oken et al, 1982) at the time points indicated in the SoA (tables 5-6).

Stage 0: is fully active and can perform all pre-disease manifestations without limitation

Stage 1: strenuous physical activity is limited but can be ambulatory and enables mild or sedentary tasks, such as mild household, office tasks

Stage 2: can be ambulatory and self-care, but cannot perform any work activities. Can get up and walk, and the awake time is more than 50 percent

Stage 3: can only carry out limited self-care, is limited on a bed or a chair, and has a waking time of more than 50 percent

Stage 4: completely disabled. No self-care can be performed. Is completely limited on the bed or the chair

Stage 5: death of

Adverse Event (AE), serious Adverse Event (SAE) and other security reports

AE is defined in the ICH GCP guidelines as "any adverse medical event that occurs in a patient administered a pharmaceutical product or in a clinical study patient, and is not necessarily causally related to the treatment.

This definition of AE was extended in this study to include the occurrence of any such event (e.g., sign, symptom or diagnosis) or worsening of a preexisting medical condition from the time the patient signed an informed consent to the time the study medication began to be used. Exacerbations indicate an increase in the severity, frequency, or duration of the condition of the preexisting medical condition (e.g., diabetes, migraine, gout, hypertension, etc.), or are associated with significantly worse results.

Serious adverse events

SAE is defined as the following AE:

leading to death;

life threatening (exposing the patient to risk of direct death);

requiring hospitalization or extending the existing hospitalization time;

hospitalization consistent with the definition of "severe" is the admission of any hospitalized patient, including at least overnight at a healthcare facility. Admission of inpatients does not include: rehabilitation facilities, point-of-care facilities, professional care facilities, nursing homes, conventional emergency room access, surgery on the same day (as outpatient/daytime/non-hospitalization) or social admission (e.g., where the patient is not sleeping).

Resulting in persistent or severe disablement/incapacitation; or (b)

Congenital abnormalities/birth defects

An important medical event that may not lead to death, life threatening or require hospitalization may jeopardize the patient and may require medical or surgical intervention to prevent occurrence of one of the results listed in this definition, if it is determined by appropriate medical judgment, the event may be regarded as SAE. Examples of such medical events include allergic reactions and allergic bronchospasms, blood cachexia or tics that require intensive treatment in an emergency room or home, but do not result in hospitalization.

Correlation of

For all AEs, enough information should be obtained to determine causal relationships of AEs (e.g., study drugs or other diseases). The AE versus study treatment was evaluated as defined below:

irrelevant: any event that does not follow the reasonable temporal order of study drug administration and that may result from the clinical state of the patient or other therapeutic pattern administered to the patient.

Unlikely to correlate: no reasonable time sequence of study drug administration is followed or any event that might result from the clinical state of the patient or other therapeutic pattern administered to the patient.

Possible correlation: any response that follows a reasonable time sequence of study drug administration or follows a known pattern of response to a suspected drug and that cannot be reasonably interpreted by known features of the patient's clinical state or other pattern of therapy administered to the patient.

Correlation: any response that follows a reasonable time sequence of study drug administration and follows a known response pattern to suspected drugs and recurs with re-challenge and/or is ameliorated by stopping the drug or reducing the dose.

Adverse event management

AE was not recorded prior to administration of the study medication of the first dose. The beginning AE or symptoms associated with medical history that worsened after study drug administration were recorded. AE should be tracked until they resolve, return to baseline or are determined to be stable or chronic. All SAE were collected until 30 days after the last dose of study medication. All SAE's associated with the study procedure must be collected from the date of patient written consent.

Immune-mediated adverse reactions

Immune Mediated Adverse Reactions (IMAR) were identified against anti-PD 1 antibodies.

The specific IMAR indicated is:

immune-mediated hepatitis

Immune-mediated nephritis

Immune mediated pneumonia

Immune-mediated colitis and diarrhea immune-mediated endocrinopathy

Immune mediated skin reactions

Other immune-mediated adverse reactions: arthritis, encephalitis, rhabdomyolysis, myositis, myocarditis, pancreatitis, and uveitis.

The monitoring program is intended to limit the severity and duration of IMAR occurring during combinatorial drug development and encompasses physical examination, vital signs, safety laboratory assessment (including hematology, biochemistry), assessment of endocrine function on each day 1 of the new dosing cycle (pre-dosing), assessment of coagulation status, and scheduled visits for urinalysis. The evaluation schedule (tables 5-6) also covers the evaluation of ejection fraction and the periodic ECG every three months.

A summary of G9.2-17 (IgG 4) -induced management of IMAR alone or in combination with other therapeutic agents is provided in tables 8-9 below.

TABLE 8 management of immune-mediated adverse reactions (IMARs) by G9.2-17 (IgG 4)

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TABLE 9 management of immune-mediated adverse reactions (IMARs) caused by combination therapy with G9.2-17 (IgG 4) +anti-PD 1 antibodies

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Dose reduction procedure for adverse event management

If dose reduction is used for AE management in part 2 of the study, two dose reductions of 50% each are allowed. Additional dose reduction is performed when clinical benefit is expected and may continue to be obtained.

Clinical laboratory abnormalities and other abnormalities assessment as AE and SAE

If the definition of AE or SAE is met, then abnormal laboratory results (e.g., clinical chemistry, hematology, and urine analysis) or other abnormal assessments (e.g., ECG or vital signs) that are judged clinically significant are recorded as AE and SAE. Clinically significant abnormal laboratory results or other abnormal assessments detected during the study or present at the time of screening and significantly worsening after study initiation were reported as AE or SAE. However, clinically significant abnormal laboratory findings or other abnormal assessments are associated with the disease being studied (unless judged to be more severe than the patient's condition is expected) or are present or detected at the beginning of the study and are not worsening, will not be reported as AE or SAE.

Laboratory measurements that deviate clinically significantly from previous measurements can be repeated. Additional or more frequent checks than prescribed in the protocol should be made if necessary to provide adequate AE recordings and AE resolution.

Time period and frequency for collecting AE and SAE information

All AEs and SAE were collected from the intervention at the time points specified in SoA (tables 5-6) until the follow-up visit.

Medical events that begin before study intervention begins but after informed consent is obtained are recorded as medical history/current medical condition, rather than AE.

All SAE are recorded and reported immediately, and should not exceed 24 hours in any case.

Follow-up for AE and SAE

After the initial AE/SAE report, each participant is required to be actively tracked at the time of subsequent visits/contacts. All SAE are tracked until resolved, stable, event gets other explanation or participants lose access.

Statistical considerations

When the last patient received the last visit, the study was completed. After the last patient had a primary endpoint event, the database was locked for primary analysis. Final study analysis was performed after study completion.

Statistical assumptions

The current study was designed to identify the MTD of G9.2-17 (IgG 4) (part 1) by evaluating DLT, followed by a two-stage optimization design of Simon to evaluate drug activity in three disease types (alone or in combination). The study of section 2 is assumed to be detailed below.

CRC and CCA G9.2-17 (IgG 4) monotherapy groups

Null hypothesis: ORR 3 is less than or equal to 5 percent

Alternative assumptions: ORR 3 is more than or equal to 15 percent

Combination treatment group of CRC and CCA G9.2-17 (IgG 4) +anti-PD 1 antibodies

Null hypothesis: ORR 3 is less than or equal to 10 percent

Alternative assumptions: ORR 3 is more than or equal to 25 percent

Analysis set

Unless otherwise specified, the intent-to-treat (ITT) population is defined as those patients who receive at least one dose of study medication. The ITT was subjected to primary efficacy analysis. Patient treatment is performed on ITT.

Efficacy populations are defined as all patients in ITT who underwent at least one measurable ORR 3 or PFS 6 assessment. This population was used for sensitivity analysis.

A population meeting regimen (PP) is defined as any patient who receives G9.2-17 (IGG 4) for at least one complete cycle without significant regimen deviation.

A safety group (SAF) is defined as all patients receiving at least one dose of study drug. Security analysis was performed on SAF.

The PK/PD population is defined as those patients who have received G9.2-17 (IGG 4) for at least one complete cycle.

Primary endpoint

Security analysis-part 1 and part 2

All security analyses were performed on SAFs unless otherwise specified.

Adverse events

Adverse Events (TEAEs) occurring during treatment are defined as events that occur at or after the first dose of study medication. The MedDRA coding dictionary is used for the coding of AE. TEAE, severe or CTCAE grade 3 or 4 TEAE and treatment-related TEAE are summarized generally in terms of system organ classification and treatment group preference. These summarize the number of events and the number and percentage of patients for a given event. In addition, the number and percentage of TEAE patients are provided in terms of maximum severity. A summary of all TEAEs (by systemic organ category and preferred terminology) occurring in ≡5% of patients in either treatment group is provided.

DLT, MTD and RP2D are summarized.

Laboratory assessment

All laboratory-based data are presented as a list of all values and abnormal results that are judged clinically significant (which are reported as AEs). The per-visit and treatment groups provided a numerical summary of all observed results and changes assessed relative to the baseline screening laboratory, including chemical, hematology, and urinalysis results. No inference comparisons were planned.

Vital signs

A summary of all observed results and numerical changes in screening vital signs from baseline, including blood pressure, heart rate, respiration rate, and body temperature, are provided by time points and treatment groups. No inference analysis of vital signs is planned.

ECG, ECHO/MUGA and physical examination

The physical examination data and changes are presented as a list. Based on the occurrence of clinically significant abnormalities, the ECG results are presented as a list and summarized by treatment group and visit. No inference comparisons across treatment groups were planned.

Principal efficacy analysis-part 2

Disease response was assessed according to RECIST v1.1 and descriptive summary was made for ITT, PP and efficacy populations.

The main curative effect end point is:

ORR 3 of CRC and CCA

PFS 6 of PDAC

Secondary endpoint

Pharmacokinetics, pharmacodynamics and immunogenicity

PK, PD and immunogenicity were summarized descriptive for PK/PD populations in part 1 and part 2.

Secondary efficacy analysis-part 2

Disease response (ORR, PFS, DCR, doR and OS) was assessed according to RECIST v1.1 and descriptive summary was made for ITT, PP and efficacy populations.

Exploratory endpoint

The analysis of exploratory endpoints is detailed in SAP.

Other analyses

Other collected data not explicitly mentioned are presented in the patient list.

Distribution, demographics, baseline characteristics, and medical history

Treatment information is summarized, including the number of patients in the group, screening failures, the number of patients treated and patients withdrawn for reasons.

Demographic data, baseline characteristics, and medical history were statistically summarized per treatment group and for ITT and PP overall usage descriptions.

Past and concomitant use of drugs

The number and percentage of patients taking past and concomitant medications were summarized in terms of treatment groups and for ITT and PP overall.

Example 2: anti-galectin-9 antibody stability Studies

Candidate IgG4 antibodies were subjected to stability analysis after storage under several different conditions and at different concentrations. Stability analysis was performed via Size Exclusion Chromatography (SEC) using a TOSOH TSKgel Super SW mAb column. SEC curves before and after storage were compared to identify any problems with protein stability (e.g., aggregation or degradation).

Materials and methods

Sample preparation

Anti-galectin-9 antibodies were stored at-80 ℃ until use. Prior to analysis, samples were thawed in a room temperature water bath and stored on ice until analysis. Absorbance at 280nm was measured using Nanodrop prior to treatment. The instrument was blank with TBS (20 mM Tris pH 8.0, 150mM NaCl). The samples were then transferred to polypropylene microcentrifuge tubes (USA Scientific, 1615-5500) and centrifuged at 16.1 kXg for 30 minutes at 4 ℃. The sample was filtered through a 0.22 μm filter (Millipore; SLGV004 SL). The absorbance after filtration was measured.

HPLC analysis

The sample conditions tested included the following: environmental stability (0 hours at room temperature, 8 hours at room temperature), refrigerated stability (0 hours at 4 ℃, 8 hours at 4 ℃, 24 hours at 4 ℃) and freeze/thaw stability (1 x freeze/thaw, 3x freeze/thaw, 5x freeze/thaw). Each condition was run in duplicate at three different concentrations: stock solution, 10x dilution and 100x dilution. Samples of 100 μl were prepared for each condition and stored in polypropylene microcentrifuge tubes. The dilutions were prepared in TBS as necessary. Absorbance at 280nm was read prior to analysis. The room temperature sample is stored on the bench for the indicated duration. The 4 ℃ samples were stored on ice or in a 4 ℃ refrigerator for the time periods indicated in table 10. The freeze-thaw samples were flash frozen in liquid nitrogen and then thawed in a room temperature water bath. The freezing and thawing process is performed once, three times or five times, and then the samples are stored at 4 ℃ until analyzed.

SEC analysis was performed on Shimadzu HPLC using a TOSOH TSKgel SuperSW mAb HR column with a 280nm UV detector. The column was loaded with 25. Mu.L of sample and run at 0.5mL/min for 40 min. KBI buffer formulation was used as mobile phase.

Results

The concentration of the antibodies was determined using UV absorbance measurements before and after filtration as shown in table 10. Two 2mL samples provided by KBI were thawed, one vial for the cold and freeze/thaw conditions and the other vial for the 4 ℃ conditions. Absorbance readings showed nearly complete recovery after filtration.

TABLE 10 protein recovery after sample preparation

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Two or three high molecular weight peaks eluting prior to the main peak were observed (fig. 2). Under each condition determined, these peaks account for about 5% of the total sample (table 11). No significant differences in protein concentration were observed under all conditions assayed.

TABLE 11 stability results

In summary, the anti-galectin-9 antibodies showed consistent stability after storage under all conditions analyzed, as indicated by no significant change in the SEC curve. There was no significant protein loss after filtration and two to three high molecular weight peaks were identified, accounting for about 5% of the total sample. The results show that the antibodies are stable under all conditions tested, and no aggregate formation or degradation is observed.

EXAMPLE 3 evaluation fractions of galectin-9 expression in tumor biopsy derived organoids

The tumor organoids may be applied to predict patient outcome because using a tumor model with similar characteristics as the original tumor may more accurately predict the patient's drug response. (see, e.g., trends in Biotechnology;36 (4): 358-371,2018, month 4, 1).

Galectin-9 levels in tumors can serve as an indicator of predicted drug responses. Biopsy-derived organoids can be used as surrogate markers for assessing galectin-9 levels in primary tumors. Thus, the ability to assess galectin-9 levels in single cell or organoid fractions was tested.

Biopsies were received from representative pancreatic adenocarcinoma and colorectal carcinoma and treated as follows. The surgically excised human tumor samples were freshly received in DMEM medium on ice and minced in 10cm petri dishes. Minced tumors were resuspended in dmem+10% FBS containing 100U/mL collagenase type IV to obtain spheroids. The partially digested samples were pelleted and then resuspended in fresh dmem+10% FBS and filtered on 100mm and 40mm filters to produce S1 (> 100 mm), S2 (40-100 mm) and S3 (< 40 mm) spheroid fractions, which were then stored in ultra low adhesion tissue culture plates.

The S2 fraction was digested with trypsin for 15 minutes to generate single cells. For flow cytometry preparation, cell pellets in S2 and S3 fractions were resuspended and after Fc receptor blocking (# 422301;BioLegend,San Diego,CA) cells were labeled by incubating with fluorescent conjugated mAbs against human CD45 (HI 30), CD3 (UCHT 1), CD11b (M1/70), epcam (9C 4) and Gal9 (9M 1-3; both from Biolegend) or Gal 9G 9.2-17Fab or Fab isoforms. Dead cells were excluded from analysis using zombie yellow (BioLegend). Flow cytometry was performed on an Attune NxT flow cytometer (Thermo Scientific). Data was analyzed using FlowJo v.10.1 (Treestar, ashland, OR).

The results are shown in FIGS. 3A-3F, 4A-4F and 5A-5F and indicate that the levels of galectin-9 detected by Gal 9G 9.2-17Fab in the S2 single cell and S3 organoid fractions are correlated. Thus, both S2 single cells and S3 organoids can be used to assess galectin-9 levels in organoids derived from tumor biopsies.

Example 4 preparation of patient-derived organotypic tumor spheroids (PDOT) for cellular analysis

Biopsy-derived organoids can be useful as measures to assess the ability of therapeutic agents to stimulate an immune response. Thus, the S2 fraction for in vitro culture described in example 3 above was treated with anti-galectin-9 antibody G9.2-17 and prepared for immune spectroscopy analysis.

An aliquot of the S2 fraction was precipitated and resuspended in type I rat tail collagen (Corning) at a concentration of 2.5mg/mL, followed by the addition of 10 x PBS with phenol red (pH adjustment using NaOH). PANPEHA Whatman paper (Sigma-Aldrich) was used to confirm pH 7.0-7.5. The spheroid-collagen mixture is then injected into the central gel region of a 3-D microfluidic culture device, such as Jenkins et al, cancer discovery.2018, month 2; 8 (2) 196-215; ex Vivo Profiling of PD-1Blockade Using Organotypic Tumor Spheroids, the contents of which are incorporated herein by reference in their entirety. After 30 minutes at 37 ℃, the collagen hydrogel containing patient-Derived Organotypic Tumor Spheroids (PDOTs) was hydrated with medium with or without anti-galectin-9 monoclonal antibody G9.2-17. PDOT was then incubated at 37 ℃ for 3 days.

The cell pellet was resuspended in FACS buffer and 1X10 ⁶ Individual cells were first stained with zombie yellow (BioLegend) to exclude dead cells. After viability staining, cells were incubated with anti-CD 16/CD32 mAb (eBiosciences, san Diego, CA) to block fcyriii/II, followed by antibody staining with 1 μg of fluorescence conjugated extracellular mAb. Intracellular staining was performed for cytokines and transcription factors using the fixation/permeabilization solution kit (eBiosciences). Useful human flow cytometry antibodies include CD45 (HI 30), CD3 (UCHT 1), CD4 (a 161 A1), CD8 (HIT 8 a), CD44 (BJ 18), tnfa (MAb 11), ifnγ (4 s.b3) and Epcam (9C 4); all from Biolegend. On an LSR-II flow cytometer (BD Biosciences) Flow cytometry was performed. Data was analyzed using FlowJo v.10.1 (Treestar, ashland, OR).

EXAMPLE 5 evaluation of galectin-9 levels in plasma and serum of cancer patients

Plasma and serum galectin-9 levels in patient samples were assessed and compared to healthy volunteers. Blood (10 ml) was drawn from the peripheral venous route of 10 healthy controls and 10 inoperable cancer patients. Serum and plasma were extracted from each blood sample. Blood was collected in standard EDTA tube PicoKine ^TM ELISA; catalog number: the EK1113 is basically used according to the manufacturer's instructions. The results of the individual values are tabulated in tables 12 and 13.

TABLE 12 patient samples

TABLE 13 healthy volunteer samples

Sample numbering	Serum	Plasma of blood
			Control 1	536.4	611.97
Control 2	476.43	592.58
			Control 3	612.66	651.43
Control 4	269.75	414.41
			Control 5	460.26	602.28
Control 6	206.66	405.8
			Control 7	385.88	439.85
Control 8	525.283	654.2
			Control 9	711.047	718.68
Control 10	296.85	349.09
			Average value of	448.122	544.029

EXAMPLE 6 evaluation of galectin-9 expression and localization Using immunohistochemical analysis

Tumor samples of paraffin embedded biopsy origin were used to assess the ability to determine galectin-9 expression levels in tumors using immunohistochemical analysis.

Briefly, slides were dewaxed (xylene: 2X3 min; absolute alcohol: 2X3 min, methanol: 1X3 min) and rinsed in cold tap water. For antigen retrieval, citrate buffer (pH 6) was preheated to 100 ℃ in a water bath and slides were incubated in citrate buffer for 5 minutes. Slides were cooled at room temperature for about 10 minutes and placed in running water. Slides were washed in PBS, a paper circle was drawn around the sections, and the sections were incubated in blocking buffer (DAKO-peroxidase blocking solution-S2023) for 5 minutes. Serum-free blocker (Novocastra serum-free protein blocker) was added and then rinsed off with PBS. Primary antibody (Sigma, anti-galectin-9 clone 1G 3) was used at 1:2000 dilution in DAKO-S2022 diluent and sections were incubated overnight at 4C. Slides were washed with PBS and then incubated with secondary antibody (anti-mouse) for 45 minutes at room temperature. Slides were washed and stained with ABC VECTOR STAIN (45 min), washed with PBS, stained with DAB (1 ml of stabilized DAB buffer+1 drop DAB) for 5 min, and washed in running water. Hematoxylin was added for 1 min and 70% etoh+1% HCL was applied to avoid over staining. Slides were placed in running water for 2-3 minutes, then immersed in water, then in absolute alcohol, then in xylene, 2 times each for 30 seconds. Coverslips and images were captured. Galectin-9 staining in chemotherapy-treated colorectal cancer and colorectal cancer liver metastases is shown in fig. 6A-6B. The results of galectin-9 negative cholangiocarcinoma are shown in fig. 6C.

EXAMPLE 7 Cross-reactivity of anti-galectin-9 antibody G9.2-17 with other galectins

To assess antibody specificity and cross-reactivity with other galectins, anti-galectin-9 antibodies G9.2-17 were tested for binding to human proteomic arrays consisting of all members of the galectin family at two working concentrations. The CDI's HuProt human proteome microarray (about 75% of human proteome) was used to evaluate antibody specificity. The microarray was incubated with primary antibody, rinsed, incubated with fluorescently labeled secondary antibody, and then analyzed for the amount of fluorescence detected for each target protein. The results are assembled into microarray images. The results indicate that anti-galectin-9 antibodies G9.2-17 are highly specific for galectin-9 and do not cross-react with any other galectin family members.

Example 8 anti-galectin-9 antibodies protect T cells from galectin-9 mediated apoptosis

To investigate the effect of anti-galectin-9 antibodies G9.2-17, apoptosis assays were performed to determine whether T cells died by the apoptotic process or other mechanisms.

Briefly, in RPMI medium supplemented with 10% FBS, 2mM L-glutamine, 10mM HEPES, 1mM sodium pyruvate, 4.5g/L glucose and 1.5g/L sodium bicarbonate at 37℃in 5% CO ₂ MOLM-13 (human leukemia) cells were cultured. The cells were then transferred to serum-free RPMI medium and suspended in serum-free medium at a concentration of 2.5e6 cells/mL. Cells were seeded into wells of a tissue culture grade 96-well plate at a density of 2e5 cells/well (80 μl cell suspension per well). Monoclonal anti-galectin-9 antibodies or matched isoforms are added to each well and incubated at 37℃with 5% CO ₂ Incubate for 30 minutes. After incubation, recombinant full-length human galectin-9 (R&D Systems2045-GA, diluted in PBS) to a final concentration of 200nM. The cells were incubated at 37℃with 5% CO ₂ Incubate for 16 hours. Cells were then stained with annexin V-488 and Propidium Iodide (PI) prior to analysis by flow cytometry. Each condition was performed in triplicate. PI is impermeable to living and apoptotic cells, but stains dead cells with red fluorescence, tightly binding to nucleic acids in the cells. Alexa in bufferAfter staining the cell population with 488 annexin V and PI, apoptotic cells showed green fluorescence and dead cells showed red and green fluorescence And living cells show little or no fluorescence. Cells were differentiated using a flow cytometer with 488nm argon ion laser line for excitation. Analysis was then performed on FlowJo software. The fraction of annexin V and Propidium Iodide (PI) positive cells was plotted as a function of the antibody concentration used in figure 7. As shown in fig. 7, the level of apoptotic T cells treated with anti-Gal 9 antibody was much lower than T cells treated with human IgG4 isotype control antibody, indicating that anti-galectin-9 antibody G9.2-17 can protect T cells from anti-galectin-9 mediated apoptosis. />

Example 9: evaluation of anti-Gal-9 antibodies alone or in combination with checkpoint inhibition in mouse models of pancreatic cancer, tumor quality and immune characteristics of mice treated with G9.2-17mIgG1

The effect of G9.2-17mIgG1 on tumor weight and immune characteristics was evaluated in a mouse model of pancreatic cancer. FC1242 PDAC cells derived from Pdx1Cre, krasG12D, trp R172H (KPC) mice (Zambiritis CP et al, TLR9 ligation in pancreatic stellate cells promotes turigenesis.J Exp Med.2015; 212:2077-94) were injected intraparenchymally into 8 week old C57BL/6 male (Jackson Laboratory, bar Harbor, ME) mice. Tumor cells were suspended in PBS containing 50% Matrigel (BD Biosciences, franklin Lakes, NJ), and 1X10 ⁵ Individual tumor cells were injected into the pancreas via laparotomy. Mice (n=10/group) received i.p. one pretreatment dose, then 3 doses (q.w.) of commercial α -galectin-9 mAb (RG 9-1, 200ug, bioxcell, lebanon, nh) or G9.2-17mIgG1 (200 μg) or the paired isotype G9.2-Iso or rat IgG2a (LTF-2, bioxcell, lebanon, nh) (200 μg) (one dose per week for three weeks). Mice were sacrificed after 3 weeks and tumors were harvested for flow cytometry analysis. Tissues were treated and prepared according to conventional practice and flow cytometry analysis was performed. See, for example, U.S. patent No. 10,450,374.

Tumor mass and immune characterization of mice treated with G9.2-17mIgG2a alone or in combination with alpha PD-1mAb

The effect of G9.2-17mIgG2a on tumor weight and immune characteristics was evaluated in a mouse model of pancreatic cancer alone or in combination with immunotherapy. FC1242 PDAC cells derived from Pdx1Cre, krasG12D, trp R172H (KPC) mice were injected intraparenchymally into 8 week old C57BL/6 male mice (Jackson Laboratory, bar Harbor, ME). Tumor cells were suspended in PBS containing 50% Matrigel (BD Biosciences, franklin Lakes, NJ), and 1x105 tumor cells were injected into the pancreas via laparotomy. Mice received i.p. one pretreatment dose and then 3 doses (q.w.) of G9.2-17mIgG2a (200 μg) or neutralized αpd-1mAb (29 f.1a12, 200 μg, bioXcell, lebanon, NH) or paired isoforms as indicated (LTF-2 and C1.18.4, bioXcell, lebanon, NH) alone or in combination. Mice were sacrificed on day 26 and tumors were harvested for analysis. Tissues were treated and prepared according to conventional practice and flow cytometry analysis was performed. See, for example, US 10,450,374. Each dot represents one mouse; * p <0.05; * P <0.01; * P <0.001; * P <0.0001; by unpaired Student t test. These results indicate that treatment with G9.2-17mIgG2a single agent reduced tumor growth at both dose levels, whereas anti-PD-1 alone had no effect on tumor size (fig. 8A-8B).

Example 10: evaluation of anti-Gal-9 antibodies in two homologous models of colorectal and melanoma cancers in immunocompetent mice

Gal-9 antibodies G9.2-17 and G9.1-8m13 were evaluated in a homologous model of colorectal and melanoma cancers in immunocompetent mice. The structure of these two antibodies is provided herein or disclosed in PCT/US2020/024767 (the relevant disclosure of which is incorporated by reference for the subject and purposes mentioned herein). Test samples were formulated and prepared weekly for the duration of the study.

Design of experiment

Pre-study animals (female C57BL/6,6-8 weeks old (Charles River Labs)) were acclimatized for 3 days, and then 5e5 b16.f10 (melanoma cell line) or MC38 cells (colorectal cancer cell line) resuspended in 100 μl PBS were subcutaneously implanted on the left flank unilaterally. Pre-study tumor volumes for each experiment were recorded beginning 2-3 days post-implantation. When the tumor reaches 50-100m ^m3 (preferably 50-75 mm) ³ ) Animals were matched to treatment or control groups for dosing and dosing started on day 0 by tumor volume.The study designs used to test anti-Gal 9 IgG1 and anti-Gal 9 IgG2 are summarized in tables 14 and 15.

TABLE 14 anti-Gal 9 IgG1 (B16F 10 and MC 38)

TABLE 15 anti-Gal 9 IgG2 (B16F 10 and MC 38)

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Tumor volumes were acquired three times a week. The final tumor volume was obtained on the day the study reached the endpoint. If the animal is found to moribund, the final tumor volume is obtained. Animals were weighed three times a week. The final body weight was weighed on the day the study reached the endpoint or if animals were found to die. Ad libitum provision to animals exhibiting weight loss of > 10% compared to day 0Any animal showed a duration of 7 days>Net weight loss of 20% or if mice showed a net weight loss compared to day 0>A net weight loss of 30% is considered moribund and euthanized. The study endpoint was set when the average tumor volume of the control group (non-deleted) reached 1500mm 3. If this occurs before day 28, the treated group and individual mice are dosed and measured until day 28. If the average tumor volume of the control group (non-deleted) did not reach 1500mm3 by day 28, then the endpoint for all animals was the day the average tumor volume of the control group (non-deleted) reached 1500mm3 up to day 60. Blood was collected from all animals in each group. For blood collection, as much blood as possible was collected to K via cardiac puncture under deep anesthesia induced by isoflurane inhalation ₂ EDTA tube (400 μl)And serum separator tube (remaining). Will collect K ₂ Blood in EDTA tubes was placed on wet ice until used to perform immunization group flow.

The blood collected in the serum separation tube was allowed to coagulate at room temperature for at least 15 minutes. The samples were centrifuged at 3500 for 10 minutes at room temperature. The resulting serum was isolated, transferred to uniquely labeled clear polypropylene tubes, and immediately frozen on dry ice or in a refrigerator set to hold-80 ℃ until shipping for bridging ADA assay (shipping within a week).

Tumors from all animals were collected as follows. Will be less than 400mm in size ³ Is flash frozen, placed on dry ice, and stored at-80 ℃ until used for RT-qPCR analysis. For a size of 400-500mm ³ Whole tumors were collected into MACS medium for Flow Panel. For sizes greater than 500mm ³ Is prepared by dividing a small piece (about 50mm ³ ) Flash frozen on dry ice and stored at-80 ℃ for RT-qPCR and the remaining tumors were collected in MACS medium for flow cytometry. For flow cytometry, tumors were placed in MACS medium and stored on wet ice until treatment.

Spleen, liver, colon, lung, heart and kidneys of all animals were kept in 10% Neutral Buffered Formalin (NBF) for 18-24 hours, transferred to 70% ethanol and stored at room temperature. Formalin-fixed samples were embedded in paraffin.

Example 11: evaluation of Gal-9 antibody in cholangiocarcinoma model

Efficacy of anti-Gal-9 antibodies was evaluated in a mouse biliary tract cancer model, as described by S.Rizvi et al (YAP-associated chromosomal instability and cholangiocarcinoma in mice, oncostarget, 9 (2018) 5892-5905), the contents of which are incorporated herein by reference in their entirety. In this transduction model, where oncogenes (AKT/YAP) are directly infused into biliary tree, tumors are produced in the biliary tract of immunocompetent hosts with species-matched tumor microenvironments. Administration is described in table 16.

TABLE 16 administration of

Briefly, murine CCA cells (described in s.rizvi et al) were harvested and washed in DMEM. Male C57BL/6 mice from Jackson Labs were anesthetized with 1.5-3% isoflurane. Under deep anesthesia, the abdominal cavity was opened through a 1cm incision under the xiphoid process. The upper outer side of the intrahepatic lobe was exposed using a sterile cotton head applicator. Using a 27 gauge needle, 40. Mu.L of standard medium containing 1X 10≡6 cells was injected to the outside of the inner leaf. The cotton head applicator is secured to the injection site to prevent cell leakage and blood loss. Subsequently, the abdominal wall and skin were closed in layers with absorbable chromium 3-0 gut suture material.

Two weeks after implantation, animals were matched to treatment or control groups for dosing based on tumor volume and dosing was started on day 0. Tumor volumes were measured and animals were weighed three times per week. The final tumor volume and weight were obtained on the day the study reached the endpoint (4 weeks or when the tumor burden of the control became 1500mm 3). Blood was collected from all animals in each group.

Example 12: in vitro and in vivo characterization of anti-Gal 9 antibody G9.2-17

In vivo and in vitro pharmacodynamic and pharmacological studies and safe pharmacology were performed as disclosed below. In vivo studies were performed using the IgG1 version of anti-galectin-9 mAb G9.2-17 for mouse studies, which is based on the fact that the antibodies were developed to have exactly the same V as G9.2-17 _H And V _L The chains and thus have exactly the same binding epitope, and the same cross-reactive properties as G9.2-17 as well as binding affinity across species and the same functional properties.

In vitro study

G9.2-17 has multi-species cross-reactivity (human, mouse, rat, cynomolgus monkey) as assessed in vitro with equivalent <1nmol binding affinity. See, for example, PCT/US2020/024767, the relevant disclosure of which is incorporated by reference for the subject matter and purposes of the present references. G9.2-17 does not cross react with the CRD1 domain of galectin-9 protein. It has excellent stability and purification characteristics and is not cross-reactive with any other galectin protein present in primates.

Table 17 below summarizes the results of the in vitro pharmacological studies.

TABLE 17 major pharmacodynamics in vitro

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Studies with knowledge of the mechanism of action include ADCC/ADCP (antibody dependent cell mediated cytotoxicity/antibody dependent cellular phagocytosis) and blocking function assessment. As expected for human IgG4 mAb, G9.2-17 did not mediate ADCC or ADCP (fig. 9A). This was tested against the IgG1 human counterparts of G9.2-17, which mediate ADCC and ADCP, as positive controls (fig. 9B).

Furthermore, the blocking function of G9.2-17 was evaluated in a competition binding ELISA assay. G9.2-17 effectively blocks the binding of the galectin-9 CRD2 domain to its binding partner CD206 human recombinant protein, confirming the expected mode of action of G9.2-17, i.e. blocking galectin-9 activity. Furthermore, we optimized the MOLM-13T apoptosis assay, in which G9.2-17 effectively rescued cells from apoptosis caused by galectin-9 protein treatment (treatment with galectin-9 resulted in about 50% apoptosis, and treatment with galectin-9+G9.2-17 resulted in about 10% apoptosis).

Further extensive in vitro characterization has been performed to compare the binding and functional characteristics of G9.2-17 to the mouse IgG 1G 9.2-17mAb, which comprises the exact same CDR domain as G9.2-17 and thus has the same binding epitope, the CRD2 galectin-9 domain. The mIgG 1G 9.2-17 was developed for use in a murine homologous pharmacological efficacy study to avoid any potential occurrence of immunogenicity with G9.2-17 itself. The mIgG 1G 9.2-17 has equivalent <1nmol cross-species affinity and the same cell-based binding affinity to the human cancer cell line CRL-2134. The data generated by mIgG 1G 9.2-17 in the MOLM-13T apoptosis assay is identical to G9.2-17 itself.

In vivo pharmacology

In vivo assays included isogenic mouse models using the mouse mAb-G9.2-17 binding epitope (G9.2-17 surrogate mAb for animal efficacy studies) cloned into the IgG1 mouse backbone, which shares the cross-responsiveness and binding affinity characteristics of G9.2-17.

The isogenic mouse model tested was:

in situ pancreatic adenocarcinoma (KPC) mouse model (single agent and in combination with anti-PD-1): survival, tumor volume assessment, and flow cytometry.

Subcutaneous melanoma B16F10 model (single agent and in combination with anti-PD-1): tumor volume assessment and flow cytometry.

Subcutaneous MC38 model (single agent and combination with anti-PD-1): tumor volume assessment

Furthermore, ex vivo patient-derived tumor cultures (organoids) treated with G9.2-17 will be used to explore the mechanism of action of G9.2-17.

Mechanistically, G9.2-17 was found to have blocking activity instead of ADCC/ADCP activity. Blocking of the interaction of galectin-9 with its binding receptor (such as CD206 on immunosuppressive macrophages) was observed. Functionally, in vivo studies showed reduced tumor growth in multiple syngeneic models (orthotopic pancreatic KPC tumor growth and subcutaneous melanoma B16F10 model) treated with G9.2-17mIgG1 surrogate antibodies. In mouse tumors treated with a single dose of anti-galectin-9 mAb and in combination with anti-PD-1, G9.2-17 reactivated effector T cells and reduced the level of immunosuppressive cytokines. Combination studies with anti-PD-1 mAb indicate higher intratumoral presence of effector T cells, supporting clinical trials of the combination approach. Importantly, the mechanistic effects of G9.2-17 have been studied and demonstrated in patient-derived tumor cultures (Jenkins et al, 2018) (tumors resected from the primary and metastatic sites of PDAC, CRC, CCA, HCC), where G9.2-17 induced reproducible and robust T cell reactivation, indicating that galectin-9 exerted in vitro intratumoral immunosuppression was reversed.

To assess the relevance of the combination anti-PD-1 and anti-galectin-9 mabs, melanoma B16 models were treated subcutaneously with single agents anti-PD-1 and anti-galectin-9, as well as the combination. In the combination group, the intratumoral presence of effector T cells was enhanced.

A significant increase in cytotoxic T cell (CD 8) levels was observed in treatment with anti-galectin-9 migg1200 μg+anti-PD-1 compared to anti-galectin-9 migg1200 μg (p < 0.01), and a significant increase in levels was observed between anti-galectin-9 igg1200 μg+anti-PD-1 compared to anti-PD-1 alone (p < 0.001). Such results indicate that the combination of anti-Gal 9 antibody and anti-PD-1 antibody is expected to achieve excellent therapeutic effects.

Table 18 below summarizes the results of the in vivo pharmacological studies.

TABLE 18 major pharmacodynamics in vivo

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In addition, tumor immune responses to treatment with G9.2-17 iggg 1 mouse mAb (also known as G9.2-17 mIgG), anti-PD-1 antibody, or a combination of G9.2-17 iggg 1 mouse mAb and anti-PD-1 antibody were studied in the B16F10 subcutaneous homology model described herein. As shown in fig. 10A-10B, the G9.2-17 and anti-PD-1 combinations showed synergy in decreasing tumor volume and increasing cd8+ cells in the mouse model. FIGS. 11A-11B show that the G9.2-17 antibody increases CD44 and TNFa expression in T cells within tumors.

Example 13 non-GLP Single dose, range explored intravenous toxicity study in Male Sprague Dawley rats with 1 week and 3 week post-dosing observation period

The study evaluated the anatomical endpoints of G9.2-17IgG4 following necropsy at week 1 (end-stage) and week 3 (recovery stage) following a single intravenous bolus administration to Sprague Dawley rats on days 8 and 22. All animals survived to the predetermined necropsy. In post-mortem animals at the end of the study or recovery period, there were no macroscopic findings, organ weight changes, or microscopic findings associated with the test samples.

The purpose of this non-GLP exploratory, single dose, range exploratory, intravenous toxicity study was to identify and characterize acute toxicity of G9.2-17IgG4 following 2 minutes of intravenous bolus administration followed by 1 week (end) and 3 weeks (recovery) of post-dosing observation period in Sprague Dawley rats.

This non-GLP single dose toxicity study was performed in 24 Sprague Dawley male rats to determine the toxicology and potential toxicity of G9.2-17 IgG4. The animals were administered vehicle or 10mg/kg, 30mg/kg or 70mg/kg G9.2-17IgG4 by slow bolus intravenous infusion over at least 2 minutes on day 1 followed by 1 week (end-stage, day 8) or 3 weeks (recovery, day 22) after administration. Study endpoints included mortality, clinical observations, weight and food consumption, clinical pathology (hematology, coagulation, clinical chemistry and urine analysis), toxico-kinetic parameters, ADA evaluation and pathology (gross necropsy, organ weight and histopathology). A summary of the experimental design is provided in table 19 below.

TABLE 19 design of experiment

Group number	Treatment of	Dosage level (mg/kg)	Number of males ^a
				1	Vehicle body ^b	0	6
2	G9.2-17 IgG4	10	6
				3	G9.2-17 IgG4	30	6
4	G9.2-17 IgG4	70	6

^a Euthanasia was performed on 3 animals/sex/group at end-stage necropsy on day 8; the remaining 3 animals/sex/group were euthanized at necropsy at day 22 recovery.

^b The vehicle was formulation buffer (20mM Tris,150mM NaCl,pH 8.0 ±0.05).

All surviving animals were necropsied on either day 8 or day 22. Complete necropsy was performed and organ weights were collected. All animals were weighed at the end and recovery stages. The tissues required for microscopic evaluation were trimmed, routinely treated, paraffin embedded and stained with hematoxylin and eosin.

There were no unexpected deaths during this study. All animals survived to end-stage or convalescent necropsy. The noted histological changes are believed to be occasional findings or associated with certain aspects of the experimental procedure rather than administration of the test article. There were no sample-related changes in the incidence, severity, or histological characteristics of these occasional tissue changes. No G9.2-17 IgG4-related findings were noted in clinical observations, body weight, food consumption, clinical pathology or anatomical pathology. In summary, single intravenous administration of 10, 30 and 70mg/kg G9.2-17 IgG4 to Sprague Dawley rats was tolerated with no adverse findings. Thus, under the conditions of this study, NOEL was 70mg/kg.

Example 14 non-GLP Single dose, range-explored intravenous infusion toxicity study of G9.2-17IgG4 in cynomolgus monkeys with 3 week post-dosing observation period

This non-GLP single dose toxicity study was performed in 8 cynomolgus monkeys to identify and characterize the acute toxicity of G9.2-17 IgG4. The animals (1 male [ M ]/1 female [ F ]/group) were administered vehicle or 30mg/kg, 100mg/kg or 200mg/kg G9.2-17IgG4 by 30 min Intravenous (IV) infusion followed by a 3 week post-administration observation period. The study endpoints included: mortality, clinical observations, body weight, and qualitative food consumption; clinical pathology (hematology, coagulation, clinical chemistry, immunophenotyping and galectin-9 expression on leukocyte subpopulations, and cytokine analysis); a toxicological kinetic parameter; serum collection for potential drug-resistant antibody evaluation (ADA); and soluble galectin-9 assays; and pathology (gross necropsy, organ weight and histopathology).

No G9.2-17IgG 4-related findings were noted in clinical observations, body weight, food consumption, clinical pathology (hematology, clinical chemistry, coagulation or cytokine analysis), immunophenotyping, galectin-9 expression on leukocyte subpopulations, soluble galectin-9 or anatomical pathology.

In summary, single intravenous infusion administration of 30, 100 and 200mg/kg G9.2-17 IgG4 to cynomolgus monkeys was tolerated without adverse findings. Thus, under the conditions of this study, the unoeheared adverse reaction level (NOAEL) was 200mg/kg, the highest dose level evaluated. The study design is shown in table 20.

TABLE 20 design of experiment

^a Group 4 was administered 1 circumferentially after group 1 to group 3 administration.

During the study, the vehicle and test article were administered by IV infusion through a catheter placed percutaneously in the saphenous vein for 30 minutes. Dosage levels were 30, 100 and 200mg/kg and were administered at a dosage volume of 20 mL/kg. The control group received vehicle in the same manner as the treatment group.

Animals were placed in sling-restraining devices during dosing. The vehicle or test article is administered based on recent body weight and using an infusion pump and sterile disposable syringe. The dosing syringe was filled with the appropriate volume of vehicle or test (20 mL/kg, 2mL additional). At the completion of dosing, animals were removed from the infusion system. The weight of each dosing syringe was recorded before the beginning and end of each infusion to determine dose liability.

Detailed clinical observations

Animals were removed from the cages and each animal was subjected to detailed clinical examinations 1 hour and 4.5 hours after the start of infusion (SOI) on day 1, after which it was performed once daily during the study. Animals were removed from the cages and each animal was subjected to detailed clinical examinations 1 hour and 4.5 hours after the start of infusion (SOI) on day 1, after which it was performed once daily during the study. Body weights of all animals were measured and recorded weekly at the time of transfer, prior to randomization, day-1 and during the study.

All animals were evaluated for clinical pathology (hematology, coagulation and clinical chemistry) prior to testing and on day 1 (pre-dosing), day 3, day 8 and day 21. Additional samples were collected 30 minutes after SOI (immediately after infusion) and 4.5, 8.5, 24.5 and 72.5 hours (relative to day 1) for determination of hematology parameters and peripheral blood lymphocyte and cytokine analysis samples. Bone marrow smears were collected and stored.

Blood samples (approximately 0.5 mL) were collected from all animals via femoral vein for determination of serum concentration of the test sample (see table 21). Animals were not fasted prior to blood collection except for the interval following the fasted for clinical pathology collection.

TABLE 21 biological analysis sample collection time Table

X = collect sample.

^a : only animals of group 1 were analyzed for test contents at the time point 0.583 hours after SOI. Additional time points may be analyzed at the discretion of the study taker.

For treatment, blood samples were collected in additive-free unobstructed microtubes and centrifuged at controlled room temperature within 1 hour after collection. The resulting serum was divided into 2 approximately equal aliquots and placed in pre-labeled freezer tubes. All aliquots were stored frozen at-60 ℃ to-90 ℃ within 2 hours after collection.

All euthanized animals were evaluated for post-mortem study at the scheduled necropsy.

Necropsy was performed under procedures approved by a veterinary pathologist. External abnormalities of the animals, including accessible bumps, were carefully examined. The skin was reflected from the ventral midline incision and any subcutaneous mass was identified and correlated with pre-mortem findings. Check if there is abnormality in abdominal cavity, thoracic cavity and cranial cavity. The organ is removed, inspected, and placed in fixative as needed. All designated tissues except the eye (including optic nerve) and testes were fixed in Neutral Buffered Formalin (NBF). Eyes (including optic nerve) and testes were placed in modified davison fixative, then transferred to 70% ethanol for up to three days, before final placement in NBF. Formalin is injected into the lungs through the trachea. Whole tissues and organs were collected from all animals.

The body weight of all animals and the organ weight specified by the protocol were recorded at the scheduled necropsy, and the appropriate organ weight ratio (relative to body weight and brain weight) was calculated. The paired organs were weighed together. The total weight of thyroid and parathyroid glands was collected.

Results

All animals survived to the scheduled necropsy on day 22. No clinical or veterinary results related to the test sample were noted in the treated animals. During the treatment or recovery period, no test-related effects on body weight were observed in the treated animals. At any dose level at any time interval, there was no G9.2-17 IgG4 related effect on hematological endpoints in either gender.

At any dose level at any time interval, there was no G9.2-17 IgG4-related effect on clotting time (i.e., activated partial thromboplastin time [ APTT ] and prothrombin time) or fibrinogen concentration in either gender. All fluctuations between individual coagulation values are considered sporadic, consistent with biologically and procedural related changes, and/or negligible in magnitude, and unrelated to G9.2-17 IgG4 administration.

At any dose level at any time interval, there was no G9.2-17 IgG4 related effect on clinical chemistry endpoint in either gender. All fluctuations between individual clinical chemistry values are considered sporadic, consistent with biological and procedural related changes, and/or negligible in magnitude, and unrelated to G9.2-17 IgG4 administration.

At any dose level at any time interval, there was no G9.2-17 IgG4-related effect on cytokine end-points in either gender. All fluctuations between individual cytokine values are considered sporadic, consistent with biologically and procedural related changes, and/or negligible in magnitude, and unrelated to G9.2-17 IgG4 administration.

Examination of the general necropsy observations showed findings that were not considered relevant to the test article. Organ weight changes not considered to be related to the test article. There were no changes associated with the test article.

In summary, single intravenous infusion administration of 30, 100 and 200mg/kg G9.2-17 IgG4 to cynomolgus monkeys was tolerated without adverse findings. Thus, under the conditions of this study, the unoeheared adverse reaction level (NOAEL) was 200mg/kg, the highest dose level evaluated.

Animals were removed from the cages and each animal was subjected to detailed clinical examinations 1 hour and 4.5 hours after the start of infusion (SOI) on day 1, after which it was performed once daily during the study.

Example 15: intravenous infusion study of G9.2-17 in cynomolgus monkeys

The objective of this study was to further characterize the toxicity and toxicology of test G9.2-17 (igg 4 monoclonal antibody conjugated to galectin-9) after 30 min Intravenous (IV) infusion once a week for 5 weeks in cynomolgus monkeys and evaluate the reversibility, progression or delayed appearance of any changes to after 3 weeks of recovery.

Design of experiment

Table 22 summarizes the study design.

TABLE 22 design of experiments

^a Based on recent actual weight measurements.

Animals used in the study (cynomolgus monkeys) were assigned to study groups according to standard, weight, randomization procedure (aimed at achieving similar group average weights). Male and female were randomized separately. The body weight assigned to the animals studied was within + -20% of the average body weight per sex.

Formulations lacking G9.2-17 ("vehicle") or comprising G9.2-17 ("test") were administered to animals by 30 minute IV infusion once a week for 5 weeks (day 1, day 8, day 15, day 22, and day 29) during the study. Dosage levels were 0, 100 and 300 mg/kg/dose and were administered at a dosage volume of 10 mL/kg. The control animal group received vehicle in the same manner as the treatment group. Doses were administered via saphenous vein through a percutaneously placed catheter, and a new sterile disposable syringe was used for each administration. Dose accountability was measured and recorded before and at the end of dosing on the pharmacokinetic sample collection days (day 1, day 15 and day 29) to ensure that ±10% of the target dose was administered. Individual doses are based on recent body weight. The last dosing site was marked for collection at end-stage and convalescent necropsy. All doses were administered within 8 hours after the test preparation.

The animals were subjected to a survival program, and measurements, as exemplified below.

Electrocardiographic examination was performed on all animals. Care was taken to avoid causing excessive excitation of the animals prior to recording the Electrocardiogram (ECG) to minimize extreme fluctuations or artifacts in these measurements. Standard ECG (10 leads) was recorded at 50 mm/s. RR, PR and QT intervals and QRS duration are measured using appropriate leads and heart rate is determined. The corrected QT (QTc) interval is calculated using a program based on the method described by Bazett (1920). All traces were evaluated and reported by the consulting veterinary cardiologist.

To aid in continuity and reliability, functional observational test combination (FOB) evaluations were performed by two independent raters for all occasions, consisting of detailed home cage and open area neurobehavioral evaluations (Gauvin and Baird, 2008). Each technician scored monkeys independently (without sharing results with each other) for each home and out-of-home score, and then assessed whether individual scores were consistent with the scores of their partners after the trial was completed. Each animal was subjected to a FOB evaluation prior to dosing (either on day-9 or day 8) to establish baseline differences, and 2 to 4 hours from the start of infusion on day 1 and day 15, and prior to end-stage and convalescent necropsy. Including but not limited to, assessment of activity level, posture, tear flow, salivation, tremor, twitching, spontaneous contractions, notch behavior, facial muscle movements, eyelid closure, pupillary response, response to stimuli (visual, auditory and food), body temperature, chaddock and Babinski reflex, proprioception, paresis, ataxia, range finding inaccuracy and slope assessment, movement and gait.

Blood pressure, including systolic, diastolic and mean arterial pressure, was measured and recorded for each animal. Blood pressure measurements were reported using three readings with Mean Arterial Pressure (MAP) within 20 mmHg.

The respiratory rate of each animal was measured and recorded 3 times at animal/collection intervals by visual assessment according to test facility SOP. The average of 3 collections is reported.

All animals were subjected to clinical pathology assessment (e.g., immunophenotyping and cytokine assessment) at predetermined time intervals. Bone marrow smears were collected and stored. Blood samples (approximately 0.5 mL) were collected from all animals via the femoral vein for determination of serum concentration of the test sample. Animals were not fasted prior to blood collection except for the interval following the fasted for clinical pathology collection. At the end of the study (day 36 or day 50), animals were euthanized and tissues were collected for histological treatment and microscopic evaluation.

Soluble galectin-9 was evaluated as follows. Blood samples (approximately 1 mL) were collected from all animals intravenously via the femoral vein 24 hours before dosing on day 1, day 8, day 15 and day 29 and beginning of infusion and before end-stage and/or convalescent necropsy for determination of serum soluble galectin-9. Animals were not fasted prior to blood collection except for the interval following the fasted for clinical pathology collection.

Soluble galectin-9 samples were treated as follows. Blood samples were collected in additive-free unobstructed tubes, allowed to coagulate at ambient temperature, and centrifuged at ambient temperature. The resulting serum was split into 2 aliquots (100 μl in aliquot 1 and the remainder in aliquot 2) and placed in pre-labeled freezer tubes. All aliquots were flash frozen on dry ice within 2 hours after collection and stored frozen at-60 to 90 ℃.

All the results shown in the reported tables are calculated from the original data rounding procedure using non-rounded values and may not be accurately reproduced from the single data provided.

Results

Mortality rate

All animals survived to a predetermined end-stage necropsy on day 36 and a convalescent necropsy on day 50.

Detailed clinical and veterinary observations

During the treatment or recovery period, no clinical or veterinary observations related to the test article were noted in the treated animals.

Functional observational test combination

During the treatment or recovery period, no test-related FOB observations were noted in the treated animals.

Body weight and weight gain

During the treatment or recovery period, no test-related effects on body weight and body weight gain were noted in the treated animals.

Ophthalmic examination

During the treatment or recovery period, no test-related effects on the ocular examination were noted in the treated animals.

Blood pressure value

During the treatment or recovery period, no test-related effects on blood pressure values were noted in the treated animals.

Respiratory rate value

During the treatment or recovery period, no test-related effects on respiratory rate values were noted in the treated animals.

Electrocardiogram

During the treatment or recovery period, no test-related effects on the electrocardiographic assessment were noted in the treated animals.

Hematology (Hematology)

At any dose level at any time point, there was no G9.2-17 related effect on hematological parameters in either gender.

Coagulation of blood

At any dose level at any time point, there was no G9.2-17 related effect on coagulation parameters in either gender.

Clinical chemistry

At any dose level at any time point, there was no G9.2-17 related effect on clinical chemistry parameters in either gender.

Urine analysis

During the middle of 13 weeks, no G9.2-17 related changes were observed among the urinalysis parameters of either sex at any dose level.

Cytokine(s)

No G9.2-17 related clear effect on cytokines was observed at any dose level or time point.

Peripheral Blood Leukocyte Analysis (PBLA)

At any dose level at any time point, there was no G9.2-17 related effect on PBLA endpoint in either gender.

Bioassay, galectin-9 and toxicokinetics evaluation

After dose administration, G9.2-17 was quantifiable in all cynomolgus monkey samples from all animals administered G9.2-17. No measurable amount of G9.2-17 was detected in the control cynomolgus monkey sample. In all cynomolgus monkey samples from all animals, soluble galectin-9 was quantifiable. In all serum samples obtained on day 1 from most of the G9.2-17 treated animals and on days 1 and 29 from control animals prior to dosing, the G9.2-17 serum concentrations were below bioassay quantification limit (LLOQ <0.04 ug/mL).

Gross pathology and organ weight

There were no clear macroscopic observations related to the test article in the main study or convalescent animals. For animals in the main study or convalescence period, there was also no sample related organ weight change.

Histopathology

There was no clear microscopic observation of the sample correlation.

In summary, once weekly intravenous infusion administration of 100 and 300mg/kg G9.2-17 to cynomolgus monkeys was tolerated for 5 weeks with no adverse findings.

Example 16: intravenous infusion study of G9.2-17 in Sprague Dawley rats

The purpose of this study was to evaluate the potential toxicity of G9.2-17 (an IgG4 human monoclonal antibody to galectin-9) when administered by intravenous infusion to Sprague Dawley rats (once a week for 4 weeks followed by a recovery period after 3 weeks of administration). In addition, the pharmacokinetic profile of G9.2-17 was determined.

Design of experiment

Table 23 summarizes the study design.

Table 23: study design

^a Individual dose volumes were calculated from the most recent body weight.

^b SSD animals: 3 animals/sex/group were used for TK collection, only after a single dose administration on day 1.

One hundred eighty six animals (Sprague Dawley rats) were randomly assigned to the treatment group by body weight. The control/vehicle, formulation buffer for test article and test article G9.2-17 were administered by a single IV injection in the tail vein at dosage levels of 0, 100 and 300mg/kg on days 1, 8, 15, 22 and 29. The test article was administered once to animals assigned to the SSD subgroup at dose levels of 100 and 300mg/kg on day 1.

Starting from the next day of environmental adaptation, a clinical observation was made every morning before cleaning the room. Mortality checks were performed twice daily to assess general animal health. Food consumption was estimated by weighing the food supply and the remaining amount in the container once a week. Average grams (g)/animal/day was calculated from weekly food consumption. Body weight was measured prior to randomization, on day-1, then weekly throughout the study, and on the day of each necropsy. Functional observation test combination (FOB) observations were recorded for SSB animals at approximately 24 hours after dose administration on days 1, 35 and 49. Urine was collected overnight using a metabolic cage. Samples were obtained on day 36 and day 50.

Animals were fasted overnight prior to each series collection including samples for serum chemistry. In these cases, the relevant clinical pathology assessment was from fasted animals. Blood is collected from the jugular vein of a restrained awake animal or from the vena cava of an anesthetized animal (at the end).

Parameters assessed during the survival check of the study included clinical observations, food consumption, body weight, functional observations and combinations of tests. Blood samples were collected at selected time points for clinical pathology (hematology, coagulation and serum chemistry) analysis. Urine samples were collected for urine analysis. Blood samples were also collected at selected time points for toxico-kinetic (TK), immunogenicity (e.g., anti-drug antibodies or ADA), and cytokine analysis. The animals were necropsied on day 36 and day 50. At each necropsy, gross observations and organ weights were recorded and tissues were collected for microscopy.

Results

Survival check

Mortality rate of: no abnormal clinical observations or body weight changes were noted in the animals during the study.

Clinical observation:no G9.2-17 related clinical observations were noted during the study.

Food consumption/body weight: no changes in G9.2-17 related food consumption, body weight or weight gain were noted during the study.

Clinical pathology: no G9.2-17 related changes were noted in the clinical pathology parameters.

Cell factorSub-analysis: there were no G9.2-17 related changes in serum concentrations of IL-2, IL-4, IFN-gamma, IL-5, IL-6, IL-10 and/or TNF-alpha, MCP-1 and MIP-1 b.

General pathology: there were no general observations related to G9.2-17. Furthermore, there was no change in absolute or relative organ weights associated with G9.2-17.

Histopathology: there was no histological findings associated with G9.2-17.

In summary, intravenous G9.2-17 administration of a total of 5 doses once a week to Sprague Dawley rats is generally well tolerated. There were no G9.2-17 related clinical observations, food consumption, body weight, FOB parameters, clinical pathology, cytokines, gross observations, or changes in organ weight.

Example 17: inhibition of polarization and repolarization of M2 macrophages

Macrophages play an indispensable role in the immune system, and have a decisive function in both innate and acquired immunity. M1 macrophages are generally thought to be potent effector cells that can kill tumor cells, while M2 polarized macrophages express a range of cytokines, chemokines and proteases to promote angiogenesis, lymphangiogenesis, tumor growth, metastasis and immunosuppression (Sica et al, 2008;Semin.Cancer Biol.2008;18:349-355). In M2 macrophages, the production of anti-inflammatory cytokines such as TGF-beta and IL-10 is enhanced (Martinez et al, front biosci.2008, 1 st, 13:453-61; mantovani et al, trends Immunol 2002, 23 (11): 549-55; zhang et al, JHematol Oncol 10,58 (2017)). Given that macrophages constitute a key component of the host immune response, inhibition of polarization or repolarization of M2 macrophages is an important therapeutic consideration in tumor immunotherapy (Poh and Ernst, front oncol.2018, day 3, month 12; 8:49).

Cd14+ monocytes were isolated using whole blood from three healthy human donors. Monocytes were allowed to differentiate into macrophages in X-VIVO-15 medium (Lonza) in 10cm tissue culture dishes for 7 days. Differentiated macrophages are used directly to assess inhibition of polarization, or they are cryopreserved and used in repolarization assays at a later time or at any other clinically indicated point in time. Prior to use in the assay, M0 macrophages were phenotyped.

Macrophage polarization was evaluated using two different polarization mixtures: a mixture containing IL-4 and IL-13, while the second contains only gal-9. The effect on M2 polarization was tested by adding G9.2-17 directly to one of these mixtures and incubating with macrophages for 48 hours. The effect of G9.2-17 on repolarization of M2 macrophages was tested via addition to M2 polarized macrophages.

The polarization state was identified by measuring secretion of IL-10 (repolarization) or TGF- β1 (polarization and inhibition of repolarization). These factors were quantified in cell culture supernatants using CytoMetric Bead Arrays according to the manufacturer's protocol.

Representative data from one donor showing the effect of G9.2-17 on the polarization of fresh monocyte-derived macrophages is in fig. 12. All donor macrophages showed similar results with reduced TGF- β1 secretion after incubation with G9.2-17 compared to isotype matched control or untreated cells. FIG. 12 shows the effect of previously frozen macrophages on TGF- β1 secretion after incubation with G9.2-17 or isotype matched controls. Treatment with 20ng/mL of the polarizing mixture significantly induced TGF- β1 secretion, whereas G9.2-17 treatment eliminated the IL-4/IL-13 dependent increase in TGF- β1 secretion. FIG. 13 shows the effect of IL-10 secretion on repolarization of cryopreserved macrophages. In the presence of both types of polarisation mixtures, treatment with G9.2-17 resulted in reduced levels of secreted IL-10 and TGF-b1 in all donors compared to untreated and IgG4 isotype control antibody controls.

This assay demonstrates that G9.2-17 can potently inhibit TGF- β1 and IL-10 at a concentration of 20 μg/ml.

Example 18: measurement of biomarkers

Multiple immunofluorescence (mIF) techniques are performed on clinical tissue from a patient. The mIF assay consisted of 10 rounds of staining, each round staining and imaging two biomarkers for a total of ten rounds. For each round, one antibody is conjugated with one of the two fluorescent dyes that will allow imaging of the biomarker, such that each round images the two biomarkers. The biomarker is stained, imaged, and then the signal quenched to allow further staining and imaging rounds to occur without exuding competing signals. When staining and imaging of the complete 19-marker panel is complete, the positives of each biomarker on the cells are classified by a deep learning algorithm trained to detect positive signals. When the analysis is complete, various data are generated, including the density and raw count of positive cells for each biomarker and co-expression of interest. Biomarkers 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.

EXAMPLE 9 evaluation of mouse galectin-9 in plasma by ELISA

This study evaluated galectin-9 in plasma of in situ pancreatic cancer xenograft model mPA6115 in female C57BL/6 mice. Mice were assigned to multiple groups and treated according to the study design shown in table 24 below. According to the protocol, plasma samples were collected from the retroorbital sinus on day 3 prior to the 1 st dose, and for euthanized mice dying (post dose), by cardiac puncture at the time of sacrifice, for all mice of groups 1-6 (pre dose) and 10 non-tumor bearing mice in group 7 (tumor implant on day 0) that were transplanted with tumors.

Table 24 study dosing schedule

P. = intraperitoneal; v. = intravenous; QW = once weekly; q4d=once every four days

Plasma samples were analyzed for galectin-9 levels by ELISA according to the following procedure:

1. all reagents and samples were left at room temperature (18-25 ℃) prior to use. All standards and samples were recommended to be run at least in duplicate.

2. According to the experiment, removable 8-hole slats were marked, where appropriate.

3. Mu.l of each standard and prepared sample were added to the appropriate wells. The wells were covered and incubated for 2.5 hours at room temperature with gentle shaking.

4. The solution was discarded and washed 4 times with 1X wash solution. Washing was performed by filling each well with wash buffer (300 μl) using a multichannel pipette or an automatic washing machine. Complete removal of liquid in each step is critical for good performance. After the last wash, any remaining wash buffer is removed by aspiration or decantation. The plate was inverted and blotted dry with a clean paper towel.

5. 100 μl of 1X prepared biotinylated antibody (reagent preparation step 3) was added to each well. Incubate with gentle shaking at room temperature for 1 hour.

6. The solution was discarded. The wash as in step 4 is repeated.

7. Mu.l of the prepared streptavidin solution was added to each well. Incubate for 45 min at room temperature with gentle shaking.

8. The solution was discarded. The wash as in step 4 is repeated.

9. Mu.l TMB one-step substrate reagent was added to each well. Incubate for 30 min at room temperature with gentle shaking in the absence of light.

10. Mu.l of stop solution was added to each well. Read immediately at 450 nm.

The results demonstrate that once tumors were transplanted in situ, galectin-9 serum levels in the mPA6115 mouse model increased, consistent with observations in pancreatic adenocarcinoma patients. This study demonstrated that galectin-9 serum levels were significantly increased in animals grown in situ with pancreatic ductal adenocarcinoma. This means that the source of this galectin-9 is indeed tumor tissue, further supporting the therapeutic approach of blocking galectin-9 in the context of this disease.

Equivalent scheme

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Accordingly, other embodiments are within the scope of the following claims.

Although several inventive embodiments have been described and illustrated herein, various other means and/or structures for performing the functions and/or obtaining the results and/or one or more advantages described herein will be apparent to those of ordinary skill in the art, and each such variation and/or modification is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, any combination of two or more such features, systems, articles, materials, kits, and/or methods is included within the scope of the present disclosure.

All definitions, as defined and used herein, should be understood to have precedence over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference to each of the cited subject matter, and in some cases, may encompass the entire document.

The indefinite articles "a" and "an" as used herein in the specification and claims should be understood to mean "at least one" unless explicitly indicated to the contrary.

As used herein in the specification and claims, the phrase "and/or" should be understood to mean "either or both" of the elements connected, i.e., the elements are in some cases present in combination and in other cases present separately. The use of "and/or" of a plurality of elements listed should be interpreted in the same manner, i.e. "one or more" of the elements so connected. In addition to the elements specifically identified by the "and/or" clause, other elements may optionally be present, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, reference to "a and/or B" when used in conjunction with an open language such as "comprising" may refer in one embodiment to a alone (optionally including elements other than B); in another embodiment may refer to B alone (optionally including elements other than a); in another embodiment, a and B (optionally including other elements) may be referred to; etc.

As used herein in the specification and claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when items are separated in a list, "or" and/or "should be understood to be inclusive, i.e., including at least one, but also including more than one of the elements or list of elements, and optionally, including additional unlisted items. Only the opposite terms, such as "only one of … …" or "exactly one of … …" or "consisting of … …" when used in a claim, will be referred to as comprising exactly one element of a plurality or list of elements. In general, the term "or" as used herein should be interpreted to mean exclusive choice (i.e., "one or the other but not both") only when crowned with exclusive terms such as "either," one of … …, "" only one of … …, "or" exactly one of … …. "consisting essentially of … …," when used in the claims, shall have its ordinary meaning as used in the patent statutes.

As used herein in the specification and claims, the phrase "at least one" with respect to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combination of elements in the list of elements. The definition also allows that elements other than the specifically identified elements within the list of elements to which the phrase "at least one" refers may optionally be present, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of a and B" (or, equivalently, "at least one of a or B," or, equivalently, "at least one of a and/or B") can refer in one embodiment to at least one (optionally including more than one) a without B present (and optionally including elements other than B); in another embodiment at least one (optionally including more than one) B is absent a (and optionally including elements other than a); in another embodiment at least one (optionally including more than one) a and at least one (optionally including more than one) B (and optionally including other elements); etc.

It should also be understood that the order of steps or acts of any method claimed herein that includes more than one step or act is not necessarily limited to the order in which the steps or acts of the method are recited, unless explicitly stated to the contrary.

Sequence listing

<110> PureTech LYT company (PureTech LYT, inc.)

<120> anti-galectin 9 antibodies and therapeutic uses thereof

<130> 112174-0211 (NP009WO1)

<140> not yet allocated

<141> along with the submission

<150> US 63/313,879

<151> 2022-02-25

<150> US 63/193,357

<151> 2021-05-26

<150> US 63/182,521

<151> 2021-04-30

<160> 35

<170> PatentIn version 3.5

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<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 6

Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp Tyr

1 5 10 15

<210> 7

<211> 124

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 7

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser

20 25 30

Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 8

<211> 108

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 8

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ser Thr Asp Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 9

<211> 20

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 9

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser

20

<210> 10

<211> 330

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 10

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 11

<211> 106

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 11

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

1 5 10 15

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

20 25 30

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

35 40 45

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

50 55 60

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

65 70 75 80

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

85 90 95

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 12

<211> 330

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 12

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 13

<211> 327

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 13

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Pro Gly Lys

325

<210> 14

<211> 327

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 14

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Pro Gly Lys

325

<210> 15

<211> 214

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 15

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ser Thr Asp Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 16

<211> 454

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 16

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser

20 25 30

Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210> 17

<211> 454

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser

20 25 30

Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210> 18

<211> 451

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 18

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser

20 25 30

Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

130 135 140

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn

195 200 205

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser

210 215 220

Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

260 265 270

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 19

<211> 451

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 19

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser

20 25 30

Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

130 135 140

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn

195 200 205

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser

210 215 220

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

260 265 270

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 20

<211> 327

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 20

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 21

<211> 327

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 21

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 22

<211> 451

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 22

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser

20 25 30

Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

130 135 140

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn

195 200 205

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser

210 215 220

Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

260 265 270

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Leu Gly Lys

450

<210> 23

<211> 451

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 23

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser

20 25 30

Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

130 135 140

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn

195 200 205

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser

210 215 220

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

260 265 270

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Leu Gly Lys

450

<210> 24

<211> 329

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 24

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 25

<211> 329

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 25

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 26

<211> 326

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 26

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Pro Gly

325

<210> 27

<211> 326

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 27

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly

325

<210> 28

<211> 326

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 28

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Pro Gly

325

<210> 29

<211> 326

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 29

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly

325

<210> 30

<211> 453

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 30

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser

20 25 30

Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly

450

<210> 31

<211> 453

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 31

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser

20 25 30

Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly

450

<210> 32

<211> 450

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 32

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser

20 25 30

Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

130 135 140

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn

195 200 205

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser

210 215 220

Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

260 265 270

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 33

<211> 450

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 33

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser

20 25 30

Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

130 135 140

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn

195 200 205

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser

210 215 220

Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

260 265 270

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Leu Gly

450

<210> 34

<211> 450

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 34

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser

20 25 30

Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

130 135 140

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn

195 200 205

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser

210 215 220

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

260 265 270

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 35

<211> 450

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Synthesis

<400> 35

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser

20 25 30

Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

130 135 140

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn

195 200 205

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser

210 215 220

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

260 265 270

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Leu Gly

450

Claims

1. A method for treating a solid tumor, the method comprising administering to a subject in need thereof an effective amount of an antibody that binds human galectin-9 (anti-galectin-9 antibody), wherein the anti-galectin-9 antibody comprises a light chain complementarity determining region 1 (CDR 1) as shown in SEQ ID NO:1, a light chain complementarity determining region 2 (CDR 2) as shown in SEQ ID NO:2, and a light chain complementarity determining region 3 (CDR 3) as shown in SEQ ID NO:3, and/or comprises a heavy chain complementarity determining region 1 (CDR 1) as shown in SEQ ID NO:4, a heavy chain complementarity determining region 2 (CDR 2) as shown in SEQ ID NO:5, and a heavy chain complementarity determining region 3 (CDR 3) as shown in SEQ ID NO:6, and wherein the anti-galectin-9 antibody is administered to the subject at a dose of about 0.2mg/kg to about 32mg/kg,

wherein the subject has one or more of the following characteristics:

(i) No resectable cancer;

(ii) Is not infected by SARS-CoV-2;

(iii) No active brain or leptomeningeal metastasis; and

(iv) With unresectable metastatic cancer, which is adenocarcinoma, optionally squamous cell carcinoma.

2. The method of claim 1, wherein the anti-galectin-9 antibody is administered to the subject at a dose of about 3mg/kg to about 15mg/kg or about 0.2mg/kg to about 16mg/kg once every two weeks to every six weeks, optionally once every two weeks.

3. The method of claim 2, wherein the anti-galectin-9 antibody is administered to the subject at a dose of about 0.2mg/kg, about 0.6mg/kg, about 0.63mg/kg, about 2mg/kg, about 4mg/kg, about 6mg/kg, about 6.3mg/kg, about 8mg/kg, about 10mg/kg, about 12mg/kg, or about 16mg/kg once every two weeks to once every six weeks, optionally once every two weeks.

4. The method of claim 1, wherein the anti-galectin-9 antibody is administered to the subject at a dose of about 650mg to about 1120mg once every two weeks to once every six weeks, optionally once every two weeks.

5. The method of claim 4, wherein the anti-galectin-9 antibody is administered to the subject at a dose of about 650mg to about 700mg once every two weeks to six weeks, optionally once every two weeks, or once every two weeks to six weeks, optionally once every two weeks at a dose of about 1040mg to about 1120 mg.

6. A method for treating a solid tumor, the method comprising administering to a subject in need thereof an effective amount of an antibody that binds human galectin-9 (an anti-galectin-9 antibody), wherein the anti-galectin-9 antibody comprises:

(a) A light chain comprising a light chain variable region (VL) comprising a Light Chain (LC) complementarity determining region 1 (CDR 1) comprising the amino acid sequence of SEQ ID NO. 1, a LC complementarity determining region 2 (CDR 2) comprising the amino acid sequence of SEQ ID NO. 2 and a LC complementarity determining region 3 (CDR 3) comprising the amino acid sequence of SEQ ID NO. 3, and

(b) A heavy chain comprising a heavy chain variable region (VH) comprising Heavy Chain (HC) complementarity determining region 1 (CDR 1) comprising the amino acid sequence of SEQ ID No. 4, HC complementarity determining region 2 (CDR 2) comprising the amino acid sequence of SEQ ID No. 5 and HC complementarity determining region 3 (CDR 3) comprising the amino acid sequence of SEQ ID No. 6;

wherein the anti-galectin-9 antibody is administered to the subject at a dose of about 0.2mg/kg to about 32mg/kg once a week.

7. The method of claim 6, wherein the anti-galectin-9 antibody is administered to the subject at a dose of about 10mg/kg to about 16mg/kg once a week.

8. The method of claim 7, wherein the anti-galectin-9 antibody is administered to the subject at a dose of 10mg/kg or 16mg/kg once a week.

9. The method of any one of claims 6-8, wherein the anti-galectin-9 antibody is administered to the subject at a dose of about 650mg to about 1120mg once a week.

10. The method of claim 9, wherein the anti-galectin-9 antibody is administered to the subject at a dose of about 650mg to about 700mg once per week or at a dose of about 1040 to about 1120mg once per week.

11. The method of any one of claims 1-10, wherein the solid tumor is an metastatic solid tumor.

12. The method of claim 11, wherein the solid tumor is 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 Gastrointestinal (GI) solid tumor.

13. The method of any one of claims 1-12, wherein the anti-galectin-9 antibody is administered to the subject by intravenous infusion.

14. The method of any one of claims 1-13, wherein V of the anti-galectin-9 antibody _L Comprising the amino acid sequence of SEQ ID NO. 8.

15. The method of any one of claims 1-13, wherein V of the anti-galectin-9 antibody _H IncludedThe amino acid sequence of SEQ ID NO. 7.

16. The method of any one of claims 1-15, wherein the anti-galectin-9 antibody is a full length antibody.

17. The method of claim 16, wherein the anti-galectin-9 antibody is an IgG1 or IgG4 molecule.

18. The method of claim 17, wherein the anti-galectin-9 antibody is a human IgG4 molecule having a modified Fc region relative to a wild-type human IgG4 counterpart.

19. The method of claim 18, wherein the modified Fc region comprises the amino acid sequence of SEQ ID No. 14.

20. The method of any one of claims 1-19, wherein 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.

21. The method of any one of claims 1-20, wherein the subject does not receive additional anti-cancer therapy concurrent with the treatment involving the anti-galectin-9 antibody.

22. The method of any one of claims 1-21, wherein the method further comprises administering to the subject an immune checkpoint inhibitor.

23. The method of claim 22, wherein the immune checkpoint inhibitor is an antibody that binds PD-1.

24. The method of claim 23, wherein the antibody that binds PD-1 is palbociclib, nivolumab, tirelib, rituximab, or cimetidine Li Shan antibody.

25. The method of any one of claims 22-24, wherein the subject is not exposed to any anti-PD-1 or anti-PD-L1 agent, does not receive microsatellite instability (MSI-H) and/or defective mismatch repair (dMMR), or a combination thereof, in any previous course of therapy.

26. The method of claim 24, wherein the antibody that binds PD-1 is nivolumab, which is administered to the subject at a dose of 240mg once every two weeks.

27. The method of any one of claims 22-26, wherein the checkpoint inhibitor is administered to the subject on the day the subject receives the anti-galectin-9 antibody.

28. The method of any one of claims 22-26, wherein the checkpoint inhibitor and the anti-galectin 9 antibody are administered to the subject for two consecutive days.

29. The method of any one of claims 22-26, wherein the administration of the checkpoint inhibitor occurs prior to the administration of the anti-galectin 9 antibody.

30. The method of any one of claims 1-29, wherein the subject has undergone one or more prior anti-cancer therapies.

31. The method of claim 30, wherein the one or more previous anti-cancer therapies comprise chemotherapy, immunotherapy, radiation therapy, a therapy involving biological agents, or a combination thereof.

32. The method of claim 30 or 31, wherein the subject has progressed on or is resistant to the one or more previous therapies.

33. The method of any one of claims 1-32, wherein the subject is a human patient having elevated galectin-9 levels relative to a control value.

34. The method of claim 33, wherein the human patient has elevated serum or plasma levels of galectin-9 relative to the control value.

35. The method of any one of claims 1-34, wherein the human patient has cancer cells that express galectin-9.

36. The method of any one of claims 1-35, wherein the human patient has immune cells expressing galectin-9.

37. The method of any one of claims 1-36, further comprising monitoring the subject for occurrence of an adverse effect.

38. The method of claim 37, further comprising reducing the dose of the anti-galectin-9 antibody, the dose of the checkpoint inhibitor, or both when adverse effects are observed.

39. The method of any one of claims 1-38, wherein multiple doses of the anti-galectin 9 antibody are administered to the subject, and a later dose is higher than an earlier dose.