Classifications

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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2851—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
  • A61K2039/507—Comprising a combination of two or more separate antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

• IO immuno-oncology
• Galectin-9 is a tandem-repeat lectin consisting of two carbohydrate recognition domains (CRDs) and was discovered and described for the first time in 1997 in patients suffering from Hodgkin’s lymphoma (HL) (Tureci et al., J. Biol. Chem. 1997, 272, 6416-6422). Three isoforms exist and can be located within the cell or extracellularly. Elevated Galectin-9 levels have been in observed a wide range of cancers, including melanoma, Hodgkin’s lymphoma, hepatocellular, pancreatic, gastric, colon and clear cell renal cell cancers (Wdowiak et al. Int. J. Mol. Sci. 2018, 19, 210).
• Galectin-9 was expressed in 57% of tumors and was significantly increased in the plasma of patients with advanced melanoma compared to healthy controls (Enninga et al., Melanoma Res. 2016 Oct; 26(5): 429-441).
• a number of studies have shown utility for Galectin- 9 as a prognostic marker, and more recently as a potential new drug target (Enninga et al., 2016; Kawashima et al. BJU Int 2014; 113: 320-332; Kageshita et al., Int J Cancer. 2002 Jun 20;99(6):809-16, and references therein).
• Galectin-9 has been described to play an important role in in a number of cellular processes such as adhesion, cancer cell aggregation, apoptosis, and chemotaxis. Recent studies have shown a role for Galectin-9 in immune modulation in support of the tumor, e.g., through negative regulation of Thl type responses, Th2 polarization and polarization of macrophages to the M2 phenotype.
• Galectin-9 has also been found to play a role in polarizing T cell differentiation into tumor suppressive phenotypes), as well as promoting tolerogenic macrophage programming and adaptive immune suppression (Daley et al., Nat Med., 2017, 23, 556-567).
• PD AC pancreatic ductal adenocarcinoma
• blockade of the checkpoint interaction between Galectin-9 and the receptor Dectin- 1 found on innate immune cells in the tumor microenvironment (TME) has been shown to increase anti-tumor immune responses in the TME and to slow tumor progression (Daley et al., Nat Med., 2017, 23, 556-567).
• Galectin-9 also has been found to bind to CD206, a surface marker of M2 type macrophages, resulting in a reduced secretion of CVL22 (MDC), a macrophage derived chemokine which has been associated with longer survival and lower recurrence risk in lung cancer (Enninga et al, J Pathol. 2018 Aug;245(4):468-477).
• the present disclosure is based, at least in part, on the development of 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, head and neck, breast cancer, lung cancer, or other GI solid tumors, involving an antibody capable of binding to human Galectin-9, either alone or in combination with a checkpoint inhibitor such as an anti-PD-1 antibody.
• PDAC pancreatic ductal adenocarcinoma
• CRC colorectal cancer
• HCC hepatocellular carcinoma
• CAA cholangiocarcinoma
• RRCC renal cell carcinoma
• urothelial head and neck, breast cancer, lung cancer, or other GI solid tumors
• the present disclosure is based, at least in part, on the unexpectedly discovery that an anti-Galectin 9 antibody G9.2-17 (IgG4) has a quicker clearance rate in human subjects as compared with other antibody therapeutics. Accordingly, a treatment regimen comprising a dosing schedule of once every week was developed to ensure a suitable plasma concentration, e.g., a therapeutic systemic exposure level, of the anti-Galectin 9 antibody for achieving therapeutic effects.
• a suitable plasma concentration e.g., a therapeutic systemic exposure level
• a method for treating a solid tumor comprising administering to a subject in need thereof (e.g., a human patient having the 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 the subject at a dose of about 0.2 mg/kg to about 32 mg/kg once every week to once every six weeks, e.g., 0.2 mg/kg, 0.63 mg/kg, 2 mg/kg, 6.3 mg/kg, 10 mg/kg, or 16 mg/kg once every week to once every six weeks.
• any of the anti-Galectin-9 antibodies disclosed herein may be administered to the subject by intravenous infusion.
• the anti-Galectin-9 antibody (e.g., G9.2-17 (IgG4)) may be administered to the subject at a dose of 0.2 mg/kg, 0.63 mg/kg, 2 mg/kg, 6.3 mg/kg, 10 mg/kg, or 16 mg/kg once every two weeks to once every four weeks.
• the anti-Galectin-9 antibody may be administered to the subject once every two weeks.
• the anti-Galectin-9 antibody e.g., G9.2-17 (IgG4)
• the anti-Gal-9 antibody such as G9.2-17 (IgG4) may be administered to a subject at a dose of about 650 mg to about 1120 mg once every 2-6 weeks, for example, once every 2 weeks, once every 3 weeks, or once every 4 weeks.
• the anti-Gal-9 antibody is administered to a subject at a dose of about 650 mg to about 700 mg once every 2-6 weeks, for example, once every 2 weeks, once every 3 weeks, or once every 4 weeks.
• the anti-Gal-9 antibody is administered to a subject at a dose of about 1040 mg to about 1120 mg once every 2-6 weeks, for example, once every 2 weeks, once every 3 weeks, or once every 4 weeks.
• the anti-Galectin-9 antibody (e.g., G9.2-17 (IgG4)) is administered to the subject at a dose of 0.2 mg/kg, 0.63 mg/kg, 2 mg/kg, 6.3 mg/kg, 10 mg/kg, or 16 mg/kg once every week.
• the anti-Galectin-9 antibody (e.g., G9.2-17 (IgG4)) is administered to the subject at a dose of 10 mg/kg or 16 mg/kg once every week.
• the anti-Gal-9 antibody disclosed herein, such as G9.2-17 (IgG4) may be administered to the subject at a dose of about 650 mg to about 1120 mg once every week.
• the anti-Gal-9 antibody can be administered to the subject at a dose of 10 mg/kg once every week or at a flat dose of about 650-700 mg once every week.
• the anti-Galectin-9 antibody can be administered to the subject at a dose of 16 mg/kg once every week or at a flat dose of about 1040- 1120 mg once every week.
• the anti-Galectin-9 antibody may comprise:
• a light chain comprising a light chain variable region (VL), which comprises a light chain (LC) complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1, a LC complementarity determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 2, and a LC complementarity determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3 and
• a heavy chain comprising a heavy chain variable region (VH), wich comprises a heavy chain (HC) complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 4, a HC complementarity determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 5, and a HC complementarity determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 6.
• HC heavy chain
• CDR1 comprising the amino acid sequence of SEQ ID NO: 4
• CDR2 HC complementarity determining region 2
• CDR3 HC complementarity determining region 3
• the VL of the anti-Galectin-9 antibody comprises the amino acid sequence of SEQ ID NO: 8.
• the VH of the anti-Galectin-9 antibody comprises the amino acid sequence of SEQ ID NO: 7.
• the anti-Galectin-9 antibody is a full-length antibody, for example, an IgGl or IgG4 molecule.
• the anti-Galectin-9 antibody is a human IgG4 molecule.
• Such an IgG4 molecule may have a modified Fc region relative to the wildtype human IgG4 counterpart.
• the modified Fc region comprises the amino acid sequence of SEQ ID NO: 14.
• the anti-Galectin-9 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 15.
• Such an anti-Galectin-9 antibody may be G9.2-17 (IgG4) as disclosed herein.
• 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, head and neck, breast cancer, lung cancer, or other GI solid tumors.
• the solid tumor is a metastatic tumor.
• the method comprises 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 anti- Galectin-9 antibody).
• the subject has one or more of the following features: (i) has no resectable cancer; (ii) has no infection by SARS-CoV-2; (iii) has no active brain or leptomeningeal metastasis; and (iv) has unresectable metastatic cancer, which is adenocarcinoma, optionally squamous cell carcinoma.
• the subject is free of other anti-cancer therapy concurrently with the anti-Galectin-9 antibody.
• the method may further comprise administering to the subject an immune checkpoint inhibitor.
• the immune checkpoint inhibitor is an antibody that binds PD-1. Examples include pembrolizumab, nivolumab, tislelizumab, dostarlimab, or cemiplimab.
• the subject is free of exposure to any anti-PD-1 or anti-PD-Ll agent in any prior lines of therapy, free of microstatellite instability (MSI-H) and/or deficient mismatch repair (dMMR), or a combination thereof.
• MSI-H microstatellite instability
• dMMR deficient mismatch repair
• the antibody that binds PD-1 is nivolumab. In some instances, nivolumab is administered to the subject at a dose of 240 mg once every two weeks. In another example, the antibody that binds PD-1 is tislelizumab. In some instances, tislelizumab is administered intravenously at a dose of about 200 mg once every 3 weeks or at a dose of about 400 mg every six weeks.
• the anti-Galectin-9 antibody is administered to the subject at a dose of about 0.2 mg/kg to about 32 mg/kg (e.g., about 3 mg/kg to about 15 mg/kg or about 2 mg/kg to about 16 mg/kg or a higher dose level, or about 0.2 mg/kg to about 15 mg/kg. or about 0.2 to about 16 mg/kg or a higher dose level) once every 2-3 weeks.
• the anti- Galectin-9 antibody is administered to the subject at a dose selected from 0.2 mg/kg, 0.63 mg/kg, 2 mg/kg, 4 mg/kg, 6 mg/kg, 6.3 mg/kg, 8 mg/kg, 10 mg/kg, 12 mg/kg, or 16 mg/kg or higher dose level In some embodiments, the anti-Galectin-9 antibody is administered to the subject at a dose selected from 2 mg/kg, 4 mg/kg, 8 mg/kg, 12 mg/kg, or 16 mg/kg or a higher dose level.
• the anti-Galectin-9 antibody is administered to the subject at a dose selected from 0.2 mg/kg, 0.63 mg/kg, 2 mg/kg, 4 mg/kg, 6 mg/kg, 6.3 mg/kg, 10 mg/kg, or 16 mg/kg or a 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 2 mg/kg, 4 mg/kg, 8 mg/kg, 12 mg/kg, or 16 mg/kg or a higher dose level once every 2 weeks.
• the anti-Galectin-9 antibody is administered to the subject at a dose selected from 0.2 mg/kg, 0.63 mg/kg, 2 mg/kg, 4 mg/kg, 6 mg/kg, 6.3 mg/kg, 10 mg/kg, or 16 mg/kg or a higher dose level once every 2 weeks.
• the anti-Galectin-9 antibody is administered once every 2 weeks for one cycle, once every 2 weeks for two cycles, once every 2 weeks for 3 cycles, once every 2 weeks for 4 cycles, or once every 2 weeks for more than 4 cycles.
• 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.
• the cycles extend for a duration of 3 months to 6 months, or 6 months to 12 months or 12 months to 24 months or longer.
• the cycle length is modified, e.g., temporarily or permanently to a longer duration, e.g., 3 weeks or 4 weeks.
• the anti-Galectin-9 antibody is administered to the subject by intravenous infusion.
• the cancer is metastatic cancer, including a metastatic cancer of any of the above-mentioned cancers.
• the method of treatment comprising administering the anti-Galectin-9 antibody does not include any other concurrent anti-cancer therapy.
• the method of treatment employing the anti-Galectin-9 antibody includes another concurrent anti-cancer therapy.
• the method of treatment employing the anti-Galectin-9 antibody further comprises administering to the subject an immune checkpoint inhibitor.
• the immune checkpoint inhibitor is an antibody that binds PD-1, for example, pembrolizumab, nivolumab, tislelizumab, dostarlimab or cemiplimab.
• the antibody that binds PD-1 is nivolumab, which is administered to the subject at a dose of 240 mg once every two weeks.
• the antibody that binds PD-1 is nivolumab, which is administered to the subject at a dose of about 240 mg every two weeks or about 480 mg once every 4 weeks. In some embodiments, the antibody that binds PD-1 is prembrolizumab, which is administered at a dose of 200 mg once every 3 weeks. In some embodiments, the antibody that binds PD-1 is cemiplimab, which is administered at a dose of about 350 mg once every 3 weeks. In some embodiments, the antibody that binds PD-1 is tislelizumab, which is administered at a dose of about 200 mg once every 3 weeks or about 400 mg every six weeks.
• the antibody that binds PD-1 is dostarlimab, which is administered at a dose of about 500 mg once every 3 weeks or at a dose of about 1000 mg every six weeks.
• the immune checkpoint inhibitor is administered by intravenous infusion.
• the subject is (v) free of exposure to any anti-PD-1 or anti-PD-Ll agent in any prior lines of therapy, free of microsatellite instability (MSI-H) and/or deficient mismatch repair (dMMR), or a combination thereof.
• the subject has microsatellite instability (MSI-H) and/or deficient mismatch repair (dMMR), or a combination thereof.
• the checkpoint inhibitor is administered to the subject on a day when the subject receives the anti-Galectin 9 antibody.
• the checkpoint inhibitor and the anti-Galectin 9 antibody are administered to the subject on two consecutive days.
• the administration of the checkpoint inhibitor is performed prior to the administration of the anti- Galectin 9 antibody or vice versa.
• the subject has undergone one or more prior anti-cancer therapies.
• the one or more prior anti-cancer therapies comprise chemotherapy, immunotherapy, radiation therapy, a therapy involving a biologic agent, or a combination thereof.
• the subject has progressed disease through the one or more prior anti-cancer therapies or is resistant to the one or more prior therapies.
• the subject is a human patient having an elevated level of Galectin-9 relative to a control value.
• the human patient has an elevated serum or plasma level of Galectin-9 relative to the control value.
• the human patient has cancer cells expressing Galectin-9.
• the human patient has immune cells expressing Galectin-9.
• the cancer cells are in tumor organoids derived from the human patient.
• the control value is based on a value obtained from a healthy human subject.
• any of the methods disclosed herein may further comprise monitoring occurrence of adverse effects in the subject.
• the method may further comprise reducing the dose of the anti-Galectin-9 antibody, the dose of the checkpoint inhibitor, or both when an adverse effect is observed.
• the subject is administered multiple doses of the anti-Galectin 9 antibody and a later dose is higher than an earlier dose.
• compositions for use in treating a solid tumor e.g., those described herein and including metastatic solid tumors
• uses of any of the anti-Galectin-9 antibodies for manufacturing a medicament for treating the solid tumor either taken alone or in combination with a checkpoint inhibitor such as any of the anti-PD-1 antibodies disclosed herein.
• FIG. 1 is a schematic depicting an exemplary study scheme.
• CRM reassessment method
• RP2D recommended Phase 2 dose
• PK pharmacokinetics
• PD pharmacodynamics
• PDAC pancreatic ductal adenocarcinoma
• CRC colorectal cancer
• CCA cholangiocarcinoma
• TBD to be decided.
• FIG. 2 is a graph showing a representative size exclusion chromatography (SEC) profile for the anti-Galectin-9 antibody. The high molecular weight peaks are labeled.
• SEC size exclusion chromatography
• FIGS. 3A-3F include bar graphs showing levels of Galectin-9 expression as measured in T cells (CD3 + ), macrophages (CDllb+,) and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from a pancreatic adenocarcinoma biopsy using anti-Galectin-9 G9.2-17 Fab fragment and a commercially available anti-Galectin-9 antibody (9M1-3).
• S2 fraction organoids.
• S3 fraction single cells.
• Corresponding isotype for G9.2-17 Fab (“Fab isotype”) and “fluorescence minus one” (FMO) 9M1-3 (“Gal9 FMO”) were used as controls for specificity, background staining and fluorescence bleed through from other channels.
• FIG. 3A shows levels of Galectin-9 in CD3 + cells as measured in the S3 fraction.
• FIG. 3B shows levels of Galectin-9 in CDllb + cells as measured in the S3 fraction.
• FIG. 3C shows levels of Galectin-9 in Epcam + cells as measured in the S3 fraction.
• FIG. 3D shows levels of Galectin-9 in CD3 + cells as measured in the S2 fraction.
• FIG. 3E shows levels of Galectin-9 in CDllb + cells as measured in the S2 fraction.
• FIG. 3F shows levels of Galectin-9 in Epcam + cells as measured in the S2 fraction.
• FIGS. 4A-4F include bar graphs showing levels of Galectin-9 expression as measured in T cells (CD3 + ), macrophages (CDllb+,) and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from a colorectal carcinoma biopsy using anti-Galectin-9 G9.2-17 Fab fragment and a commercially available anti-Galectin-9 antibody (9M1-3).
• S2 fraction organoids.
• S3 fraction single cells.
• Corresponding isotype for G9.2-17 Fab (“Fab isotype”) and FMO 9M1-3 (“Gal9 FMO”) were used controls for specificity, background staining and fluorescence bleed through from other channels.
• FIG. 4A shows levels of Galectin-9 in CD3 + cells as measured in the S3 fraction.
• FIG. 4B shows levels of Galectin -9 in GDI lb + cells as measured in the S3 fraction.
• FIG. 4C shows levels of Galectin-9 in Epcam + cells as measured in the S3 fraction.
• FIG. 4D shows levels of Galectin -9 in CD3 + cells as measured in the S2 fraction.
• FIG. 4E shows levels of Galectin-9 in CDllb + cells as measured in the S2 fraction.
• FIG. 4F shows levels of Galectin -9 in Epcam + cells as measured in the S2 fraction.
• FIGS. 5A-5F include bar graphs showing levels of Galectin-9 expression as measured in T cells (CD3 + ), macrophages (CDllb+,) and tumor cells (Epcam+) in S2 and S3 organoid fractions derived from a second pancreatic adenocarcinoma biopsy using anti-Galectin-9 G9.2-17 Fab fragment and a commercially available Galectin-9 antibody (9M1-3).
• S2 fraction organoids.
• S3 fraction single cells.
• Corresponding isotype for G9.2-17 Fab (“Fab isotype”) and FMO 9M1-3 (“Gal9 FMO”) were used as controls for specificity, background staining and fluorescence bleed through from other channels.
• FIG. 5A shows levels of Galectin-9 in CD3 + cells as measured in the S3 fraction.
• FIG. 5B shows levels of Galectin-9 in CDllb + cells as measured in the S3 fraction.
• FIG. 5C shows levels of Galectin-9 in Epcam + cells as measured in the S3 fraction.
• FIG. 5D shows levels of Galectin-9 in CD3 + cells as measured in the S2 fraction.
• FIG. 5E shows levels of Galectin-9 in CDllb + cells as measured in the S2 fraction.
• FIG. 5F shows levels of Galectin-9 in Epcam + cells as measured in the S2 fraction.
• FIGS. 6A-6C include photographs of immunohistochemical analysis of various tumors using anti-Galectin-9 antibody 1G3. All magnifications are 200X.
• FIG. 6A shows chemotherapy- treated colorectal cancer with heterogeneous intensity score 2 and 3 (moderate and high) Galectin-9 expression. Galectin-9 staining was observed at the cell membrane in particular; additionally, intraglandular macrophages are moderately positive and stromal reaction in tumor shows multinucleated macrophage giant cells with moderately strong Galectin-9 expression.
• FIG. 6B shows liver metastasis of colorectal carcinoma with high (intensity score 3) Galectin-9 expression. Staining is located on the membrane and in the cytoplasm.
• FIG. 6C shows Galectin- 9 positive (intensity score 2) entrapped bile ducts and Galectin-9 negative cancer.
• FIG. 7 includes a graph showing the fraction of annexin V- and propidium iodide (Ex- positive cells plotted as a function of antibody concentration used.
• MOLM-13 cells were co- incubated with varying concentrations of either G9.2-17 or human IgG4 isotype antibody and recombinant human Galectin-9 for 16 hours.
• Cells were stained with annexin V and propidium iodide prior to analysis by flow cytometry. Each condition was performed in triplicate. Analysis was performed on FlowJo software.
• FIGS. 8A-8B depict graphs showing results of a study in which mice treated with G9.2- 17 mlgG2a alone or in combination with oPD-1 mAb.
• FIG. 8B depicts bar graphs showing tumors were excised from control and treated animals at the end of experiment (Day 18) and processed for flow cytometry of intra-tumoral immune cells and related activation and immunosuppressive markers. Mouse tumors were digested before flow. Flow cytometry was carried out on the Attune NxT flow cytometer (ThermoFisher Scientific, Waltham, MA). Data were analyzed using FlowJo v.10.1 (Treestar, Ashland, OR)
• FIGS. 9A-9B depict graphs showing the results of ADCC assays performed with the IgGl form of G9.2-17 (FIG. 9 A) and the IgG4 form of G9.2-17 (FIG. 9B).
• G9.2-17 does not mediate ADCC (FIG. 9B).
• FIGS. 10A-10B depict graphs showing the effect of 9.2-17 in a B16F10 subcutaneous syngeneic model. Tumors were engrafted subcutaneously and treated with G9.2-17 IgGl mouse mAb, anti-PD-1 antibody or a combination of G9.2-17 IgGl mouse mAb and anti-PD-1 antibody.
• FIG. 10A depicts a graph showing the effect on tumor volume.
• FIG. 10B depicts a graph showing intratumoral CD8 T cell infiltration. Results show that intra-tumoral presence effector T cells were enhanced in the combination arm.
• FIGS. 11A-11B include charts showing cholangiocarcinoma patient-derived tumor cultures ex vivo (organoids) treated with G9.2-17.
• Patient derived tumor cultures ex vivo (organoids) were treated with G9.2-17 or isotype control for three days.
• Expression of CD44 (FIG. 11A), and TNFa (FIG. 11B) in CD3+ T cells from PDOTS was assessed.
• FIG. 12 includes a graph showing the effect of G2.9-17 on TGF-betal secretion measurements in whole blood of an exemplary healthy human donor.
• IgG4 isotype is a negative control antibody.
• Data represent mean + SEM of triplicate measures. Significance was determined by two-way ANOVA with Dunnett’s multiple comparison test. * p ⁇ 0.05
• FIG. 13 includes a graph showing the effect of G2.9-17 on IL-10 secretion in whole blood of an exemplary healthy human donor.
• IgG4 isotype is a negative control antibody.
• Data represent the mean ( ⁇ SEM) of triplicate. Significance was determined by two-way ANOVA with Tukey’s multiple comparisons test, * P ⁇ 0.05.
• anti-Galectin-9 antibodies e.g., G9.2-17 (IgG4)
• solid tumors for example, pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), cholangiocarcinoma (CAA), renal cell carcinoma (RCC), urothelial, head and neck, breast cancer, lung cancer, or other GI solid tumors.
• PDAC pancreatic ductal adenocarcinoma
• CRCC colorectal cancer
• HCC hepatocellular carcinoma
• CAA cholangiocarcinoma
• RRCC renal cell carcinoma
• urothelial head and neck
• breast cancer breast cancer
• lung cancer or other GI solid tumors.
• the cancers are metastatic.
• the methods disclosed herein provide specific doses and/or dosing schedules, for example, 0.2 mg/kg to 16 mg/kg of the antibody once every week (e.g., 10 mg/kg or 16 mg/kg once every week). It was discovered that G9.2-17 (IgG4) has an unexpectedly quick clearance rate in human subjects as compared with conventional antibody therapeutics. Accordingly, a treatment regimen comprising a dosing schedule of once very week was developed to ensure a systemic exposure level of the anti- Galectin 9 antibody that achieves therapeutic effect. In some instances, the methods disclosed herein target specific patient populations, for example, patients who have undergone prior treatment and show disease progression through the prior treatment, or patients who are resistant (de novo or acquired) to the prior treatment.
• Galectin-9 a tandem-repeat lectin, is a beta-galactoside-binding protein, which has been shown to have a role in modulating cell-cell and cell-matrix interactions. It is found to be strongly overexpressed in Hodgkin’s disease tissue and in other pathologic states. It has in some instances also been found circulating in the tumor microenvironment (TME).
• TAE tumor microenvironment
• Galectin-9 is found to interact with Dectin- 1, an innate immune receptor which is highly expressed on macrophages in PDAC, as well as on cancer cells (Daley, et al. Nat Med. 2017;23(5):556-6). Regardless of the source of Galectin-9, disruption of its interaction with Dectin- 1 has been shown to lead to the reprogramming of CD4 + and CD8 + cells into indispensable mediators of anti-tumor immunity. Thus, Galectin-9 serves as a valuable therapeutic target for blocking the signaling mediated by Dectin- 1. Accordingly, in some embodiments, the anti-Galectin-9 antibodies describe herein disrupt the interaction between Galectin-9 and Dectin- 1.
• Galectin-9 is also found to interact with TIM-3, a type I cell surface glycoprotein expressed on the surface of leukemic stem cells in all varieties of acute myeloid leukemia (except for M3 (acute promyelocytic leukemia)), but not expressed in normal human hematopoietic stem cells (HSCs).
• TIM-3 a type I cell surface glycoprotein expressed on the surface of leukemic stem cells in all varieties of acute myeloid leukemia (except for M3 (acute promyelocytic leukemia)), but not expressed in normal human hematopoietic stem cells (HSCs).
• TIM- 3 signaling resulting from Galectin-9 ligation has been found to have a pleiotropic effect on immune cells, inducing apoptosis in Thl cells (Zhu et al., Nat Immunol., 2005, 6:1245-1252) and stimulating the secretion of tumor necrosis factor-a (TNF-a), leading to the maturation of monocytes into dendritic cells, resulting in inflammation by innate immunity (Kuchroo et al., Nat Rev Immunol., 2008, 8:577-580).
• Galectin-9/TIM-3 signaling has been found to co-activate NF-KB and P-catenin signaling, two pathways that promote LSC self- renewal (Kikushige et al., Cell Stem Cell, 2015, 17(3):341-352).
• An anti-Galectin-9 antibody that interferes with Galectin-9/TIM-3 binding could have a therapeutic effect, especially with respect to leukemia and other hematological malignancies. Accordingly, in some embodiments, the anti- Galectin-9 antibodies described herein disrupt the interaction between Galectin-9 and TIM-3.
• Galectin-9 is found to interact with CD206, a mannose receptor highly expressed on M2 polarized macrophages, thereby promoting tumor survival (Enninga et al., J Pathol. 2018 Aug;245(4):468-477).
• Tumor-associated macrophages expressing CD206 are mediators of tumor immunosuppression, angiogenesis, metastasis, and relapse (see, e.g., Scodeller et al., Set Rep. 2017 Nov 7;7(1): 14655, and references therein).
• Ml also termed classically activated macrophages
• Thl-related cytokines and bacterial products express high levels of IL- 12, and are tumoricidal.
• M2 macrophages
• Th2-related factors express high level of anti-inflammatory cytokines, such as IL- 10, and facilitate tumor progression (Biswas and Mantovani; Nat Immunol. 2010 Oct; ll(10):889-96).
• the pro-tumoral effects of M2 include the promotion of angiogenesis, advancement of invasion and metastasis, and the protection of the tumor cells from chemotherapy-induced apoptosis (Hu et al., Tumour Biol. 2015 Dec; 36(12): 9119-9126, and references therein).
• Tumor-associated macrophages are thought be of M2-like phenotype and have a protumor role.
• Galectin-9 has been shown to mediate myeloid cell differentiation toward an M2 phenotype (Enninga et al., Melanoma Res. 2016 Oct; 26(5):429-41). It is possible that Galectin-9 binding CD206 may result in reprogramming TAMs towards the M2 phenotype, similar to what has been previously shown for Dectin- 1. Without wishing to be bound by theory, blocking the interaction of Galectin-9 with CD206 may provide one mechanism by which an anti- Galectin-9 antibody, e.g., a G9.2-17 antibody, can be therapeutically beneficial. Accordingly, in some embodiments, the anti-Galectin-9 antibodies described herein disrupt the interaction between Galectin-9 and CD206.
• Galectin-9 has also been shown to interact with protein disulfide isomerase (PDI) and 4- 1BB (Bi S, et al. Proc Natl Acad Set USA. 2011; 108(26): 10650-5; Madireddi et al. J Exp Med. 2014;211(7): 1433-48).
• PDI protein disulfide isomerase
• 4- 1BB Bi S, et al. Proc Natl Acad Set USA. 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 a Galectin-9 signaling plays a role).
• an anti-Galectin-9 antibody may block a signaling pathway mediated by Galectin-9.
• the antibody may interfere with the interaction between Galectin-9 and its binding partner (e.g., Dectin- 1, TIM-3 or CD206), thereby blocking the signaling triggered by the Galectin-9/Ligand interaction.
• an anti-Galectin-9 antibody may also exert its therapeutic effect by inducing blockade and/or cytotoxicity, for example, ADCC, CDC, or ADCP against pathologic cells that express Galectin-9.
• a pathologic cell refers to a cell that contributes to the initiation and/or development of a disease, either directly or indirectly. See, e.g., WO2019/084553, WG2020/198390, WG2020/0223702, and WO2021022256, the relevant disclosures of each of which are incorporated by reference for the subject matter and purpose referenced herein.
• the anti-Galectin-9 antibodies disclosed herein are capable of suppressing the signaling mediated by Galectin-9 (e.g., the signaling pathway mediated by Galectin-9/Dectin-l or Galectin- 9/Tim-3) or eliminating pathologic cells expressing Galectin-9 via, e.g., ADCC. Accordingly, the anti-Galectin-9 antibodies described herein can be used for inhibiting any of the Galectin-9 signaling and/or eliminating Galectin-9 positive pathologic cells, thereby benefiting treatment of diseases associated with Galectin-9.
• Anti-Galectin-9 antibodies such as G9.2-17 were found to be effective in inducing apoptosis against cells expressing Galectin-9. Further, the anti-tumor effects of anti-Galectin-9 antibodies such as G9.2-17 were demonstrated in a mouse model, either by itself, or in combination with a checkpoint inhibitor (e.g., an anti-PD-1 antibody). 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 (PDOTs).
• PDOTs patient derived organoid tumor models
• the orthotopic PDAC KPC mouse model (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-l-Cre) that was used recapitulates many features of human disease, including unresponsiveness to approved checkpoint inhibitors (Bisht and Feldmann G; Animal models for modeling pancreatic cancer and novel drug discovery; Expert Opin Drug Discov. 2019;14(2):127-142; Weidenhofer et al., Animal models of pancreatic cancer and their application in clinical research; Gastrointestinal Cancer: Targets and Therapy 2016;6).
• the B16F10 melanoma mouse model has been a long-standing standard to test immunotherapies (Curran et al., PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors; Proc Natl Acad Sci U S A. 2010; 107(9):4275-4280).
• PDOTs isolated from fresh human tumor samples retain autologous lymphoid and myeloid cell populations, including antigen-experienced tumor infiltrating CD4 and CDS T lymphocytes, and respond to immune therapies in short-term ex vivo culture (Jenkins et al. Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids. Cancer Discov.
• G9.2-17 mouse IgGl (G9.2-17 mlgGl contains the exact same binding epitope as G9.2-17 human IgG4 and has the same effector function), which achieves significant reduction of tumor growth already as a single agent in the orthotopic KPC model, where approved checkpoint inhibitors do not work.
• G9.2-17 significantly exceeds the efficacy of anti-PD-1.
• modulation of the intra-tumoral immune microenvironment using G9.2-17 mlgGl through the upregulation of effector T cell activity and inhibition of immunosuppressive signals, as well as the augmentation of intra- tumoral CD8 T cell infiltration was demonstrated.
• anti-Galectin-9 antibodies for treating certain cancers as disclosed herein.
• the present disclosure provides anti-Galectin-9 antibody G9.2-17 and functional variants thereof for use in the treatment methods disclosed herein.
• An antibody is an immunoglobulin molecule capable of specific 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.
• antibody e.g., anti-Galectin-9 antibody
• An antibody e.g., anti-Galectin-9 antibody
• an antibody of any class such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class.
• immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.
• the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
• the subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known.
• a typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are usually involved in antigen binding.
• VH and VL regions can be further subdivided into regions of hypervariability, also known as “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as “framework regions” (“ER”).
• CDR complementarity determining regions
• ER framework regions
• Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
• the extent of the framework region and CDRs can be precisely identified using methodology known in the art, for example, by the Kabat definition, the Chothia definition, the AbM definition, the EU definition, the “Contact” numbering scheme, the IMGT” numbering scheme, the “AHo” numbering scheme, and/or the contact definition, all of which are well known in the art. See, e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department 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.
• the anti-Galectin-9 antibody described herein is a full-length antibody, which contains two heavy chains and two light chains, each including a variable domain and a constant domain.
• the anti-Galectin-9 antibody can be an antigen- binding fragment of a full-length antibody.
• binding fragments encompassed within the term “antigen-binding fragment” of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) that retains functionality.
• a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CHI domains
• F(ab')2 fragment a bivalent fragment including two Fab fragment
• the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv).
• scFv single chain Fv
• any of the antibodies described herein can be either monoclonal or polyclonal.
• a “monoclonal antibody” refers to a homogenous antibody population and a “polyclonal antibody” refers to a heterogeneous antibody population. These two terms do not limit the source of an antibody or the manner in which it is made.
• Reference antibody G9.2-17 refers to an antibody capable of binding to human Galectin-9 and comprises a heavy chain variable region of SEQ ID NO: 7 and a light chain variable domain of SEQ ID NO: 8, both of which are provided below.
• the anti-Galectin-9 antibody for use in the methods disclosed herein is the G9.2-17 antibody.
• the anti-Galectin-9 antibody for use in the methods disclosed herein is an antibody having the same heavy chain complementarity determining regions (CDRs) as reference antibody G9.2-17 and/or the same light chain complementarity determining regions as reference antibody G9.2-17.
• Two antibodies having the same VH and/or VL CDRS means that their CDRs are identical when determined by the same approach (e.g., the Kabat approach, the Chothia approach, the AbM approach, the Contact approach, or the IMGT approach as known in the art. See, e.g. , bioinf.org.uk/abs/).
• the heavy and light chain CDRs of reference antibody G9.2-17 is provided in Table 1 below (determined using the Kabat methodology):
• the anti-Galectin-9 antibody for use in the methods disclosed herein may comprise (following the Kabat scheme) a heavy chain complementarity determining region 1 (CDR1) set forth as SEQ ID NO: 4, a heavy chain complementarity determining region 2 (CDR2) set forth as SEQ ID NO: 5, and a heavy chain complementarity determining region 3 (CDR3) set forth as SEQ ID NO: 6 and/or may comprise a light chain complementarity determining region 1 (CDR1) set forth as SEQ ID NO: 1, a light chain complementarity determining region 2 (CDR2) set forth as SEQ ID NO: 2, and a light chain complementarity determining region 3 (CDR3) set forth as SEQ ID NO: 3.
• CDR1 heavy chain complementarity determining region 1
• CDR2 light chain complementarity determining region 2
• CDR3 light chain complementarity determining region 3
• the anti- Galectin-9 antibody can be in any format as disclosed herein, for example, a full-length antibody or a Fab.
• G9.2-17(IgG4) used herein refers to a G9.2-17 antibody which is an IgG4 molecule.
• G9.2-17 (Fab) refers to a G9.2-17 antibody, which is a Fab molecule.
• the 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 increment 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.
• the 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 increment therein) sequence identity to the heavy chain variable region CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NO: 4, 5, and 6, respectively.
• Galectin-9 antibodies e.g., which bind to the CRD1 and/or CRD2 region of Galectin-9 are described in co-owned, co-pending US Patent Application 16/173,970 and in co- owned, co-pending International Patent Applications PCT/US 18/58028 and PCT/US2020/024767, the contents of each of which are herein incorporated by reference in their entireties.
• the anti-Galectin-9 antibody disclosed herein comprises light chain CDRs that have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity, individually or collectively, as compared with the corresponding VL CDRS of reference antibody G9.2-17.
• the anti-Galectin-9 antibody comprises heavy chain CDRs that have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity, individually or collectively, as compared with the corresponding VH CDRS of reference antibody G9.2-17.
• Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997.
• the default parameters of the respective programs e.g., XBLAST and NBLAST.
• the anti-Galectin-9 antibody described herein comprises a VH that comprises the HC CDR1, HC CDR2, and HC CDR3, which collectively contain up to 8 amino acid residue variations (8, 7, 6, 5, 4, 3, 2, or 1 variation(s), including additions, deletions, and/or substitutions) relative to the HC CDR1, HC CDR2, and HC CDR3 of reference antibody G9.2-17.
• the anti-Galectin-9 antibody described herein comprises a VH that comprises the LC CDR1, LC CDR2, and LC CDR3, which collectively contain up to 8 amino acid residue variations (8, 7, 6, 5, 4, 3, 2, or 1 variations(s) including additions, deletions, and/or substitutions) relative to the LC CDR1, LC CDR2, and LC CDR3 of reference antibody G9.2-17.
• amino acid residue variations are conservative amino acid residue substitutions.
• a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.
• Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
• amino acids include substitutions made amongst 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.
• the anti-Galectin-9 antibodies disclosed herein, having the heavy chain CDRs disclosed herein contains framework regions derived from a subclass of germline VH fragment.
• germline VH regions are well known in the art. See, e.g., the IMGT database (www.imgt.org) or at www.vbase2.org/vbstat.php.
• IGHV1 subfamily e.g., IGHV1-2, IGHV1-3, IGHV1-8, IGHV1-18, IGHV1-24, IGHV1-45, IGHV1-46, IGHV1-58, and IGHV1-69
• the IGHV2 subfamily e.g., IGHV2-5, IGHV2-26, and IGHV2-70
• the IGHV3 subfamily e.g., IGHV3-7, IGHV3-9, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-20, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-33, IGHV3-43, IGHV3-48, IGHV3-49, IGHV3-53, IGHV3-64, IGHV3-66, IGHV3-72, and IGHV3-73, IGHV3-74), the IGHV4 subfamily (e.g., IGHV4-4, IGHV4-28, IGH
• the anti-Galectin-9 antibody having the light chain CDRs disclosed herein, contains framework regions derived from a germline VK fragment.
• framework regions derived from a germline VK fragment examples include an IGKV1 framework (e.g., IGKV1-05, IGKV1-12, IGKV1-27, IGKV1-33, or IGKV1-39), an IGKV2 framework (e.g., IGKV2-28), an IGKV3 framework (e.g., IGKV3-11, IGKV3-15, or IGKV3-20), and an IGKV4 framework (e.g., IGKV4-1).
• IGKV1 framework e.g., IGKV1-05, IGKV1-12, IGKV1-27, IGKV1-33, or IGKV1-39
• an IGKV2 framework e.g., IGKV2-28
• an IGKV3 framework e.g
• the anti-Galectin-9 antibody comprises a light chain variable region that contains a framework derived from a germline VX fragment.
• a framework derived from a germline VX fragment examples include an IGX1 framework (e.g., IGAV1-36, IGXV1-40, IGXVL44, IGXV1-47, IGXV1-51), an IGX2 framework (e.g., IGXV2-8, IGXV2-11, IGXV2-14, IGXV2-18, IGXV2-23,), an IGX3 framework (e.g., IGXV3-1, IGXV3-9, IGXV3-10, IGXV3-12, IGXV3-16, IGXV3-19, IGXV3-21, IGXV3-25, IGXV3-27,), an IGX4 framework (e.g., IGXV4-3, IGXV4-60, IGXV4-69,), an IGX5 framework (
• the anti-Galectin-9 antibody for use in the method disclosed herein can be an antibody having the same heavy chain variable region (VH) and/or the same light chain variable region (VL) as reference antibody G9.2-17, the VH and VL region amino acid sequences are provided below:
• ADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSS (SEQ ID NO : 7 )
• 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.
• 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.
• the anti-Galectin-9 antibody disclosed herein is a functional variant of reference antibody G9.2-17.
• a functional variant can be structurally similar as the reference antibody (e.g., comprising the limited number of amino acid residue variations in one or more of the heavy chain and/or light chain CDRs as G9.2-17 as disclosed herein, or the sequence identity relative to the heavy chain and/or light chain CDRs of G9.2-17, or the VH and/or VL of G9.2-17 as disclosed herein) with substantially similar binding affinity (e.g., having a KD value in the same order) to human Galectin-9.
• the anti-Galectin-9 antibody as described herein can bind and inhibit the activity of Galectin-9 by at least 20% (e.g., 31%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).
• the apparent inhibition constant (Ki app or Ki,a PP ) which provides a measure of inhibitor potency, is related to the concentration of inhibitor required to reduce enzyme activity and is not dependent on enzyme concentrations.
• the inhibitory activity of an anti-Galectin-9 antibody described herein can be determined by routine methods known in the art.
• the Ki, app value of an antibody may be determined by measuring the inhibitory effect of different concentrations of the antibody on the extent of the reaction (e.g., enzyme activity); fitting the change in pseudo-first order rate constant (v) as a function of inhibitor concentration to the modified Morrison equation (Equation 1) yields an estimate of the apparent Ki value.
• the Ki app can be obtained from the y-intercept extracted from a linear regression analysis of a plot of Ki, app versus substrate concentration.
• the anti-Galectin-9 antibody described herein has a Ki app value of 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 pM or less for the target antigen or antigen epitope.
• the anti-Galectin-9 antibody has a lower Ki app for a first target (e.g., the CRD2 of Galectin-9) relative to a second target (e.g., CRD1 of the Galectin-9).
• Differences in Ki app can 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 fold.
• the anti-Galectin-9 antibody inhibits a first antigen (e.g., a first protein in a first conformation or mimic thereof) greater relative to a second antigen (e.g. , the same first protein in a second conformation or mimic thereof; or a second protein).
• any of the anti- Galectin-9 antibodies is further affinity matured to reduce the Ki app of the antibody to the target antigen or antigenic epitope thereof.
• the anti-Galectin-9 antibody suppresses Dectin- 1 signaling, e.g., in tumor infiltrating immune cells, such as macrophages. In some embodiments, the anti-Galectin-9 antibody suppresses Dectin- 1 signaling triggered by Galectin-9 by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein). Such inhibitory activity can be determined by conventional methods, such as routine assays. Alternatively or in addition, the anti-Galectin-9 antibody suppresses the T cell immunoglobulin mucin- 3 (TIM-3) signaling initiated by Galectin-9.
• TIM-3 T cell immunoglobulin mucin- 3
• the anti-Galectin-9 antibody suppresses the T cell immunoglobulin mucin-3 (TIM-3) signaling, e.g., in tumor infiltrating immune cells, e.g., in some embodiments, by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).
• TIM-3 T cell immunoglobulin mucin-3
• Such inhibitory activity can be determined by conventional methods, such as routine assays.
• the anti-Galectin-9 antibody suppresses the CD206 signaling, e.g., in tumor infiltrating immune cells. In some embodiments, the anti-Galectin-9 antibody suppresses the CD206 signaling triggered by Galectin-9 by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein). Such inhibitory activity can be determined by conventional methods, such as routine 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 greater, including any increment therein). Such inhibitory activity can be determined by conventional methods, such as routine assays.
• the anti-Galectin-9 antibody induces cell cytotoxicity, such as ADCC, in target cells expressing Galectin-9, e.g., wherein the target cells are cancer cells or immune suppressive immune cells.
• the anti-Galectin-9 antibody induces apoptosis in immune cells, such as T cells, or cancer cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).
• Such inhibitory activity can be determined by conventional methods, such as routine assays.
• any of the anti-Galectin-9 antibodies described herein induce cell cytotoxicity such as complement-dependent cytotoxicity (CDC) against target cells expressing Galectin-9.
• CDC complement-dependent cytotoxicity
• ADCP Antibody-dependent cell-mediated phagocytosis
• the anti-Galectin-9 antibody induces cell phagocytosis of target cells, e.g., cancer cells or immune suppressive immune cells expressing Galectin-9 (ADCP).
• the anti-Galectin-9 antibody increases phagocytosis of target cells, e.g., cancer cells or immune suppressive immune cells, by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).
• the anti-Galectin-9 antibody described herein induces cell cytotoxicity such as complement-dependent cytotoxicity (CDC) against target cells, e.g., cancer cells or immune suppressive immune cells.
• CDC complement-dependent cytotoxicity
• the anti-Galectin-9 antibody increases CDC against target cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).
• the anti-Galectin-9 antibody induces T cell activation, e.g., in tumor infiltrating T cells, i.e., suppress Galectin-9 mediated inhibition of T cell activation, either directly or indirectly.
• the anti-Galectin-9 antibody promotes T cell activation by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).
• T cell activation can be determined by conventional methods, such as using well-known assays for measuring cytokines and checkpoint inhibitors (e.g., measurement of CD44, TNF alpha, IFNgamma, and/or PD-1).
• the anti- Galectin-9 antibody promotes CD4+ cell activation by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).
• the anti-Galectin antibody induces CD44 expression in CD4+ cells.
• 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 greater, including any increment therein).
• the anti-Galectin antibody induces IFNgamma expression in CD4+ cells.
• the anti-Galectin-9 antibody increases IFNgamma expression in CD4+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).
• the anti-Galectin antibody induces TNFalpha expression in CD4+ cells.
• the anti-Galectin-9 antibody increases TNFalpha expression in CD4+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).
• the anti-Galectin-9 antibody promotes CD8+ cell activation by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater), including any increment therein).
• the anti-Galectin antibody induces CD44 expression in CD8+ cells.
• 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 greater, including any increment therein).
• the anti-Galectin antibody induces IFNgamma expression in CD8+ cells.
• the anti-Galectin-9 antibody increases IFNgamma expression in CD8+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).
• the anti-Galectin antibody induces TNFalpha expression in CD8+ cells.
• the anti-Galectin-9 antibody increases TNFalpha expression in CD8+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increment therein).
• an anti-Galectin-9 antibody as described herein has a suitable binding affinity for the target antigen (e.g., Galectin-9) or antigenic epitopes thereof.
• binding affinity refers to the apparent association constant or KA.
• the KA is the reciprocal of the dissociation constant (KD).
• the anti-Galectin-9 antibody described herein may have a binding affinity (KD) of at least 10" 5 , 10" 6 , 10" 7 , 10" 8 , 10" 9 , 10" 10 M, or lower for the target antigen or antigenic epitope.
• KD binding affinity
• An increased binding affinity corresponds to a decreased KD.
• Binding affinity 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 fluorescence assay). Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v/v) Surfactant P20).
• KA affinity-binding affinity
• a quantitative measurement of affinity e.g., determined using a method such as ELISA or FACS analysis
• KA proportional to KA, and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g. , by activity in a functional assay, e.g., an in vitro or in vivo assay.
• the in vitro binding assay is indicative of in vivo activity.
• the in vitro binding assay is not necessarily indicative of in vivo activity.
• tight binding is beneficial, but in other cases tight binding is not as desirable in vivo, and an antibody with lower binding affinity is more desirable.
• the heavy chain of any of any of the anti-Galectin-9 antibodies as described herein further comprise a heavy chain constant region (CH) or a portion thereof (e.g., CHI, CH2, CH3, or a combination thereof).
• the heavy chain constant region can be of any suitable origin, e.g., human, mouse, rat, or rabbit.
• the heavy chain constant region is from a human IgG (a gamma heavy chain) of any IgG subfamily as described herein.
• the heavy chain constant region of the antibodies described herein comprise a single domain (e.g., CHI, CH2, or CH3) or a combination of any of the single domains, of a constant region (e.g., SEQ ID NO: 4, 5, 6).
• the light chain constant region of the antibodies described herein comprise a single domain (e.g., CL), of a constant region.
• Exemplary light and heavy chain sequences are listed below.
• Exemplary light and heavy chain sequences are listed below.
• the hlgGl LALA sequence includes two mutations, L234A and L235A (EU numbering), which suppress FcgR binding as well as a P329G mutation (EU numbering) to abolish complement Clq binding, thus abolishing all immune effector functions.
• the hIgG4 Fab Arm Exchange Mutant sequence includes a mutation to suppress Fab Arm Exchange (S228P; EU numbering).
• An IL2 signal sequence (MYRMQLLSCIALSLALVTNS; SEQ ID NO: 9) can be located N-terminally of the variable region. It is used in expression vectors, which is cleaved during secretion and thus not in the mature antibody molecule.
• the mature protein (after secretion) starts with "EVQ" for the heavy chain and "DIM” for the light chain.
• Amino acid sequences of exemplary heavy chain constant regions are provided below: hlgGl Heavy Chain Constant Region (SEQ ID NO: 10)
• the heavy chain constant region in an anti-Galectin-9 antibody disclosed herein may have the C-terminal Lysine (K) residue removed for, e.g., manufacturing purposes.
• K Lysine
• K residue are provided below: hlgGl Heavy Chain Constant Region with No C-Terminal Lysine (SEQ ID NO: 24)
• anti-Galectin-9 antibodies having any of the above heavy chain constant regions are paired with a light chain having the following light chain constant region:
• the anti-Galectin-9 antibody comprises a heavy chain IgGl constant region that has at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 10.
• the constant region of the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO: 10.
• the constant region of the anti- Galectin-9 antibody comprises a heavy chain IgGl constant region consisting of SEQ ID NO: 10.
• the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region that has at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 20.
• the constant region of the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO: 20.
• the constant region of the anti- Galectin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO: 20.
• the constant region is from human IgG4.
• the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region that has at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 13.
• the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO: 13.
• the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO: 13.
• the constant region is from human IgG4.
• the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region that has at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 20.
• the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO: 20.
• the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO: 20.
• the anti-Galectin-9 antibody comprises a light chain constant region that has at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 11.
• the anti-Galectin-9 antibody comprises a light chain constant region comprising SEQ ID NO: 11.
• the anti-Galectin-9 antibody comprises a light chain constant region consisting of SEQ ID NO: 11.
• the IgG is a mutant with minimal Fc receptor engagement.
• the constant region is from a human IgGl LALA.
• the anti- Galectin-9 antibody comprises a heavy chain IgGl constant region that has at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 12.
• the anti-Galectin-9 antibody comprises a heavy chain IgGl constant region comprising SEQ ID NO: 12.
• the anti-Galectin-9 antibody comprises a heavy chain IgGl constant region consisting of SEQ ID NO: 12.
• the anti-Galectin-9 antibody comprises a modified constant region.
• the anti-Galectin-9 antibody comprise a modified constant region that is immunologically inert, e.g., does not trigger complement mediated lysis, or does not stimulate antibody-dependent cell mediated cytotoxicity (ADCC). ADCC activity can be assessed using methods disclosed in U.S. Pat. No. 5,500,362.
• the constant region is modified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCT Application No. PCT/GB99/01441; and/or UK Patent Application No. 9809951.8.
• the IgG4 constant region is a mutant with reduced heavy chain exchange.
• the constant region is from a human IgG4 Fab Arm Exchange mutant S228P.
• the constant region of the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region that has at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 14.
• the constant region of the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO: 14.
• the constant region of the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO: 14.
• the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region that has at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 21.
• the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO: 21.
• the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO: 21.
• the anti-Galectin -9 antibody has chains corresponding to SEQ ID NO: 15 for the light chains; and the amino acid sequences of exemplary heavy chains correspond to SEQ ID NO: 10 (hlgGl); 12 (hlgGl LALA); 13 (hIgG4); 20 (hIgG4); 14 (hIgG4 mut); and 21 (hIgG4 mut).
• 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 NO: 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.
• 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.
• 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.
• the anti-Galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 16.
• the anti- Galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO: 16.
• the anti-Galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 16.
• the anti-Galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 17.
• the anti- Galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO: 17.
• the anti-Galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 17.
• the anti-Galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 18.
• the anti- Galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO: 18.
• the anti-Galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 18.
• the anti-Galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 22.
• the anti- Galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO: 22.
• the anti-Galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 22.
• the anti-Galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 19.
• the anti- Galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO: 19.
• the anti-Galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 19.
• the anti-Galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 23.
• the anti- Galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO: 23.
• the anti-Galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 23.
• the anti-Galectin-9 antibody comprises a light chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity to SEQ ID NO: 15.
• the anti-Galectin-9 antibody comprises a light chain sequence comprising SEQ ID NO: 15.
• the anti-Galectin-9 antibody comprises a light chain sequence consisting of SEQ ID NO: 15.
• the anti-Galectin-9 antibody used in the treatment methods disclosed herein has a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 15.
• the anti-Galectin-9 antibody used in the treatment methods disclosed herein is G9.2-17 IgG4. In some examples, such an anti-Galectin-9 antibody does not have the C-terminal lysine residue in its heavy chain.
• Antibodies capable of binding Galectin-9 as described herein can be made by any method known in the art, including but not limited to, recombinant technology. One example is provided below.
• Nucleic acids encoding the heavy and light chain of an anti-Galectin-9 antibody as described herein can be cloned into one expression vector, each nucleotide sequence being in operable linkage to a suitable promoter.
• each of the nucleotide sequences encoding the heavy chain and light chain is in operable linkage to a distinct promoter.
• the nucleotide sequences encoding the heavy chain and the light chain can be in operable linkage with a single promoter, such that both heavy and light chains are expressed from the same promoter.
• an internal ribosomal entry site IRS
• the nucleotide sequences encoding the two chains of the antibody are cloned into two vectors, which can be introduced into the same or different cells.
• the two chains are expressed in different cells, each of them can be isolated from the host cells expressing such and the isolated heavy chains and light chains can be mixed and incubated under suitable conditions allowing for the formation of the antibody.
• a nucleic acid sequence encoding one or all chains of an antibody can be cloned into a suitable expression vector in operable linkage with a suitable promoter using methods known in the art.
• the nucleotide sequence and vector can be contacted, under suitable conditions, with a restriction enzyme to create complementarity ends on each molecule that can pair with each other and be joined together with a ligase.
• synthetic nucleic acid linkers can be ligated to the termini of a gene. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector. The selection of expression vectors/promoter would depend on the type of host cells for use in producing the antibodies.
• promoters can be used for expression of the antibodies described herein, including, but not limited to, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, E. colt lac UV5 promoter, and the herpes simplex tk virus promoter.
• CMV cytomegalovirus
• a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR
• SV40 simian virus 40
• E. colt lac UV5 promoter E. colt lac UV5 promoter
• herpes simplex tk virus promoter the herpes simplex tk virus promoter.
• Regulatable promoters can also be used.
• Such regulatable promoters include those using the lac repressor from E. colt as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters [Brown, M. et al., Cell, 49:603-612 (1987)], those using the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Set. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci.
• Regulatable promoters that include a repressor with the operon can be used.
• the lac repressor from E. colt can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters (M. Brown et al., Cell, 49:603- 612 (1987); Gossen and Bujard (1992); M. Gossen et al., Natl. Acad. Sci.
• tetracycline repressor tetR
• VP 16 transcription activator
• tetR-mammalian cell transcription activator fusion protein tTa (tetR- VP 16)
• hCMV human cytomegalovirus
• a tetracycline inducible switch is used.
• tetracycline repressor alone, rather than the tetR-mammalian cell transcription factor fusion derivatives can function as potent trans-modulator to regulate gene expression in mammalian cells when the tetracycline operator is properly positioned downstream for the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy, 10(16): 1392-1399 (2003)).
• tetracycline inducible switch is that it does not require the use of a tetracycline repressor-mammalian cells transactivator or repressor fusion protein, which in some instances can be toxic to cells (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)), to achieve its regulatable effects.
• the vector can contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA.
• a selectable marker gene such as the neomycin gene for selection of stable or transient transfectants in mammalian cells
• enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription
• transcription termination and RNA processing signals from SV40 for mRNA stability
• SV40 polyoma origins of replication and ColEl for proper episomal replication
• polyadenylation signals useful to practice the methods described herein include, but are not limited to, human collagen I polyadenylation signal, human collagen II polyadenylation signal, and SV40 polyadenylation signal.
• One or more vectors comprising nucleic acids encoding any of the antibodies may be introduced into suitable host cells for producing the antibodies.
• the host cells can be cultured under suitable conditions for expression of the antibody or any polypeptide chain thereof.
• Such antibodies or polypeptide chains thereof can be recovered by the cultured cells (e.g., from the cells or the culture supernatant) via a conventional method, e.g., affinity purification.
• polypeptide chains of the antibody can be incubated under suitable conditions for a suitable period of time allowing for production of the antibody.
• methods for preparing an antibody described herein involve a recombinant expression vector that encodes both the heavy chain and the light chain of an anti- Galectin-9 antibody, as also described herein.
• the recombinant expression vector can be introduced into a suitable host cell (e.g., a dhfr- CHO cell) by a conventional method, e.g., calcium phosphate-mediated transfection.
• a suitable host cell e.g., a dhfr- CHO cell
• Positive transformant host cells can be selected and cultured under suitable conditions allowing for the expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or from the culture medium.
• the two chains recovered from the host cells can be incubated under suitable conditions allowing for the formation of the antibody.
• two recombinant expression vectors are provided, one encoding the heavy chain of the anti-Galectin-9 antibody and the other encoding the light chain of the anti- Galectin-9 antibody.
• Both of the two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr- CHO cell) by a conventional method, e.g., calcium phosphate- mediated transfection.
• each of the expression vectors can be introduced into a suitable host cell. Positive transformants can be selected and cultured under suitable conditions allowing for the expression of the polypeptide chains of the antibody.
• the antibody produced therein can be recovered from the host cells or from the culture medium.
• the polypeptide chains can be recovered from the host cells or from the culture medium and then incubated under suitable conditions allowing for formation of the antibody.
• the two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cells or from the corresponding culture media. The two polypeptide chains can then be incubated under suitable conditions for formation of the antibody.
• Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recovery of the antibodies from the culture medium.
• some antibodies can be isolated by affinity chromatography with a Protein A or Protein G coupled matrix.
• nucleic acids encoding the heavy chain, the light chain, or both of an anti- Galectin-9 antibody as described herein vectors (e.g., expression vectors) containing such; and host cells comprising the vectors are within the scope of the present disclosure.
• Anti-Galectin-9 antibodies thus prepared can be characterized using methods known in the art, whereby reduction, amelioration, or neutralization of Galectin-9 biological activity is detected and/or measured.
• an ELISA-type assay is suitable for qualitative or quantitative measurement of Galectin-9 inhibition of Dectin- 1 or TIM-3 signaling.
• the bioactivity of an anti-Galectin-9 antibody can verified by incubating a candidate antibody 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 the signaling transduction mediated by the binding; (b) preventing, ameliorating, or treating any aspect of a solid tumor; (c) blocking or decreasing Dectin- 1 activation; (d) inhibiting (reducing) synthesis, production or release of Galectin-9.
• TIM- 3 can be used to verify the bioactivity of an anti-Galectin-9 antibody using the protocol described above.
• CD206 can be used to verify the bioactivity of an anti-Galectin-9 antibody using the protocol described above.
• bioactivity or efficacy is assessed in a subject, e.g., by measuring peripheral and intra-tumoral T cell ratios, T cell activation, or by macrophage phenotyping.
• Additional assays to determine bioactivity of an anti-Galectin-9 antibody include measurement of CD8+ and CD4+ (conventional) T-cell activation (in an in vitro or in vivo assay, e.g., by measuring inflammatory cytokine levels, e.g., IFNgamma, TNFalpha, CD44, ICOS granzymeB, Perforin, IL2 (upregulation); CD26L and IL-10 (downregulation)); measurement of reprogramming of macrophages (in vitro or in vivo), e.g., from the M2 to the Ml phenotype (e.g., increased MHCII, reduced CD206, increased TNF-alpha and iNOS), Alternatively, levels of ADCC can be assessed, e.g., in an in vitro assay, as described herein.
• inflammatory cytokine levels e.g., IFNgamma, TNFalpha, CD44, ICOS granzymeB
• the present disclosure provides methods for treating solid tumors, including, but not limited to, PDAC, CRC, HCC, and cholangiocarcinoma, renal cell carcinoma, urothelial cancer, head and neck cancer, breast cancer, or other GI solid tumors, using any of the anti-Galectin antibodies, for example G9.2-17, e.g., G9.2-17 IgG4, either 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.
• the anti-Galectin-9 antibody is G9.2-17 (e.g., G9.2-17 (IgG4)).
• Such antibodies can be used for treating diseases associated with Galectin-9.
• the present disclosure provides methods of treating cancer.
• the disclosure provides a method for treating a solid tumor in a subject, the method comprising administering to a subject in need thereof an effective amount of an anti-Galectin-9 antibody described herein, including but not limited to, G9.2-17 IgG4.
• the method disclosed herein is applied to a human patient having pancreatic cancer, for example, ductal adenocarcinoma (PDAC).
• the PDAC patient may have a metastatic cancer.
• the method disclosed herein is applied to a human patient having colorectal cancer (CRC).
• the colorectal cancer is metastatic.
• the method disclosed herein is applied to a human patient having hepatocellular carcinoma.
• the hepatocellular carcinoma is metastatic.
• the method disclosed herein is applied to a human patient having cholangiocarcinoma.
• the cholangiocarcinoma is metastatic.
• Pancreatic ductal adenocarcinoma is a devastating disease with few long-term survivors (Yadav et al., Gastroenterology, 2013, 144, 1252-1261). Inflammation is paramount in PDAC progression as oncogenic mutations alone, in the absence of concomitant inflammation, are insufficient for tumorigenesis (Guerra et al., Cancer Cell, 2007, 11, 291-302). Innate and adaptive immunity cooperate to promote tumor progression in PDAC. In particular, specific innate immune subsets within the tumor microenvironment (TME) are apt at educating adaptive immune effector cells towards a tumor-permissive phenotype.
• TAE tumor microenvironment
• Antigen presenting cell (APC) populations including M2-polarized tumor-associated macrophages (TAMs) and myeloid dendritic cells (DC), induce the generation of immune suppressive Th2 cells in favor of tumor- protective Thl cells (Ochi et al., J of Exp Med.., 2012, 209, 1671-1687; Zhu et al., Cancer Res., 2014, 74, 5057-5069) .
• APC Antigen presenting cell
• MDSC myeloid derived suppressor cells negate anti-tumor CD8 + cytotoxic T-Lymphocyte (CTL) responses in PDAC and promote metastatic progression (Connolly et al., J Leak Biol., 2010, 87, 713-725; Pylayeva-Gupta et al., Cancer Cell, 2012, 21, 836-847; Bayne et al., Cancer Cell, 2012, 21, 822-835).
• CTL cytotoxic T-Lymphocyte
• Pancreatic cancer remains a disease that is difficult to treat due to a typically late presentation, relatively high resistance to chemotherapy, and lack of effective immune and targeted therapies.
• the median life expectancy for patients with metastatic pancreatic cancer is less than 1 year with current treatment, while most patients (as many as 80%) present at an advanced/metastatic stage, when the disease is beyond curative resection.
• metastatic pancreatic cancer Despite advancements in the detection and management of pancreatic cancer, the five-year survival rate of metastatic disease remains at ten percent.
• the current standard of care for metastatic pancreatic cancer is predominantly chemotherapy, while a distinct minority of patients (under ten percent) with BRCA1/2 mutations and mismatch repair deficient tumors may benefit from PARP inhibitors and potentially anti-PD-1 therapy.
• currently approved immunotherapies have been generally unsuccessful due to a highly immunosuppressive environment.
• CRC Colorectal cancer
• bowel cancer also known as bowel cancer, colon cancer, or rectal cancer
• CRC is any cancer affecting the colon and the rectum.
• CRC is known to be driven by genetic alterations of tumor cells and is also influenced by tumor-host interactions. Recent reports have demonstrated a direct correlation between the densities of certain T lymphocyte subpopulations and a favorable clinical outcome in CRC, supporting a major role of T-cell-mediated immunity in repressing tumor progression of CRC.
• CRC presents one of the largest cancer burdens in the world.
• 147,950 cases are predicted in 2020 with 53,200 estimated deaths (Colorectal Cancer Stats).
• the five-year survival rate for metastatic CRC remains around ⁇ 20%. Death from CRC is expected to nearly double within the next 20 years.
• the current standard of care for CRC are chemotherapy regimens, combined and/or sequenced with anti-angiogenic therapy and anti-epidermal growth factor receptor modalities in selected patients.
• Hepatocellular carcinoma is the most common type of primary liver cancer. Hepatocellular carcinoma occurs most often in people with chronic liver diseases, such as cirrhosis caused by hepatitis B or hepatitis C infection. HCC is usually accompanied by cirrhotic liver with extensive lymphocyte infiltration due to chronic viral infection. Many studies have demonstrated that tumor-infiltrating effector CD8+ T cells and T helper 17 (Thl7) cells correlate with improved survival after surgical resection of tumors. However, tumor-infiltrating effector T cells fail to control tumor growth and metastasis (Pang et al., Cancer Immunol Immunother 2009; 58:877-886).
• Cholangiocarcinoma is a group of cancers that begin in the bile ducts. Cholangiocarcinoma is commonly classified by its location in relation to the liver. For example, intrahepatic cholangiocarcinoma, accounting for less than 10% of all cholangiocarcinoma cases, begins in the small bile ducts within the liver. In another example, perihilar cholangiocarcinoma (also known as a Klatskin tumor), accounting for more than half of the cholangiocarcinoma cases, begins in hilum, where two major bile ducts join and leave the liver. Others are classified as distal cholangiocarcinomas, which begin in bile ducts outside the liver.
• CCAs are aggressive tumors, and most patients have advanced-stage disease at presentation.
• the incidence of CCA is rising, and effective therapies are urgently needed.
• Gemcitabine plus cisplatin remains the standard first-line systemic therapy for advanced CCA, although it leaves much to be desired, as median survival is less than 1 year. Beyond failure of first line therapy, available evidence to guide therapeutic decisions is scarce.
• Food and Drug Administration (FDA) only recently approved the first targeted therapy in this indication for patients harboring fibroblast growth factor receptor 2 gene fusions and other rearrangements in their tumors.
• FDA Food and Drug Administration
• Suboptimal response rates to immunotherapy in human clinical trials imply that the preponderance of CCAs are immune ‘cold’ tumors with a non-T -cell infiltrated microenvironment.
• immunotherapy to date has produced response rates not exceeding 17% and as of the date of this prospectus, no immune oncology agents have been approved (Zayac and Almhanna, 2020).
• methods are provided to increase anti-tumor activity (e.g., reduce cell proliferation, tumor growth, tumor volume, and/or tumor burden or load or reduce the number of metastatic lesions over time) by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to levels prior to treatment or in a control subject.
• reduction is measured by comparing cell proliferation, tumor growth, and/or tumor volume in a subject before and after administration of the pharmaceutical composition.
• the method is provided to improve one or more symptoms of the cancer by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.
• cancerous cells and/or biomarkers in a subject are measured in a biological sample, such as blood, serum, plasma, urine, peritoneal fluid, and/or a biopsy from a tissue or organ.
• the methods include administration of the compositions of the invention to reduce tumor volume, size, load or burden in a subject to an undetectable size, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the subject's tumor volume, size, load or burden prior to treatment.
• methods are provided for reducing the cell proliferation rate or tumor growth rate in a subject to an undetectable rate, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the rate prior to treatment.
• methods include administration of the compositions of the invention to reduce the development of or the number or size of metastatic lesions in a subject to an undetectable rate, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the rate prior to treatment.
• the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which are dependent in part on how the value is measured or determined, i.e., the limitations of the measurement system.
• “about” can mean within an acceptable standard deviation, per the practice in the art.
• “about” can mean a range of up to ⁇ 20 %, preferably up to ⁇ 10 %, more preferably up to ⁇ 5 %, and more preferably still up to ⁇ 1 % of a given value.
• the term can mean within an order of magnitude, preferably within 2-fold, of a value.
• treating refers to the application or administration of a composition including one or more active agents to a subject, who has a target disease or disorder, a symptom of the disease/disorder, or a predisposition toward the disease/disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, a symptom of the disease or disorder, or the predisposition toward the disease or disorder.
• Alleviating a target disease/disorder includes delaying the development or progression of the disease or reducing disease severity or prolonging survival. Alleviating the disease or prolonging survival does not necessarily require curative results.
• "delaying" the development of a target disease or disorder means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated.
• a method that “delays” or alleviates the development of a disease, or delays the onset of the disease is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.
• “Development” or “progression” of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a target disease or disorder includes initial onset and/or recurrence.
• a subject having any of the above noted cancers can be identified by routine medical examination, e.g., laboratory tests, organ functional tests, genetic tests, interventional procedure (biopsy, surgery) any and all relevant imaging modalities.
• the subject to be treated by the method described herein is a human cancer patient who has undergone or is subjected to an anti-cancer therapy regimen delivered systemically and/or locally, for example, chemotherapy, radiotherapy, tumor-treating fields (TTFields), immunotherapy, biological therapy, small molecule inhibitors, anti-hormonal therapy, cell-based therapy, and/or surgery, in any combination or sequence of the outlined therapeutic modalities.
• subjects have received prior immune-modulatory or any other anti-tumor agents or treatment modalities listed above.
• Non-limiting examples of such immune- modulatory agents include, but are not limited to as anti-PD-1, anti-PD-Ll, anti-CTLA-4, anti- TIGIT, anti-PVRIG, anti-LAG-3, anti-CD47, anti-CD40, anti-CSFRl, anti-CD73, anti-SIRP, anti-A2AR, anti-OX40, anti-CD137, platinum-based agent, etc.
• platinum-based agents include cisplatin, carboplatin, oxaliplatin, nedaplatin, and lobaplatin.
• the subject shows disease progression through the treatment. In other embodiments, the subject is resistant to the treatment (either de novo or acquired).
• such a subject is demonstrated as having advanced malignancies (e.g., inoperable or metastatic).
• advanced malignancies e.g., inoperable or metastatic.
• the subject has no standard therapeutic options available or ineligible for standard treatment options, which refer to therapies commonly used in clinical settings for treating the corresponding solid tumor.
• Tumor-treating fields are a cancer treatment modality that uses alternating electric fields of intermediate frequency ( ⁇ 100-500 kHz) and low intensity (1-3 V/cm) to disrupt cell division.
• the anti-Galectin-9 antibody alone or in combination with a checkpoint inhibitor, such as an anti-PD-1 antibody, may be administered prior to, concurrent with, or after a tumor-treating fields (TTFields) regimen.
• the subject may be a human patient having a refractory disease, for example, a refractory PDAC, a refractory CRC, a refractory HCC, or a refractory cholangiocarcinoma.
• refractory refers to the tumor that does not respond to or becomes resistant to a treatment.
• the subject may be a human patient having a relapsed disease, for example, a relapsed PDAC, a relapsed CRC, a relapsed HCC, or a relapsed cholangiocarcinoma.
• relapsed or “relapses” refers to the tumor that returns or progresses following a period of improvement (e.g., a partial or complete response) with treatment.
• the 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.
• the human patient may be 18 or older; having histologically confirmed unresectable metastatic or inoperable cancer (e.g., without standard therapeutic options), having a life expectancy > 3 months, having recent archival tumor sample available for biomarker analysis (e.g., an archival species for Galectin-9 tumor tissue expression levels assessed by IHC); having a measurable disease, according to RECIST vl.l, having Eastern Cooperative Oncology Group (ECOG) performance status 0-1 or Kamofsky score >70; having no available standard of care options, having MSI-H (Microsatellite instability high and MSS ( Microsatellite Stable); received at least one line of systemic therapy in the advanced/metastatic setting; having adequate hematologic and end organ function (defined in Example 1 below; e.g., e.g.
• the subject suitable for the treatment disclosed herein may not have one or more of the following: diagnosed with metastatic cancer of an unknown primary; any active uncontrolled bleeding, and any patients with a bleeding diathesis (e.g., active peptic ulcer disease); receiving any other investigational agents within 4 weeks or 5 half-lives of anti-galectin- 9 antibody administration; receiving radiation therapy within 4 weeks of the first dose of the anti- Galectin-9 antibody, except for palliative radiotherapy to a limited field, such as for the treatment of bone pain or a focally painful tumor mass; having fungating tumor masses; for PDAC patients, having prior gemcitabine containing regimen less than 6 months from the begin of the treatment, patients having locally advanced PDAC; having active clinically serious infection > grade 2 NCL CTCAE version 5.0; having symptomatic or active brain metastases; having ⁇ CTCAE grade 3 toxicity (see details and exceptions in Example 1); having history of second malignancy (see exceptions in Example 1); having evidence of severe or uncontrolled systemic diseases,
• Leptomeningeal disease active or previously treated; having significant vascular disease; having active auto-immune disorder (see exceptions in Example 1); require systemic immunosuppressive treatment; having tumor-related pain (> grade 3) unresponsive to broad analgesic interventions (oral and/or patches); having uncontrolled hypercalcemia, despite use of bisphosphonates; having any history of an immune-related Grade 4 adverse event attributed to prior checkpoint inhibitor therapy (CIT); received an organ transplant(s); and/or on undergoing dialysis; for HCC patients and/or CCA patients, having any ablative therapy prior to the treatment; hepatic encephalopathy or severe liver adenoma; and/or having Child-Pugh score ⁇ 7.
• CIT checkpoint inhibitor therapy
• the human patient may not have metastatic hepatocellular carcinoma that progressed while receiving at least one previous line of systemic therapy; have refuse or not tolerated sorafenib; or have had standard therapy considered ineffective, intolerable, or inappropriate or for which no effective standard therapy is available.
• the 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.
• the human patient may be older than 18 and have histologically confirmed unresectable metastatic cancer (e.g., adenocarcinomas and squamous cell carcinomas).
• the patient may have measurable disease, according to RECIST v. 1.1.
• the human patient may have recent archival tumor sample (e.g., obtained within 5 years) available for biomarker analyses (e.g., galectin-9 tumor tissue expression, which may be assessed by IHC).
• the human patient is a PDAC patient who has received at least one line of systemic therapy in the metastatic cancer setting.
• the human patient is a metastatic PDAC patient who has or has not received systemic therapy before receiving an anti-Galectin-9 containing regimen.
• Such a patient may either be gemcitabine-containing regimen naive or at least 6 months out of having been treated using a gemcitabine-containing regimen in a previous disease stage setting.
• the patient may have Eastern Cooperative Oncology Group (ECOG) performance status 0-1 and/or Karnofsky score > 70.
• ECOG Eastern Cooperative Oncology Group
• the patient may also have adequate hematologic and end organ function, e.g., neutrophil count ⁇ 1 x 10 9 /L, platelet count ⁇ 100 x 10 9 /L, for HCC in Part 1 ⁇ 50 x 10 9 /L; hemoglobin ⁇ 9.0 g/dL without transfusion in the previous week, Creatinine ⁇ 1.5 x ULN, AST (SGOT) ⁇ 3 x ULN ( ⁇ 5 x ULN when HCC or hepatic metastases are present), ALT (SGPT) ⁇ 3 x ULN ( ⁇ 5 x ULN when HCC or hepatic metastases present), Bilirubin ⁇ 1.5 x ULN (patients with known Gilbert's disease may have a bilirubin ⁇ 3.0 x ULN), Albumin ⁇ 3.0 g/dL, INR and PTT ⁇ 1.5 x ULN; and/or amylase and lipase ⁇ 1.5 x UL
• the human patient shows no evidence of active infection or infections requiring parenteral antibiotics, and no serious infection within 4 weeks before the treatment starts.
• Pancreatic, biliary, or enteric fistulae allowed, provided they are controlled with an appropriate non-infected and patent drain.
• the human patient subject to any treatment disclosed herein may be free of: (i) metastatic cancer of an unknown primary, (ii) clinically significant, active uncontrolled bleeding, any bleeding diathesis (e.g., active peptic ulcer disease); (iii) radiation therapy within 4 weeks of the first dose of the treatment, (iv) with fungating tumor masses; (v) ⁇ CTCAE grade 3 toxicity (except alopecia and vitiligo) due to prior cancer therapy; (v) history of second malignancy, (vi) evidence of severe or uncontrolled systemic diseases, congestive cardiac failure > New York Heart Association (NYHA) class 2, or myocardial infarction (MI) within 6 months, (vii) serious non-healing wound, active ulcer, or untreated bone fracture; (viii) uncontrolled pleural effusion, pericardial effusion, or ascites requiring recurrent drainage procedures; (ix) history of severe allergic, anaphylactic, or other hypersensitivity reactions to chimeric or humanized antibodies or
• the subject is a human patient having an elevated level of Galectin-9 as relative to a control level.
• the level of Galectin-9 can be a plasma or serum level of Galectin-9 in the human patient.
• the level of Galectin-9 is the level of Galectin-9 of cancer cells within the tumor.
• the level of Galectin-9 is the level of Galectin-9 of immune cells within the tumor.
• the level of Galectin-9 can be the level of cell- surface Galectin-9, for example the level of Galectin-9 on cancer cells.
• the level of Galectin-9 can be the level of Galectin-9 expressed cancer cells, e.g., on the surface of cancer cells, or Galectin-9 expressed in immune cells, measured in patient-derived organotypic tumor spheroids (PDOT), which can be prepared by, e.g., the method disclosed in Examples below.
• a control level may refer to the level of Galectin-9 in a matched sample of a subject of the same species (e.g., human) who is free of the solid tumor.
• the control level represents the level of Galectin-9 in healthy subjects.
• the control level may be a baseline level prior to treatment.
• a suitable biological sample can be obtained from a subject who is suspected of having the solid tumor and the biological sample can be analyzed to determine the level of Galectin-9 contained therein (e.g., free, cell-surface expressed, or total) using conventional methods, e.g., ELISA or FACS.
• organoid cultures are prepared, e.g., as described herein, and used to assess Galectin-9 levels in a subject. Single cells derived from certain fractions obtained as part of the organoid preparation process are also suitable for assessment of Galectin-9 levels in a subject.
• an assay for measuring the level of Galectin-9 involves the use of an antibody that specifically binds the Galectin-9 (e.g., specifically binds human Galectin- 9).
• an antibody described herein e.g., a G9.2-17 antibody
• an antibody described in US Patent No. 10,344,091 and WO2019/084553 the relevant disclosures of each of which are incorporated by reference for the purpose and subject matter referenced herein.
• the anti-Galectin-9 antibody is a Fab molecule. Assay methods for determining Galectin-9 levels as disclosed herein are also within the scope of the present disclosure.
• the antibodies described herein are administered to a subject in need of the treatment at an amount sufficient to inhibit the activity of Galectin-9 (and/or Dectin- 1 or TIM- 3 or CD206) in immune suppressive immune cells in a tumor by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) in vivo.
• the antibodies described herein are administered in an amount effective in reducing the activity level of Galectin-9 (and/or Dectin- 1 or TIM- 3 or CD206) in immune suppressive immune cells in a tumor by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) (as compared to levels prior to treatment or in a control subject).
• the antibodies described herein are administered to a subject in need of the treatment at an amount sufficient to promote Ml-like programming in TAMs by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) in vivo (as compared to levels prior to treatment or in a control subject).
• the anti-Galectin-9 antibody can be administered to a subject by intravenous infusion.
• Injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like).
• water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused.
• Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer’s solution or other suitable excipients.
• Intramuscular preparations e.g., a sterile formulation of a suitable soluble salt form of the antibody
• a pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or 5% glucose solution.
• an effective amount of the pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of the treatment via a suitable route, systemically or locally.
• the anti-Galectin-9 antibodies are administered by intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-arterial, intra-articular, intrasynovial, intrathecal, intratumoral, sub-urothelial, oral, inhalation or topical routes.
• the anti-Galectin-9 antibody is administered to the subject by intravenous infusion.
• the anti-galectin-9 antibody is administered to the subject intraperitoneally.
• an effective amount refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents.
• the therapeutic effect is reduced Galectin-9 activity and/or amount/expression, reduced Dectin- 1 signaling, reduced TIM- 3 signaling, reduced CD206 signaling, or increased anti-tumor immune responses in the tumor microenvironment.
• increased anti-tumor responses include increased activation levels of effector T cells or switching of the TAMs from the M2 to the Ml phenotype.
• the anti-tumor response includes increased ADCC responses. Determination of whether an amount of the antibody achieved the therapeutic effect would be evident to one of skill in the art.
• Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific 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 no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.
• Empirical considerations such as the half-life, generally contribute to the determination of the dosage.
• antibodies that are compatible with the human immune system such as humanized antibodies or fully human antibodies, are in some instances used to prolong half-life of the antibody and to prevent the antibody being attacked by the host's immune system.
• Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of a target disease/disorder.
• sustained continuous release formulations of an antibody may be appropriate.
• formulations and devices for achieving sustained release are known in the art.
• dosages for an antibody as described herein are determined empirically in individuals who have been given one or more administration(s) of the antibody. Individuals are given incremental dosages of the antagonist. To assess efficacy of the antagonist, an indicator of the disease/disorder can be followed.
• the anti-Galectin-9 antibodies described herein are be used for treating the target cancer disclosed herein, i.e., free of other anti-cancer therapy concurrently with the therapy using the anti-Galectin-9 antibody.
• the anti-Galectin-9 antibody such as G9.2-17(IgG4) disclosed herein may be used in a monotherapy (i.e., with the anti- Galectin-9 antibody as the sole active agent).
• the anti-Galectin-9 antibody such as G9.2-17(IgG4) disclosed herein may be used in a combined therapy, e.g., in combination with a PD-1 inhibitor such as those disclosed herein.
• the disclosure provides a method for treating a solid tumor in a subject, the method comprising administering to a subject in need thereof effective amount of an anti-Galectin-9 antibody or an effective amount of a pharmaceutical composition comprising an anti-Galectin-9 antibody described herein or antigen binding fragment thereof.
• an anti-Galectin-9 antibody or an effective amount of a pharmaceutical composition comprising an anti-Galectin-9 antibody described herein or antigen binding fragment thereof.
• Any of the anti- Galectin-9 antibodies disclosed herein can be used in the methods disclosed herein, e.g., antibody G9.2-17 (IgG4) (e.g., having a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 15).
• the antibody is administered once every two to six weeks, e.g., via intravenous infusion.
• the antibody may be administered once every 2-4 weeks, for example, every 2 weeks.
• the antibody may be administered once every week.
• the anti-Galectin 9 antibody disclosed herein e.g., G9.2-17 IgG4 is administered via a 30-minute to 6-hour infusion period intravenously.
• the intravenous infusion of the anti-Galectin 9 antibody may be performed for 30 minutes to 2 hours.
• the anti-Galectin 9 antibody may be administered via a long infusion period, for example, about 2-6 hours, e.g., about 2-4 hours or about 4-6 hours.
• examples anti-Galectin 9 antibody may be infused intravenous in a period of about 3 hours, about 4 hours, about 5 hours, or about 6 hours.
• the anti-Galectin-9 antibody disclosed herein for use in treating a solid tumor (e.g., those disclosed herein) can be administered to the subject at a dose of about 0.2 mg/kg to about 32 mg/kg, e.g., the dose may be selected from 0.2 mg/kg, 0.63 mg/kg, 2 mg/kg, 4 mg/kg, 6 mg/kg, 6.3 mg/kg, 8 mg/kg, 10 mg/kg, 12 mg/kg, and 16 mg/kg or a higher dose level.
• the dose may be selected from 0.2 mg/kg, 0.63 mg/kg, 2 mg/kg, 4 mg/kg, 6 mg/kg, 6.3 mg/kg, 8 mg/kg, 10 mg/kg, 12 mg/kg, and 16 mg/kg or a higher dose level.
• the anti-Galectin-9 antibody may be administered to the subject at a dose of about 1 mg/kg to about 32 mg/kg, e.g., the dose may be selected from 2 mg/kg, 4 mg/kg, 8 mg/kg, 12 mg/kg, and 16 mg/kg or a higher dose level.
• the anti-Galectin-9 antibody may be administered to the subject at a dose of about 0.2 mg/kg to about 32 mg/kg, e.g., the dose may be selected from 0.2 mg/kg, 0.63 mg/kg, 2 mg/kg, 4 mg/kg, 6 mg/kg, 6.3 mg/kg, 10 mg/kg, or 16 mg/kg or a higher dose level.
• 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 cycle, once every 2 weeks for two cycles, once every 2 weeks for 3 cycles, once every 2 weeks for 4 cycles, or once every 2 weeks for more than 4 cycles. 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 cycles extend for a duration of 3 months to 6 months, or 6 months to 12 months or 12 months to 24 months or longer.
• the cycle length is modified, e.g., temporarily or permanently to a longer duration, e.g., 3 weeks or 4 weeks.
• the use further comprises administering to the subject an immune checkpoint inhibitor, e.g., an anti-PD-1 antibody, as described herein, e.g., administered according to a regimen described herein.
• the interval or cycle is one week. In some embodiments, the interval or cycle is 2 weeks. In specific embodiments, the interval or cycle is 2 weeks. In specific embodiments, the interval or cycle is 3 weeks. In specific embodiments, the interval or cycle is 4 weeks.
• Solid tumor is selected from pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), cholangiocarcinoma (CAA), renal cell carcinoma (RCC), urothelial, head and neck, breast cancer, lung cancer, and other GI solid tumors, and in some embodiments, the regimen or dosing schedule is 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.
• PDAC pancreatic ductal adenocarcinoma
• CRC colorectal cancer
• HCC hepatocellular carcinoma
• CAA cholangiocarcinoma
• RRCC renal cell carcinoma
• urothelial head and neck
• breast cancer breast cancer
• lung cancer and other GI solid tumors
• the regimen or dosing schedule is once every 2 weeks for one cycle, once every 2 weeks for two cycles, once every
• the treatment is once every 2 weeks for 1 to 3 months, once every 2 weeks for 3 to 6 months, once every 2 weeks for 6 to 12 months, or once every 2 weeks for 12 to 24 months, or longer.
• the antibody is administered via intravenous infusion.
• the regimen or dosing schedule is once every 3 weeks or 4 weeks for one cycle, once every 3 weeks or 4 weeks for two cycles, once every once every 3 weeks or 4 weeks for three cycles, once every 3 weeks or 4 weeks for four cycles, or once every 3 weeks or 4 weeks for more than four cycles.
• the treatment is once every 3 weeks or 4 weeks for 1 to 3 months, once every 3 weeks or 4 weeks for 3 to 6 months, once every 3 weeks or 4 weeks for 6 to 12 months, or once every 3 weeks or 4 weeks for 12 to 24 months, or longer.
• the treatment is once every 3 weeks or 4 weeks for 1 to 3 months, once every 6 weeks for 3 to 6 months, once every 3 weeks or 4 weeks for 6 to 12 months, or once every 3 weeks or 4 weeks for 12 to 24 months, or longer. In some embodiments, the treatment is longer than 24 months when clinically indicated. In some embodiments, the antibody is administered via intravenous infusion.
• the anti-Galectin-9 antibody such as G9.2-17 (IgG4) may be administered to a human patient at a suitable dose (e.g., the doses disclosed herein) once every week.
• a suitable dose e.g., the doses disclosed herein
• 2.0 mg/kg of G9.2-17(IgG4) may be administered to the human patient once every week.
• 6.3 mg/kg of G9.2-17(IgG4) may be administered to the human patient once every week.
• 10 mg/kg of G9.2-17(IgG4) may be administered to the human patient once every week.
• 12 mg/kg of G9.2-17(IgG4) may be administered to the human patient once every week.
• 16 mg/kg of G9.2-17(IgG4) may be administered to the human patient once every week.
• the anti-Galectin-9 antibody may be given to the human patient for at least 2 cycles, at least 3 cycles, at least 4 cycles, at least 5 cycles, at least 6 cycles, or more.
• the treatment period may be 6 months to 12 months. In other instances, the treatment period may be 12 months to 24 months. In other instances, the treatment period may be longer than 24 months.
• the anti-Gal-9 antibody such as G9.2-17(IgG4) disclosed herein may be administered to a subject at a flat dose, e.g., about 650 mg to about 1120 mg, once every week to once every 4 weeks. In some examples, the anti-Gal-9 antibody is administered to a subject at a about 650 mg to about 700 mg once every week. In some examples, the anti-Gal-9 antibody is administered to a subject at a about 650 mg to about 700 mg once every two weeks. In some examples, the anti-Gal-9 antibody is administered to a subject at a about 1040 mg to about 1120 mg once every week. In some examples, the anti-Gal-9 antibody is administered to a subject at a about 1040 mg to about 1120 mg once every two weeks.
• the dosage(s) is adjusted in accordance with the patient’s response to treatment. In some embodiments, the dosages are altered between treatment intervals. In some embodiments, the treatment may be temporarily stopped. In some embodiments, the treatment may be temporarily stopped. In some embodiments, anti-Galectin-9 therapy is temporarily stopped. In some embodiments, a checkpoint inhibitor therapy employed in combination with the anti-Galectin-9 antibody is temporarily stopped. In some embodiments, both are temporarily stopped.
• a human patient may start with a low dose of the anti-Galectin-9 antibody such as G9.2-17 (IgG4) disclosed herein, for example, 0.2 mg/kg, 0.63 mg/kg, or 2 mg/kg.
• the dose of the antibody may be elevated, for example, to 6.3 mg/kg, 10 mg/kg, or 16 mg/kg.
• Galectin-9 Given that pro-tumor action of Galectin-9 is mediated through interaction with immune cells (e.g., interactions with lymphoid cells via TIM-3, CD44, and 41BB, and with macrophages via dectin- 1 and CD206) and given that Galectin-9 is expressed in a large number of tumors, targeting Galectin-9, e.g., using a Galectin-9 binding antibody to inhibit interaction with its receptors provides a therapeutic approach that can be applied across a variety of different tumor types.
• immune cells e.g., interactions with lymphoid cells via TIM-3, CD44, and 41BB, and with macrophages via dectin- 1 and CD206
• Galectin-9 is expressed in a large number of tumors
• targeting Galectin-9 e.g., using a Galectin-9 binding antibody to inhibit interaction with its receptors provides a therapeutic approach that can be applied across a variety of different tumor types.
• any of the anti-Galectin-9 antibodies described herein can be used in any of the methods described herein, administered in combination with a second therapeutic, e.g., a checkpoint inhibitor, such as an anti-PD-1 antibody or an anti-PD-Ll antibody.
• a second therapeutic e.g., a checkpoint inhibitor, such as an anti-PD-1 antibody or an anti-PD-Ll antibody.
• checkpoint inhibitors and administration regimen are provided elsewhere.
• the treatment method disclosed herein may further comprise administering to the subject an inhibitor of a checkpoint molecule, for example, PD-1.
• PD-1 inhibitors include anti-PD-1 antibodies, such as pembrolizumab, nivolumab, tislelizumab, dostarlimab, and cemiplimab.
• Such checkpoint inhibitors can be administered simultaneously or sequentially (in any order) with the anti-Galectin-9 antibody according to the present disclosure.
• the checkpoint molecule is PD-L1.
• PD-L1 inhibitors include anti-PD-Ll antibodies, such as durvalumab, avelumab, and atezolizumab.
• the checkpoint molecule is CTLA-4.
• An example of a CTLA-4 inhibitor is the anti-CTLA-4 antibody ipilimumab.
• the inhibitor targets a checkpoint molecule selected from CD40, GUR, LAG-3, 0X40, TIGU and TIM-3.
• the anti-Galectin-9 antibody improves the overall response, e.g., at 3 months, relative to a regimen comprising the inhibitor of the checkpoint molecule (e.g., anti- PD-1, for example, nivolumab) alone.
• the inhibitor of the checkpoint molecule e.g., anti- PD-1, for example, nivolumab
• the anti-PD-1 antibody is PD-1 is nivolumab
• the method described herein comprises administration of nivolumab to the subject at a dose of 240 mg intravenously once every two weeks.
• the antibody that binds PD-1 is co-used with the anti-Galectin-9 antibody disclosed herein (e.g., G9.2-17(IgG4)).
• the anti-PD-1 antibody can be is administered using a flat dose.
• the antibody that binds PD-1 is nivolumab, which can be administered to the subject at a dose of about 240 mg every two weeks or about 480 mg once every 4 weeks.
• the antibody that binds PD-1 is prembrolizumab, which can be administered at a dose of about 200 mg once every 3 weeks.
• the antibody that binds PD-1 is cemiplimab, which can be administered at a dose of about 350 mg intravenously once every 3 weeks. In some embodiments, the antibody that binds PD-1 is tislelizumab, which can be administered at a dose of about 200 mg intravenously once every 3 weeks or at a dose of about 400 mg intravenously once every 6 weeks. In some embodiments, the antibody that binds PD-1 is dostarlimab, which can be administered at a dose of about 500 mg intravenously every three weeks or about 1000 mg intravenously every six weeks.
• the antibody that binds PD-L1 is co-used with the anti-Galectin-9 antibody disclosed herein (e.g., G9.2-17(IgG4)).
• the antibody that binds PD-L1 is administered using a flat dose.
• the anti-PD-Ll antibody is atezolizumab, which may be administered at a dose of 1200 mg intravenously once every 3 weeks.
• the anti-PD-Ll antibody antibody is Avelumab, which may be administered at a dose of 10 mg/kg intravenously every 2 weeks.
• the anti- PD-Ll antibody is durvalumab, which may be administered at a dose of 1500 mg intravenously every 4 weeks.
• any of the methods disclosed herein comprise (i) administering 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 tumors) any of the anti-Galectin-9 antibodies disclosed herein (e.g., G9.2-17 such as the the antibody having the heavy chain of SEQ ID NO: 19 and the light chain of SEQ ID NO:5) at a dose of about 0.2 to about 32 mg/kg (e.g., about 3 mg/kg or about 15 mg/kg) once every two weeks; and (ii) administering to the human patient an effective amount of an anti-PD-1 antibody (e.g., nivolumab, prembrolizumab, tislelizumab, or cemiplimab, dostarlimab, durvaluma
• any of the methods disclosed herein comprise (i) administering 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 tumors) any of the anti-Galectin-9 antibodies disclosed herein (e.g., G9.2-17 such as the the antibody having the heavy chain of SEQ ID NO: 19 and the light chain of SEQ ID NO:5) at a dose of about 0.2 to about 32 mg/kg (e.g., about 10 mg/kg or about 16 mg/kg) once every week; and (ii) administering to the human patient an effective amount of an anti-PD-1 or anti-PD-Ll antibody (e.g., nivolumab, prembrolizumab, tislelizumab, or cemiplimab, dostarlim
• anti-Galectin-9 antibodies through their inhibition of Dectin-1, can reprogram immune responses against tumor cells via, e.g., inhibiting the activity of y ⁇ T cells infiltrated into tumor microenvironment, and/or enhancing immune surveillance against tumor cells by, e.g., activating CD4+ and/or CD8+ T cells.
• an anti-Galectin-9 antibody and an immunomodulatory agent such as those described herein would be expected to significantly enhance anti-tumor efficacy.
• the methods are provided, wherein the anti-Galectin-9 antibody is administered concurrently with a checkpoint inhibitor.
• the anti-Galectin-9 antibody is administered before or after a checkpoint inhibitor.
• the checkpoint inhibitor is administered systemically.
• the checkpoint inhibitor is administered locally.
• 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, intracerebrospinal, subcutaneous, intra-arterial, intra-articular, intravesical, intrasynovial, intrathecal, intratumoral, or sub-urothelial route.
• the checkpoint inhibitor is administered to the subject by intravenous infusion.
• the checkpoint inhibitor such as any of the anti-PD-1 antibodies disclosed herein and any of the anti-Galectin 9 antibodies disclosed herein such as G9.2-17 (e.g., G9.2-17(IgG4)) may have same day administration.
• the checkpoint inhibitor can be administered to a subject prior to administration of the anti-Galectin 9 antibody.
• the administration of the checkpoint inhibitor, e.g., anti-PD-1 antibody, and the administration of the anti-Galectin 9 antibody are performed on two consecutive days.
• the checkpoint inhibitor, e.g., anti-PD-1 antibody may be administered to the subject on the first day of dosing and the anti-Galectin-9 antibody can be administered to the subject on the subsequent day.
• the 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 the anti-Galectin 9 antibodies disclosed herein such as G9.2-17.
• the anti-Galectin 9 antibody can be administered to a subject prior to administration of the checkpoint inhibitor, e.g., an anti-PD-1 antibody.
• the administration of the anti-Galectin 9 antibody and the administration of the checkpoint inhibitor, e.g., anti-PD-1 antibody are performed on two consecutive days.
• the anti-Galectin-9 antibody may be administered to the subject on the first day of dosing and checkpoint inhibitor, e.g., anti- PD-1 antibody, can be administered to the subject on the subsequent day.
• the anti-Galectin-9 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 the checkpoint inhibitor, such as any of the anti-PD-1 antibodies disclosed herein.
• the anti-galectin-9 antibody can be administered (alone or in combination with an anti-PD-1 antibody) 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.
• the treatment is 1 to 3 months, 3 to 6 months, 6 to 12 months, 12 to 24 months, or longer.
• the treatment is once every 2 weeks for 1 to 3 months, once every 2 weeks for 3 to 6 months, once every 2 weeks for 6 to 12 months, or once every 2 weeks for 12 to 24 months, or longer.
• the anti-Galectin-9 antibody may be used in combination with a regimen comprising UGN-102, UGN-201, or UGN-302.
• UGN-102, UGN- 201, or UGN-302 are formulated in a hydrogel e.g., a reverse-thermal hydrogel technology-based hydrogel.
• the anti-Galectin-9 antibody can be administered prior to UGN-102, UGN-201, or UGN-302.
• the anti-Galectin-9 antibody can be administered concurrently with UGN-102, UGN-201, or UGN-302.
• the anti-Galectin-9 antibody may be administered after UGN-102, UGN-201, or UGN-302.
• a response to treatment can be assessed according to RECIST or the RECIST 1.1 criteria and /or irRC, irRECIST, iRECIST, imRECISTPDAC, as described in Example 1 below and Eisenhower et al., New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1); European Journal Of Cancer 45 (2009) 228 - 247; or Borcoman et al., Annals of Oncology 30: 385-396, 2019;Nishino et al., Clin Cancer Res 2013; 19(14): 3936-3943, the contents of each of which is herein incorporated by reference in its entirety.
• methods are provided for improving and or controlling the overall response/tumor burden/tumor size (e.g., at approximately 2, 3, 6 or 12 months, or a later time) comprising administering an anti-Galectin-9 antibody described herein, e.g., as compared to a baseline level obtained prior to initiation of G9.2-17 IgG4 treatment regimen.
• the methods are for improving and or controlling the overall response/tumor burden/tumor size at approximately 2 months.
• treating can improve or control the overall response /tumor burden/tumor size (e.g., at approximately 2, 3, 6 or 12 months, or a later time), e.g., as compared to a baseline level obtained prior to initiation of treatment.
• methods are provided, which result in a complete response, a partial response or stable disease (e.g., as measured at approximately 2 months, 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point), comprising administering an anti-Galectin-9 antibody described herein.
• a response can be temporary over a certain time period or permanent.
• the methods improve the likelihood of a complete response, a partial response or stable disease (e.g., as measured at approximately 2 months, 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point), e.g., as compared to a baseline level obtained prior to initiation of G9.2-17 IgG4 treatment regimen.
• a response can be temporary over a certain time period or permanent.
• treating can result in reduced or attenuated progressive disease (e.g., as measured at approximately 2 months, 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point), e.g., as compared to a baseline level obtained prior to initiation of G9.2-17 IgG4 treatment regimen.
• Such an attenuation may be temporary or permanent.
• anti-Galectin-9 antibody may be administered in combination with a checkpoint inhibitor, e.g., an anti-PD-1 antibody.
• the disclosure provides methods for attenuating disease progression or reducing progressive disease (e.g., as measured at approximately 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point).
• the method comprising administering to the subject a therapeutically effective amount of an anti- Galectin-9 antibody as disclosed herein.
• the anti-Galectin-9 antibody may be administered in combination with a checkpoint inhibitor, e.g., an anti-PD-1 antibody.
• Partial response, stable disease, complete response, a partial response, stable disease, progressive disease, disease progressing can be assessed according to irC criteria, RECIST criteria, RECIST1.1., irRECIST or iRECIST, or imRECIST criteria, or other criteria known in the art (see, e.g., Borcoman et al., Annals of Oncology 30: 385-396, 2019’ iRC: Hoos et al., J. Immunother. 30 (1): 1-15).
• a partial response is a decrease in the size of a tumor, or in the extent of cancer in the body, i.e., the tumor burden, in response to treatment as compared to a baseline level before the initiation of the treatment.
• a partial response is defined as at least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum diameters.
• Progressive disease is a disease that is growing, spreading, or getting worse.
• progressive disease includes disease in which at least a 20% increase in the sum of diameters of target lesions is observed, and the sum must also demonstrate an absolute increase of at least 5 mm. Additionally, the appearance of one or more new lesions is also considered progression.
• a tumor that is neither decreasing nor increasing in extent or severity as compared to a baseline level before initiation of the treatment is considered stable disease.
• stable disease occurs when there is neither sufficient shrinkage to qualify for partial response nor sufficient increase to qualify for progressive disease, taking as reference the smallest sum diameters while on study.
• the disclosure provides methods for reducing or maintaining tumor size in a subject, including a human subject (e.g., as measured at approximately 2 months, 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point), either permanently or over a minimum time period, relative to a baseline tumor size prior to initiation of the treatment in the subject, the method comprising administering to the subject a therapeutically effective amount of an anti-Galectin-9 antibody alone or in combination with a checkpoint inhibitor, e.g., an anti-PD-1 antibody.
• a checkpoint inhibitor e.g., an anti-PD-1 antibody.
• Tumor size e.g., the diameters of tumors
• tumor size reduction, maintenance of tumor size refers to the size of target lesions. In some embodiments, tumor size reduction, maintenance of tumor size refers to the size of non-target lesions.
• all lesions up to a maximum of five lesions total (and a maximum of two lesions per organ) representative of all involved organs should be identified as target lesions. All other lesions (or sites of disease) including pathological lymph nodes should be identified as non-target lesions.
• the disclosure provides methods for increasing the likelihood of reducing or maintaining a tumor burden (e.g., as measured at approximately 2 months, 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point), the methods comprising administering to the 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.
• treating can result in in a greater likelihood of a reduction of tumor burden, or maintenance of tumor burden, (e.g., as measured at approximately 2 months, 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point).
• tumor burden refers to amount of cancer, the size or the volume of the tumor in the body of a subject, accounting for all sites of disease.
• 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, comprising bioluminescence imaging (BLI) and fluorescence imaging (ELI).
• FDG-PET FDG positron emission tomography
• MRI magnetic resonance imaging
• ELI fluorescence imaging
• the methods described herein increase in the time to disease progression or in progression free survival (e.g., as measured at approximately 2 months, 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point post initiation of treatment).
• Progression free survival can be either permanent or progression free survival over a certain amount of time.
• the methods provide a greater likelihood of progression free survival (either permanent progression free survival or progression free survival over a certain amount of time, e.g., 3, 6 or 12 months or e.g., as measured at approximately 2 months, 3 months, 6 months, or 12 months, at a later time, or at any other clinically indicated time point post initiation of treatment).
• Progression-free survival is defined as the time from random assignment in a clinical trial, e.g., from initiation of a treatment to disease progression or death from any cause.
• the methods achieve longer survival or greater likelihood of survival, e.g., at a certain time, e.g., at 6 or 12 months.
• a response to treatment can be assessed according to iRECIST criteria, as described in Seymour et al, iRECIST: guidelines for response criteria for use in trials; The Lancet, Voll8, March 2017, the contents of which is herein incorporated by reference in its entirety.
• iRECIST was developed for the use of modified RECIST 1.1 criteria specifically in cancer immunotherapy trials, to ensure consistent design and data collection and can be used as guidelines to a standard approach to solid tumor measurements and definitions for objective change in tumor size for use in trials in which an immunotherapy is used.
• iRECIST is based on RECIST 1.1.
• iRECIST Responses assigned using iRECIST have a prefix of “i” (i.e., immune)- e.g., “immune” complete response (iCR) or partial response (iPR), and unconfirmed progressive disease (iUPD) or confirmed progressive disease (iCPD) or stable disease (iSD) to differentiate them from responses assigned using RECIST 1.1, and all of which are defined in Seymour et al. RECIST 1.1.
• criteria can be compared to baseline levels prior to initiation of treatment.
• the 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.
• the disclosure provides methods for improving overall response (iOR) or achieving “immune” complete response (iCR), a partial response (iPR) or stable disease (iSD) (e.g., as measured at approximately 2 months, 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point), as compared to the baseline level of disease prior to initiation of the treatment.
• the reduction in the “immune” response, e.g., iCR, iPR, or iSD can be temporary over a certain time period or permanent.
• treating can improve the likelihood of a complete response (iCR), a partial response (iPR) or stable disease (iSD) (e.g., as measured at approximately 2 months, 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point), e.g.,
• the disclosure provides methods for attenuating disease progression or reducing progressive disease, e.g., reducing unconfirmed progressive disease (iUPD) or reducing confirmed progressive disease (iCPD)) (e.g., as measured at approximately 2 months, 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point), the method comprising administering to the subject a therapeutically effective amount of an anti-Galectin-9 antibody as disclosed herein.
• any of these above mentioned iRECIST criteria can be compared to baseline levels prior to initiation of treatment.
• the anti-Galectin-9 antibody may be administered alone or in combination with a checkpoint inhibitor, e.g., an anti- PD-1 antibody.
• the reduction in iUPD or iCPD can be temporary over a certain time period or permanent.
• treating can result in greater likelihood of overall reduction in unconfirmed progressive disease (iUPD) or confirmed progressive disease (iCPD) (e.g., as measured at approximately 2 months , 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point
• iUPD unconfirmed progressive disease
• iCPD confirmed progressive disease
• the disclosure provides methods for reducing the number of new lesions in a subject, including a human subject, according to iRECIST criteria (e.g., as measured at approximately 2 months , 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point), the methods comprising administering to the subject a therapeutically effective amount of an anti-Galectin-9 antibody as disclosed herein.
• the anti-Galectin-9 antibody may be administered in combination with a checkpoint inhibitor, e.g., an anti-PD-1 antibody or anti-PD-Ll antibody.
• tumor burden can be measured according to the irRC criteria (Hoos et al., 2007).
• irRC tumor burden is measured by combining “index” lesions with new lesions, i.e., new lesions are considered a change in tumor burden.
• an immune-related Complete Response irCR
• irPR immune-related Partial Response
• irPD immune-related Progressive Disease
• irSD immune-related Stable Disease
• Immune-related RECIST (irRECIST) is based on unidimensional measurements of RECIST, and Specific immune-related criteria were further redefined in the irRECIST. Recently, new criteria were evaluated based on atezolizumab data in NSCLC, the immune-modified RECIST (imRECIST), requiring a confirmation of disease progression at least 4 weeks after initial assessment (Hodi et al, ICO 2018; 36(9): 850-858).
• a subject being treated by any of the anti-galectin-9 antibodies disclosed herein may be monitored for occurrence of adverse effects (for example, severe adverse effects).
• a checkpoint inhibitor e.g., an anti-PD-1 or anti- PD-L1 antibody
• exemplary adverse effects to monitor are provided in Example 1 below. If occurrence of adverse effects is observed, treatment conditions may be changed for that subject. For example, the dose of the anti-galectin-9 antibody may be reduced and/or the dosing interval may be extended. Suitability and extent of reduction may be assessed by a qualified clinician. In one embodiment, a reduction level of 30 or 50% of the previous dose level is implemented.
• a reduction level as per clinician’s assessment or at least by 30% is implemented (to dose level 1, the level at first dose reduction). If required, one more dose reduction by 30% of dose level -1 is implemented (dose level -2, the level at second dose reduction). In another example, one more dose reduction by 50% of dose level -1 is implemented (dose level -2). In some embodiments, one or more dose reductions by about 10% to about 80% of a previous dose level are implemented. In some embodiments, one or more dose reductions 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% of a previous dose level are implemented.
• one or more dose reductions by 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, or 70% to 80% of a previous dose level are implemented.
• one or more dose reductions by about 10%, by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, by about 70%, or by about 80% of a previous dose level are implemented.
• one or more dose reductions by 10%, by 20%, by 30%, by 40%, by 50%, by 60%, by 70%, or by 80% of a previous dose level are implemented.
• the dose of the checkpoint inhibitor can be reduced and/or the dosing interval of the checkpoint inhibitor may be extended. In some instances (e.g., occurring of life - threatening adverse effects), the treatment may be terminated.
• Response to treatment can also be characterized by one or more of immunophenotype in blood and tumors, cytokine profile (serum), soluble galectin-9 levels in blood (serum or plasma), galectin-9 tumor tissue expression levels and pattern of expression by immunohistochemistry (tumor, stroma, immune cells), tumor mutational burden (TMB), PD-L1 expression (e.g., by immunohistochemistry), mismatch repair status, or tumor markers relevant for the disease (e.g., as measured at approximately 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point).
• tumor markers include, but are not limited to, CA15-3, CA-125, CEA, CA19-9, alpha fetoprotein.
• 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-Ll antibody.
• the subject may examined for one or more of the following features before, during, and/or after the treatment: (a) one or more tumor markers in blood samples from the 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/excisional specimen multiplex immunophenotyping, f) tumor tissue biopsy/excisional specimen galectin-9 expression levels and pattern, g) any other immune score test such as: PD-L1 immunohistochemistry, tumor mutational burden (TMB), tumor microsatellite instability status, as well as panels such as: Immunoscore®- HalioDx, ImmunoSeq- Adaptive Biotechnologies, TIS, developed on the NanoString
• the methods are described herein for changing levels of immune cells and immune cell markers in the blood or in tumors, e.g., immune activation, comprising an anti-Gal-9 antibody is administered alone or in combination with a checkpoint inhibitor, e.g., an anti-PD-1 antibody.
• a checkpoint inhibitor e.g., an anti-PD-1 antibody.
• Such changes can be measured in patient blood and tissue samples using methods known in the art, such as multiplex flow cytometry and multiplex immunohistochemistry.
• a panel of phenotypic and functional PBMC immune markers can be assessed at baseline prior to commencement of the treatment and at various time point during treatment. Table 2 lists non-limiting examples of markers useful for these assessment methods.
• FC Flow cytometry
• modulation comprises in one or more of (1) an increase in more CDS cells in plasma or tumor tissue, (2) a reduction in T regulatory cells (Tregs) in plasma or tumor tissue, (3) an increase in Ml macrophages in plasma or tumor tissue and (4) a decrease in MDSCs in plasma or tumor tissue, and (5) a decrease in M2 macrophages in plasma or tumor tissue (e.g., as measured at approximately 2 months , 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point).
• the markers that are assessed using the techniques described above or known in the art are selected from CD4, CDS CD 14, CDllb/c, and CD25. These parameters can be compared to baseline levels prior to initiation of treatment.
• PBMC phenotyping markers comprising administering an anti- gal9 alone or in combination with a checkpoint inhibitor therapy, for modulating proinflammatory and anti-inflammatory cytokines.
• methods are provided for one or more of (1) increasing levels of IFNgamma in plasma or tumor tissue; (2) increasing levels of TNFalpha in plasma or tumor tissue; (3) decreasing levels of IL- 10 in plasma or tumor tissue (e.g., as measured at approximately 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point). These parameters can be compared to baseline levels prior to initiation of treatment.
• cytokine levels or immune cell levels may be assessed between a pre dose 1 tumor biopsy and repeat biopsy conducted at a feasible time. In some embodiments, cytokine levels or immune cell levels may be assessed between 2 repeat biopsies. In some embodiments, methods are provided for modulating one or more of soluble galectin-9 levels in blood (serum or plasma), or galectin-9 tumor tissue expression levels and pattern of expression by immunohistochemistry (tumor, stroma, immune cells), (e.g., as measured at approximately 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point).
• the methods decrease soluble galectin-9 levels in blood (serum or plasma), or galectin-9 tumor tissue expression levels or pattern of expression by immunohistochemistry (tumor, stroma, immune cells) (e.g., as measured at approximately 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 a control group not receiving the treatment or healthy subjects.
• the anti-Galectin-9 antibody may be administered alone or in combination with a checkpoint inhibitor, e.g., an anti-PD-1 antibody.
• methods for modulating PD-L1 expression comprising administering an anti-Galectin-9 antibody, alone or in combination with a checkpoint inhibitor, e.g., an ant-Galectin-9 antibody.
• the methods modulate in one or more tumor markers (increase or decrease) relevant for the disease (e.g., as measured at approximately 2 months, 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point).
• 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.
• the anti-Galectin-9 antibody may be administered alone or in combination with a checkpoint inhibitor, e.g., an anti- PD-1 antibody.
• the disclosure provides methods of modulating an immune response in a subject.
• the term “immune response” includes T cell-mediated and/or B cell-mediated immune responses that are influenced by modulation of immune cell activity, for example, T cell activation.
• an immune response is T cell mediated.
• the term “modulating” means changing or altering, and embraces both upmodulating and downmodulating.
• modulating an immune response means changing or altering the status of one or more immune response parameters).
• Exemplary parameters of a T cell mediated immune response include levels of T cells (e.g., an increase or decrease in effector T cells) and levels 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 levels of B cells, B cell activation and B cell mediated antibody production.
• modulating the immune response causes an increase (or upregulation) in one or more immune response parameters and a decrease (or downregulation) 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 an inflammatory immune response.
• modulating the immune response causes an increase (or upregulation) in one or more immune response parameters and a decrease (or downregulation) 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 an inflammatory response.
• an increase in an overall immune response i.e., an increase in an overall inflammatory immune response
• an increase in an overall immune response is determined by a reduction in tumor weight, tumor size or tumor burden or any RECIST or iRECIST criteria described herein.
• an increase in an overall immune response is determined by increased level(s) of one or more proinflammatory cytokine(s), e.g., including two or more, three or more, etc. or a majority of proinflammatory cytokines (one or more, two or more, etc. or a majority of anti-inflammatory and/or immune suppressive cytokines and/or one or more of the most potent anti-inflammatory or immune suppressive cytokines either decrease or remain constant).
• an increase in an overall immune response is determined by increased levels of one or more of the most potent proinflammatory cytokines (one or more anti-inflammatory and/or immune suppressive cytokines including one or more of the most potent cytokines either decrease or remain constant). In some embodiments an increase in an overall immune response is determined by decreased levels of one or more, including a majority of, immune suppressive and/or anti-inflammatory cytokines (the levels of one or more, or a majority of, proinflammatory cytokines, including e.g., the most potent proinflammatory cytokines, either increase or remain constant).
• an increase in an overall immune response is determined by increased levels of one or more of the most potent anti-inflammatory and/or immune suppressive cytokines (one or more, or a majority of, proinflammatory cytokines, including, e.g., the most potent proinflammatory cytokines either increase or remain constant).
• an increase in an overall immune response is determined by a combination of any of the above.
• an increase (or upregulation) of one type of immune response parameter can lead to a corresponding decrease (or downregulation) in another type of immune response parameter.
• an increase in the production of certain proinflammatory cytokines can lead to the downregulation of certain anti-inflammatory and/or immune suppressive cytokines and vice versa.
• the disclosure provides methods for modulating an immune response (e.g., as measured at approximately 2 months, 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point) in a subject, including a human subject, comprising administering to the subject a therapeutically effective amount of an anti-Galectin-9 antibody as disclosed herein.
• the disclosure provides methods for modulating levels of immune cells and immune cell markers, including but not limited to those described herein in Table 2, e.g., as compared to baseline levels prior to initiation of treatment, e.g., as compared to a baseline level obtained prior to initiation of the anti-Gal9 antibody treatment regimen, , in the blood or in tumors of a subject, including a human subject, comprising administering to the subject a therapeutically effective amount of an anti-Galectin-9 antibody as disclosed herein.
• the overall result of modulation is upregulation of proinflammatory immune cells and/or down regulation of immune-suppressive immune cells.
• the disclosure provides methods for modulating levels of immune cells, wherein the modulating encompasses one or more of (1) increasing CDS cells in plasma or tumor tissue, (2) reducing Tregs in plasma or tumor tissue, (3) increasing Ml macrophages in plasma or tumor tissue and (4) decreasing MDSC in plasma or tumor tissue, and (5) decreasing in M2 macrophages in plasma or tumor tissue, and wherein the methods comprise administering to the subject a therapeutically effective amount of an anti-Galectin-9 antibody as disclosed herein.
• the markers to assess levels of such immune cells include but are not limited to CD4, CDS CD14, CDllb/c, and CD25.
• the disclosure provides methods for modulating levels of proinflammatory and immune suppressive cytokines (e.g., as measured at approximately 2 months , 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point), e.g., as compared to baseline levels prior to initiation of treatment, in the blood or in tumors of a subject, including a human subject, comprising administering to the subject a therapeutically effective amount of an anti-Galectin-9 antibody as disclosed herein.
• the overall result of modulation is upregulation of proinflammatory cytokines and/or down regulation of immune-suppressive cytokines.
• the disclosure provides methods for modulating levels of cytokines cells, wherein the modulating encompasses one or more of (1) increasing levels of IFNgamma in plasma or tumor tissue; (2) increasing levels of TNFalpha in plasma or tumor tissue; (3) decreasing levels of IL-10 in plasma or tumor tissue.
• the disclosure provides methods for changing one or more of soluble galectin-9 levels in blood (serum or plasma), or in galectin-9 tumor tissue expression levels and pattern of expression by immunohistochemistry (tumor, stroma, immune cells) (e.g., as measured at 2 weeks, 4 weeks, 1 month, 2 month , 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point), comprising administering to the subject a therapeutically effective amount of an anti-Galectin-9 antibody as disclosed herein.
• immunohistochemistry tumor, stroma, immune cells
• one or more of soluble galectin-9 levels in blood (serum or plasma), or in galectin-9 tumor tissue expression levels and pattern of expression by immunohistochemistry (tumor, stroma, immune cells) remain unchanged.
• the methods provided herein decrease one or more of soluble galectin-9 levels in blood (serum or plasma), or in galectin-9 tumor tissue expression levels and pattern of expression by immunohistochemistry (tumor, stroma, immune cells) (e.g., e.g., as measured at 2 weeks, 4 weeks, 1 month, 2 month, 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, the Galectin-9 levels may be compared to healthy subjects. In some embodiments, treating results in a change in PD-L1 expression, e.g., by immunohistochemistry. 16 mg/kg or higher dose level 16 mg/kg or higher dose level 16 mg/kg or a higher dose level.
• the disclosure provides methods for changing PD-L1 expression, e.g., as assessed by immunohistochemistry (e.g., as measured at 2 weeks, 4 weeks, 1 month, 2 month, 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point), comprising administering to the subject a therapeutically effective amount of an anti-Galectin-9 antibody as disclosed herein.
• PD-L1 expression e.g., as assessed by immunohistochemistry
• PD-L1 levels can be compared to baseline levels prior to initiation of treatment. .
• the methods provided herein decrease PD-L1 expression, e.g., as assessed by immunohistochemistry.
• PD-L1 levels may be measured using routine methods known in the art.
• PD-L1 SP263 (Roche, Ventana) can be used for detection of PD-L1 in cancer tissues using immunohistochemistry.
• the disclosure provides methods for changing one or more tumor markers (increasing or decreasing) relevant for the 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 time point), comprising administering to the subject a therapeutically effective amount of an anti- Galectin-9 antibody as disclosed herein.
• one or more tumor markers (increasing or decreasing) relevant for the disease remain unchanged. Examples of such tumor markers include, but not limited to CA15-3, CA-125, CEA, CA19-9, alpha fetoprotein. Levels of tumor markers can be compared to baseline levels prior to initiation of treatment.
• the methods provided herein decrease the occurrence of one or more tumor markers relevant for the disease.
• the disclosure provides methods for changing one or more biomarkers (increasing or decreasing) relevant for the 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 time point), comprising administering to the subject a therapeutically effective amount of an anti-Galectin-9 antibody as disclosed herein.
• levels of biomarkers in clinical tissues from patients can be measured using routine methods, such as multiplex Immunofluorescence (mlF) technology, as described herein in the examples.
• An exemplary panel of biomarkers may include CD3, CD4, CDS, CD45RO, FoxP3, GDI lb, CD14, CD15, CD16, CD33, CD68, CD163, HLA-DR, Arginasel, Granzyme B, Ki67, PD-1, PD-L1, F4/80, Ly6G/C and PanCK.
• the antibody comprises a light chain complementarity determining region 1 (CDR1) set forth as SEQ ID NO: 1, a light chain complementarity determining region 2 (CDR2) set forth as SEQ ID NO: 2, and a light chain complementarity determining region 3 (CDR3) set forth as SEQ ID NO: 3 and/or comprises a heavy chain complementarity determining region 1 (CDR1) set forth as SEQ ID NO: 4, a heavy chain complementarity determining region 2 (CDR2) set forth as SEQ ID NO: 5, and a heavy chain complementarity determining region 3 (CDR3) set forth as SEQ ID NO: 6.
• CDR1 light chain complementarity determining region 1
• CDR2 light chain complementarity determining region 2
• CDR3 light chain complementarity determining region 3
• 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.
• the antibody is G9.2-17 IgG4.
• the anti- Galectin-9 antibody is administered to the subject at a dose of about 0.2 mg/kg to about 32 mg/kg, e.g., the dose may be selected from 0.2 mg/kg, 0.63 mg/kg, 2 mg/kg, 4 mg/kg, 6 mg/kg, 6.3 mg/kg, 8 mg/kg, 10 mg/kg, 12 mg/kg, and 16 mg/kg or a higher dose level.
• the anti-Galectin-9 antibody is administered to the subject at a dose of about 1 mg/kg to about 32 mg/kg, e.g., the dose may be selected from 2 mg/kg, 4 mg/kg, 8 mg/kg, 12 mg/kg, and 16 mg/kg or a higher dose level.
• the anti-Galectin-9 antibody is administered to the subject at a dose of about 0.2 mg/kg to about 32 mg/kg, e.g., the dose may be selected from 0.2 mg/kg, 0.63 mg/kg, 2 mg/kg, 4 mg/kg, 6 mg/kg, 6.3 mg/kg, 10 mg/kg, or 16 mg/kg or a higher dose level.
• the antibody is administered once every two weeks, e.g., via intravenous infusion.
• the method further comprises administering to the subject an immune checkpoint inhibitor, e.g., an anti-PD-1 antibody or an anti-PD-Ll antibody.
• the solid tumor is selected from pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), cholangiocarcinoma (CAA), renal cell carcinoma (RCC), urothelial, head and neck, breast cancer, lung cancer, and other GI solid tumors, and in some embodiments, the solid tumor is a metastatic tumor.
• the disclosure provides methods for improving quality of life and/or improving symptom control (e.g., as measured at 1 month, 3 months, 6 months or 12 months, or at a later time or at any other clinically indicated time point) in a subject, including a human subject, 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 may be compared to baseline prior to initiation of treatment.
• improvements can be measured on the ECOG scale.
• kits for use in treating or alleviating a disease associated with Galectin-9 for example associated with Galectin-9 binding to a cell surface glycoprotein (e.g., Dectin-1, TIM3, CD206, etc.), or pathologic cells (e.g., cancer cells) expressing Galectin-9.
• a cell surface glycoprotein e.g., Dectin-1, TIM3, CD206, etc.
• pathologic cells e.g., cancer cells
• examples include solid tumors such as PDAC, CRC, HCC, cholangiocarcinoma and other GI solid tumors, and others described herein.
• kits can include one or more containers comprising an anti-Galectin-9 antibody, e.g., any of those described herein, and optionally a second therapeutic agent (e.g., a checkpoint inhibitor such as an anti-PD- 1 antibody as disclosed herein) to be co-used with the anti-Galectin-9 antibody, which is also described herein.
• the kit can comprise instructions for use in accordance with any of the methods described herein. The included instructions can comprise a description of administration of the anti-Galectin-9 antibody, and optionally the second therapeutic agent, to treat, delay the onset, or alleviate a target disease as those described herein.
• the kit further comprises a description of selecting an individual suitable for treatment based on identifying whether that individual has the target disease, e.g., applying the diagnostic method as described herein.
• the instructions comprise a description of administering an antibody to an individual at risk of the target disease.
• the instructions relating to the use of an anti-Galectin-9 antibody generally include information as to dosage, dosing schedule, and route of administration for the intended treatment.
• the containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.
• Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
• the label or package insert indicates that the composition is used for treating, delaying the onset and/or alleviating the disease associated with Galectin-9 (e.g., Dectin- 1, TIM-3, or CD206 signaling).
• Galectin-9 e.g., Dectin- 1, TIM-3, or CD206 signaling.
• instructions are provided for practicing any of the methods described herein.
• kits of this invention are in suitable packaging.
• suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g. , sealed Mylar or plastic bags), and the like.
• packages for use in combination with a specific device such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump.
• a kit has a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
• the container also has a sterile access port (for example the container is an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
• a sterile access port for example 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 those described herein.
• Kits may optionally provide additional components such as buffers and interpretive information.
• the kit comprises a container and a label or package insert(s) on or associated with the container.
• the invention provides articles of manufacture comprising contents of the kits described above.
• Example 1 A Phase 1/2 Open-label, Multi-center Study of the Safety, Pharmacokinetics, and Anti-tumor Activity of Anti-Galectin-9 Monoclonal Antibody Alone and in Combination with an Anti-PD-1 Antibody in Patients with Metastatic Solid Tumors
• Galectin-9 is a molecule overexpressed by many solid tumors, including those in pancreatic cancer, colorectal cancer, and hepatocellular carcinoma. Moreover, Galectin-9 is expressed on tumor-associated macrophages, as well as intra-tumoral immunosuppressive gamma delta T cells, thereby acting as a potent mediator of cancer-associated immunosuppression. As described herein, monoclonal antibodies targeting Galectin-9 (e.g., G9.2-17, IgG4) have been developed. Data have demonstrated that the G9.2-17 halts pancreatic tumor growth by 50% in orthotopic KPC models and extended the survival of KPC animals by more than double.
• anti-Galectin-9 antibodies reverse the M2 to Ml phenotype, facilitating intra-tumoral CD8 + T cell activation.
• antibody G9.2-17 (having a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 15) has been found to synergize with anti-PD-1.
• G9.2-17 is a fully human IgG4 monoclonal antibody (mAb) targeting galectin-9 (- gal-9) protein.
• Gal-9 functions as an immuno-suppressor, conferring immune privilege to tumor cells and disabling immune mediated cancer attack by regulating macrophages, T-cells, myeloid derived suppressor cells as well as cancer cell susceptibility to cytotoxic T-cell-induced death.
• G9.2-17 (IgG4) blockade of gal-9 interferes with the immunosuppressive functions of gal-9 resulting in effective immune activation and tumor growth inhibition across 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 cell and small cell lung cancer, nasopharyngeal cancer, malignant melanoma, ovarian cancer etc., and high levels of tissue and/or circulating gal-9 correlate with aggressive tumor features and adverse survival outcome.
• CCA cholangiocarcinoma
• CRC colorectal cancer
• breast cancer bladder cancer
• ovarian cancer non-small cell and small cell lung cancer
• nasopharyngeal cancer malignant melanoma
• ovarian cancer etc. and high levels of tissue and/or circulating gal-9 correlate with aggressive tumor features and adverse survival outcome.
• the target indications of G9.2-17 (IgG4) are relapsed or refractory, metastatic solid tumors, where G9.2-17 (IgG4) is investigated both as a single agent, or and in combination with a checkpoint inhibitor (e.g., a programmed cell death 1 [PD 1] antibody such as nivolumab, pembrolizumab, cemiplimab, dostarlimab, or tislelizumab).
• a checkpoint inhibitor e.g., a programmed cell death 1 [PD 1] antibody such as nivolumab, pembrolizumab, cemiplimab, dostarlimab, or tislelizumab.
• Dose escalation is conducted in all solid tumor types in order to establish the safety and tolerability profile of G9.2-17 (IgG4), assess its immunogenicity potential, establish the pharmacokinetic (PK) and pharmacodynamic (PD) profile, and arrive at the recommended Phase 2 dose (RP2D). This may be the maximal tolerated dose (MTD).
• the expansion cohorts are planned in: first line metastatic pancreatic ductal adenocarcinoma (PDAC), as well as CRC and CCA, both as a single agent and in combination with an anti-PDl antibody.
• G9.2-17 (IgG4) has been shown to be highly specific for gal-9 and has been demonstrated to be efficacious in multiple animal models of cancer.
• the patient populations targeted for enrollment are at late stages in their disease and have failed at standard of care treatments prior to enrollment in this study.
• G9.2-17 (IgG4), either taken alone or in combination with a checkpoint inhibitor such as an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, tislelizumab, dostarlimab, or cemiplimab) would be expected to benefit treatment of malignant tumors such as malignant solid tumors.
• a checkpoint inhibitor such as an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, tislelizumab, dostarlimab, or cemiplimab)
• an anti-PD-1 antibody e.g., nivolumab, pembrolizumab, tislelizumab, dostarlimab, or cemiplimab
• Part 1 This is an open-label, uncontrolled, multicenter Phase 1/2 study with a dose escalation phase (Part 1) and a cohort expansion phase (Part 2) in patients with relapsed/refractory metastatic solid tumors.
• Part 2 This study is conducted at up to 20 sites in the United States. The study duration is estimated to be 12-24 months.
• follow-up for survival continues for up to 2 years.
• a study schema is presented in FIG. 1.
• G9.2-17 This study includes both monotherapy of G9.2-17 (IgG4) and combination of G9.2-17 and an anti-PD-1 antibody such as nivolumab.
• Doses of G9.2-17 may range from about 3 mg/kg to 15 mg/kg once every two weeks. In an alternative embodiment, doses of G9.2-17 may range from about 0.2 mg/kg to 16 mg/kg or higher dose level once every two weeks.
• the antibody is administered by intravenous infusion. Treatment Duration and Treatment Periods
• Study drug administration continues until progression of disease, unacceptable toxicity, or withdrawal from the study. Patients who discontinue the study drug prior to disease progression and are not being treated with other systemic anti-cancer therapy(ies), are followed on the study until the time of disease progression.
• Treatment period 28-day treatment cycles as presented in the Schedule of Assessments (SoA; Tables 5-6 below)
• Post-treatment period 30 days after last treatment (End of Treatment Visit/Early Termination Visit)
• IMAR follow-up period 90-days after last treatment (G9.2-17 (IgG4) + an anti-PD-1 antibody arm)
• a dose-finding study is conducted using a continuous reassessment method (CRM) (O’Quigley et al., 1990) to establish DLTs and the RP2D.
• CCM continuous reassessment method
• Two to six patients per treatment cohort 1-6 are assigned to receive sequentially higher IV injections of G9.2-17 (IGG4) every two weeks (Q2W) on Day 1 and Day 15 of each 28-day cycle, starting at a dose of 0.2 mg/kg.
• Patients assigned to a specific dose escalation cohort receive the corresponding study dose for that cohort. They receive study drug at one of 8 dose levels until progression of disease, unacceptable toxicity, or withdrawal from the study for other reasons. Patients who withdraw for reasons other than toxicity or tolerability issues during the first treatment cycle only are replaced.
• cohorts 1-6 two patients at a time are dosed under the CRM design. Dose escalations are based on analysis of patient safety data focusing on occurrences of DLTs at previous dose levels and other relevant safety and dosing data from previous cohorts. Dose escalations may occur after a minimum of 28 days (1 cycle). No dose level skipping is allowed. Following the completion of cohort 6 under the CRM design, a once weekly (QW) G9.2- 17 (IGG4) dosing schema is evaluated, provided the RP2D has not been reached within the CRM design. Cohorts 7 and 8 are not evaluated with the CRM design. Patients are only allowed to enter Cohort 7 once no DLT has been identified.
• QW once weekly
• G9.2- 17 (IGG4) dosing schema is evaluated, provided the RP2D has not been reached within the CRM design.
• Cohorts 7 and 8 are not evaluated with the CRM design. Patients are only allowed to enter Cohort 7 once no DLT has been identified.
• cohorts 7 and 8 four patients at a time are dosed per cohort.
• Four patients per dose level in cohorts 7 and 8 are assigned to receive sequentially higher IV injections of G9.2-17 (IGG4) every week (QW) on Days 1, 8, 15, and 22 of each 28-day cycle.
• IGF4 G9.2-17
• Patients treated in early cohorts prior to identification of the RP2D are allowed to dose escalate up to the highest dose level cleared. After a complete cycle, dose escalations may occur after a minimum of 28 days (1 cycle). Dose escalations may not occur in the middle of a cycle. Patients can continue to dose escalate to the highest approved dose level until they are discontinued for toxicity or disease progression, or for other reasons (e.g., a patient elects to discontinue from the study).
• Dose escalations are based on the development of DLTs in patients treated at previous dose levels.
• prior DLT probabilities are specified from GLP-compliant toxicity studies as well as from preclinical models.
• the prior distribution on the parameter “a” has a mean zero normal distribution with the least informative prior variance.
• the trial is stopped for safety if the lower limit of an Agresti and Coull binomial confidence interval (CI) for the lowest study dose level exceeds the target DLT rate (Agresti and Coull, 1998).
• the RP2D is the MTD dose derived from Part 1.
• G9.2-17 IgG4
• the dose of G9.2-17 (IgG4) is initially reduced by 50%, and potentially by a further reduction of 50%, as defined by the dose modification guidance provided in Table 3. No further dose reductions are allowed.
• Part 1 is completed when up to six patients have received the dose that has been identified as RP2D.
• the RP2D is based, in part, on the continual reassessment method (CRM) study design, PK and PD data parameters, additional safety and efficacy data and any other factors to be considered.
• CRM continual reassessment method
• backfill cohorts The purpose of backfill cohorts is to assess the safety, tolerability, and the biological effect of G9.2-17 (IGG4) in patients whose tumors are gal-9 positive.
• the gal-9 status of the RP2D cohort is retrospectively determined. If fewer than 6 patients with gal-9 positive tumors are treated at the RP2D, patients designated for the backfill cohort require prospective assessment of gal-9 tumor status by IHC. Up to 6 additional patients, whose tumors are gal-9 positive, may be enrolled to backfill cohorts at the RP2D dose level.
• the second part of the protocol adopts a Simon’s two-stage optimal design and includes approximately 223 patients. It is planned to expand cohorts for PDAC, CRC and CCA and/or potentially other solid tumor types which are based on implementing tumor-specific consideration for expansion cohorts and clinical trial endpoints. The rationale behind this approach is to ensure recruitment feasibility, as well as to capture the clinical need for specific indications.
• CRC and CCA patients receive one of two treatments (4 treatment arms total):
• the anti-PD-1 antibody should be administered prior to G9.2-17 (IgG4). If for any reason same-day administration cannot be accomplished, an anti-PD-1 antibody should be administered on the first day, and G9.2-17 (IgG4) on the subsequent day.
• this study may investigate the use of the anti-Galectin-9 antibody G9.2- 17 (IgG4) alone (single agent arms of the study) or in conjunction with nivolumab (e.g., at a 240 mg flat dose administered once every two weeks).
• IgG4 anti-Galectin-9 antibody G9.2- 17
• nivolumab e.g., at a 240 mg flat dose administered once every two weeks.
• Treatment of single agent cohorts or combination agent cohorts for CRC and CCA patients may be executed in parallel.
• the starting dose of G9.2-17 (IgG4) in the single treatment is the RP2D identified in Part 1.
• the optimal two-stage design (Stages I and II) are used to test the null hypothesis that the ORR3 is ⁇ 5% versus the alternative hypothesis that the 0RR3 is ⁇ 15% within the single-agent arms.
• this trial arm is terminated if ⁇ 1 patient responds. If the trial goes on to the Stage II of Simon’s optimal design, approximately 33 patients are treated additionally in each of the single-agent arms. If the total number of responding patients is ⁇ 5, the investigational drug within that arm is rejected. If ⁇ 6 patients have a confirmed ORR 3, the Part 3 expansion cohort for that arm is activated and described in an amendment to the protocol.
• Dose reduction is allowed only if the clinical benefit is expected and may continue to be expected to derive with lower doses of G9.2-17 (IgG4).
• the dose of G9.2-17 (IgG4) is initially reduced by 50%, and potentially by a further reduction of 50%, as defined by the dose modification guidance provided in the protocol. No further dose reductions are allowed.
• the dose of G9.2-17 (IGG4) in the combination treatment with an anti-PD-1 antibody is the RP2D-1, which is the dose immediately preceding the RP2D dose identified in Part 1.
• the optimal two-stage design is also used to test the null hypothesis that the ORR 3 is ⁇ 10% versus the alternative hypothesis that the ORR 3 is ⁇ 25%.
• a safety run-in is performed in which the first 8 patients are dosed. This arm continues to enroll only if ⁇ 2 patients develop a DLT, which is below the target toxicity level (TTL) of 25%. If 3 or more patients develop a DLT this combination arm is terminated for the cancer type being treated. In this combination treatment run-in cohort, patients who withdraw for reasons other than toxicity or tolerability issues during the first treatment cycle only are replaced. If a DLT occurs, in any of the 8 safety run in patients, during the first 28 days of treatment, that patient is permanently discontinued from study drug administration.
• TTL target toxicity level
• G9.2-17 For patients who experience toxicities outside of the DLT window, dose reduction is allowed when clinical benefit is expected and may continue to be derived with lower doses of G9.2-17 (IgG4).
• the dose of G9.2-17 (IgG4) is initially reduced by 50%, and potentially by a further reduction of 50%, as defined by the dose modification guidance provided in the protocol. No further dose reductions are allowed. Dose modifications for an anti-PD-1 antibody are allowed. If an IMAR occurs/recurs that is not managed by dose reduction of either agent, both study medications must be discontinued.
• the respective trial arm is terminated if ⁇ 2 patients respond. If the trial goes on to Stage II of Simon’s optimal design, approximately 25 patients are treated additionally in each of the combination arms. If the total number of responding patients is ⁇ 7, the combination within that arm is rejected. If ⁇ 8 patients have a confirmed ORR 3, the expansion cohort for that arm is activated and described in an amendment to the protocol.
• the Part 2 cohort for patients with metastatic PDAC entails combination treatment of G9.2-17 (IgG4) in the first line metastatic setting.
• the dose of G9.2-17 is the RP2D-1 dose, which is the dose level in the cohort immediately preceding the RP2D dose identified in Part 1.
• a safety run- in is performed in which the first 8 patients are dosed and that arm is continued only if ⁇ 2 patients develop a DLT, which is below the target toxicity level (TTL) of 25%. If 3 or more patients develop a DLT, this combination treatment arm is terminated.
• TTL target toxicity level
• G9.2-17 IgG4
• the dose of G9.2-17 (IgG4) is initially reduced by 50%, and potentially by a further 50%. No further dose reductions are allowed.
• the primary efficacy endpoint is patient PFS6.
• Part 2 Completion of Part 2 is dependent upon patient ORR 3 for CRC and CCA patients, and PFS 6 for PDAC.
• Part 3 Expansion
• an expansion cohort is launched to confirm the finding as described above.
• the sample size for each of the expansion arms is determined based on the point estimates determined in Part 2, in combination with a predetermined level of precision for the 95% CI around the ORR/OS and PFS.
• a protocol amendment is submitted with details around the expansion population, treatment regimen, and statistical analysis plan prior to initiating Part 3.
• Dose-limiting toxicities assessed in this trial are defined as a clinically significant hematologic and/or non-hematologic AE or abnormal laboratory value assessed as unrelated to metastatic tumor disease progression, intercurrent illness, or concomitant medications and is possibly related or related to the study drug and occurring during the first cycle (28 days) on study. Any patient that experiences a DLT in Part 1 or Part 2 during the first 28 days of treatment is permanently discontinued from study drug administration.
• a DLT is a toxicity that meets any of the following criteria:
• End of study for Part 1 of the study is defined at the point when the RP2D has been identified and all patients have been treated with G9.2-17 (IGG4) until confirmed disease progression.
• the end of the study is defined as the date of the last patient’s last visit.
• the trial is stopped for safety if the lower limit of an Agresti and Coull binomial CI for the lowest study dose level exceeds the target DLT rate (Agresti and Coull, 1998).
• the respective trial arm is stopped if ⁇ 1 patient responds. If the trial goes on to the Stage II of Simon’s optimal design, a trial arm is stopped if the total number of responding patients is ⁇ 5 within that arm.
• a safety run-in is performed in which the first 8 patients are dosed.
• enrollment continues only if ⁇ 2 patients develop a DLT, which is below the target toxicity level (TTL) of 25%. If 3 or more patients with a given cancer type develop a DLT in a combination treatment arm, enrollment for that cancer type in that arm is terminated.
• TTL target toxicity level
• alanine aminotransferase ⁇ 3 x ULN ( ⁇ 5 x ULN when HCC or hepatic metastases present)
• g. bilirubin ⁇ 1.5 x ULN patients with known Gilbert’s disease may have a bilirubin ⁇ 3.0 x ULN) h. albumin ⁇ 3.0 g/dL i. international normalized ratio (INR) and partial thromboplastin time (PTT) ⁇ 1.5 x ULN j. amylase and lipase ⁇ 1.5 x ULN
• Women of child-bearing potential must have a negative pregnancy test within 72 h prior to start of treatment.
• a woman is of childbearing potential if she is post-menarche, has not reached a postmenopausal state ( ⁇ 12 continuous months of amenorrhea with no identified cause other than menopause), and has not undergone surgical sterilization (removal of ovaries and/or uterus).
• contraceptive methods with a failure rate of ⁇ 1% per year include bilateral tubal ligation, male sterilization, hormonal contraceptives that inhibit ovulation, hormone-releasing intrauterine devices and copper intrauterine devices.
• the reliability of sexual abstinence should be evaluated in relation to the duration of the clinical trial and the preferred and usual lifestyle of the patient.
• Periodic abstinence e.g., calendar, ovulation, symptom-thermal, or post ovulation methods
• withdrawal are not acceptable methods of contraception.
• Fertile men must practice effective contraceptive methods during the study, unless documentation of infertility exists.
• Biliary or gastric outlet obstruction allowed, provided it is effectively drained by endoscopic, operative, or interventional means
• pancreatic, biliary, or enteric fistulae allowed, provided they are controlled with an appropriate non-infected and patent drain (if any drains or stents are in situ, patency needs to be confirmed before study start) Additionally, for Part 1 only:
• Patients a. who has already received at least one prior line of systemic therapy for metastatic disease, or b. who has a tumor type for which there are no available standard of care options.
• PDAC expansion cohort 1st line metastatic patients who are either gemcitabine- containing regimen naive or at least 3 months out of having been treated using a gemcitabine- containing regimen previously in a neoadjuvant or adjuvant/locally advanced setting
• Grade 4 immune-mediated toxicides with a prior checkpoint inhibitor Grade 2 or Grade 3 pneumonitis or any other Grade 3 checkpoint inhibitor-related toxicity that led to immunotherapy treatment discontinuation.
• Low-grade ( ⁇ Grade 3) toxicides, such as neuropathy from prior treatments, manageable electrolyte abnormalities and lymphopenia, alopecia and vitiligo are allowed.
• recurrent is defined as ⁇ 3 drains in the previous 30 days.
• Active auto-immune disorder except type VII diabetes, hypothyroidism requiring only hormone replacement, vitiligo, psoriasis, or alopecia areata
• Requires systemic immunosuppressive treatment including, but not limited to cyclophosphamide, azathioprine, methotrexate, thalidomide, and anti-TNF agents.
• Patients who have received or are receiving acute, low dose systemic immunosuppressant medications e.g., ⁇ 10 mg/day of prednisone or equivalent
• Replacement therapy e.g., thyroxine, insulin, physiologic corticosteroid replacement therapy [eg, ⁇ 10 mg/day of prednisone equivalent] for adrenal or pituitary insufficiency
• inhaled corticosteroids and mineralocorticoids eg, fludrocortisone
• topical steroids eg, intranasal steroids, intra-articular, and ophthalmic steroids is allowed.
• Study intervention(s) is/are defined as any investigational agent(s), marketed product(s), placebo, or medical device(s) intended to be administered/used to/in a study participant according to the study protocol.
• Nivolumab (OPDIVO®) is a programmed death receptor- 1 (PD-1) blocking antibody indicated for the treatment of multiple tumor types. Nivolumab can be used as an exemplary anti-PD-1 antibody in combination with the anti-Galectin-9 antibody disclosed herein such as
• Nivolumab can be administered as an intravenous infusion over 30 minutes (unless guided otherwise) at 240 mg every 2 weeks, in a 28-day cycle. As per the FDA label, there are no contraindications for administrations of nivolumab.
• Nivolumab AEs are presented in the Tables below according to their frequency of occurrence.
• G9.2-17 (IgG4) is administered via IV infusion, weekly, or every 2 weeks, until progression of disease, unacceptable toxicity, or withdrawal of consent.
• patients receive the RP2D of G9.2-17 (IgG4) (as determined in Part 1) as a single agent or the G9.2-17 (IgG4) RP2D-1 in combination with an anti-PD-1 antibody as follows:
• Patients who experience a DLT in Part 1 do not resume treatment. Patients who experience a DLT in Part 2 have their treatment interrupted. Their treatment may resume at the same or reduced dose of G9.2-17 (IgG4) if they are experiencing a clinical benefit.
• IgG4 G9.2-17
• RP2D-1 dose level in cohort immediately preceding the RP2D
• Manufacture and packaging of the investigational medicinal product (IMP) G9.2-17 (IgG4) is in accordance with applicable current Good Manufacturing Practice (cGMP) and the product meets applicable criteria for use in humans.
• cGMP Current Good Manufacturing Practice
• G9.2-17 (IgG4) drug product is diluted to the target dose prior to administration. All dilutions should be performed in a controlled and sterile environment (patient dose is prepared for and delivered via an approximately 60 minutes IV infusion).
• G9.2-17 (IgG4) is a sterile liquid and is stored at 2°C to 8°C and protected from light.
• G9.2-17 may be continued if:
• Nab-paclitaxel is not recommended in patients who have total bilirubin >5 x ULN or AST >10 x ULN.
• Nab-paclitaxel is not recommended in patients with metastatic adenocarcinoma of the pancreas who have moderate to severe hepatic impairment (total bilirubin >1.5 x ULN and AST ⁇ 10 x ULN). The starting dose should be reduced for patients with moderate or severe hepatic impairment.
• nivolumab modifications based on specific AEs are provided below. There are no recommended dose modifications of nivolumab for hypothyroidism or hyperthyroidism.
• study intervention is permanently discontinued due to reasons other than disease progression, and the patient is not being treated with other anti-cancer therapy(ies), the patient continues to be evaluated for disease progression for up to 2 years. See the SoA for data to be collected at the time of discontinuation of study intervention and follow-up and for any further evaluations that need to be completed.
• a patient may be discontinued prior to disease progression for any of the following reasons:
• Any medication or vaccine (including over-the-counter or prescription medicines, recreational drugs, vitamins, and/or herbal supplements) that the participant is receiving at the time of enrollment or receives during the study must be recorded along with:
• inhaled corticosteroids and mineralocorticoids e.g., fludrocortisone
• topical steroids e.g., topical steroids
• intranasal steroids e.g., intranasal steroids
• intra-articular e.g., intra-articular
• ophthalmic steroids e.g., corticosteroids, mineralocorticoids, topical steroids, intranasal steroids, intra-articular, and ophthalmic steroids
• Vaccination for CO VID- 19 common flu and/or other common clinically required indications (e.g., tetanus, pneumococcus, HBV, etc.) is allowed before or during the study period. The timing and type of vaccine must be recorded.
• Systemic immunosuppressive treatment including, but not limited to cyclophosphamide, azathioprine, methotrexate, thalidomide, and anti-TNF agents.
• systemic immunosuppressant medications e.g., ⁇ 10 mg/day of prednisone or equivalent.
• Replacement therapy e.g., thyroxine, insulin, physiologic corticosteroid replacement therapy [eg, ⁇ 10 mg/day of prednisone equivalent] for adrenal or pituitary insufficiency
• thyroxine e.g., insulin, physiologic corticosteroid replacement therapy [eg, ⁇ 10 mg/day of prednisone equivalent] for adrenal or pituitary insufficiency
• physiologic corticosteroid replacement therapy eg, ⁇ 10 mg/day of prednisone equivalent] for adrenal or pituitary insufficiency
• Patients should receive full supportive care during the study, including transfusions of blood and blood products, and treatment with antibiotics, antiemetics, antidiarrheals, and analgesics, and other care as deemed appropriate, and in accordance with institutional guidelines
• ADA anti-drug antibodies
• AE adverse event
• ALT alanine aminotransferase
• APTT activated partial thromboplastin time
• AST aspartate aminotransferase
• C cycle
• CPK creatine phosphokinase
• COVID19 Coronavirus SARS-CoV-2
• CRP C -reactive protein
• CT computed tomography
• D or d day(s)
• ECG electrocardiogram
• ECOG Eastern Cooperative Oncology Group
• ECHO echocardiography/cardiac ultrasound
• FSH follicle-stimulating hormone
• IMAR immune-mediated adverse reaction
• INR international normalized ratio
• LDH lactate dehydrogenase
• LH luteinizing hormone
• PD pharmacodynamics
• PK pharmacokinetics
• PTH parathyroid hormone
• PTT partial thromboplastin time
• G9.2-17 (IGG4) treatment is administered, on CXD1, CXD8, CXD15, and CXD22 on every weekly cycle (Cohorts 7-8).
• an anti-PDl antibody i administered on Day 1 of every cycle on the G9.2-17 (IGG4) combination regimen.
• Study drug may be administered on Days 1, 8 and 15 +/- 3 days from C2 onwards.
• Demographics Data include age, gender, race, and ethnicity.
• Medical history In addition to general medical history, data collection also includes oncology history, surgical/transplant and radiation therapy history and COVID-19 history testing.
• Previous and concomitant medications including vaccines and complementary treatments/supplements: Data to include name, indication, dose, route, start and end dates for each. Allergies and intolerances, dose modifications while on study, schedule of dosing changes and reasons for them should also be obtained.
• Adverse events Any AEs starting or worsening after study drug administration is recorded. AEs should be followed until resolved to one of the following: baseline, stabilized deemed irreversible. All SAEs are to be collected until 30 days after last dose of study medication. All study -procedure-related SAEs must be collected from the date of patient’s written consent.
• ECHOZMUGA This assessment of heart function is conducted at Screening and repeated on Day 1 of Cycle 4; the assessment window is +/- 5 days. It should be conducted m frequently when clinically indicated and once every 3 months.
• G Physical exam: Include height at screening for determination of body surface area. Include weight at all scheduled exam times. A Neurological exam is conducted only on pal who have stable and/or pre-treated brain metastases.
• Hematology Analysis includes complete blood count, differential, platelets, hemoglobin. Collect blood samples pre-dose.
• Serum chemistry Analysis includes albumin, alkaline phosphatase, bilirubin (total, direct), blood urea nitrogen, calcium, CPK, creatinine, electrolytes (sodium, potassium, 3 o chloride, magnesium, phosphorus), gamma glutamyl transferase (gamma GT), glucose, hemoglobin Ale (HgbAlc) (only if history of Type 1 or Type 2 diabetes mellitus), LDH, O SGPT (ALT) or SCOT (AST), total protein. Fasting glucose to be assessed only if clinically indicated. Collect blood samples pre-dose. b5
• Blood Coagulation Collect blood samples pre-dose. Analysis includes APTT, PT, PIT, and INR (if on allowable anti-coagulants), CRP, and troponin.
• Urinalysis Analysis includes color, appearance, dipstick for specific gravity, protein, white blood cell-esterase, glucose, ketones, urobilinogen, nitrite, WBC, RBC, pH. (Urir culture and sensitivity to be run only if patient is clinically symptomatic.)
• Tumor biopsies If patient MMR/MSI status is unknown at screening, the test should be run at the local laboratory. In Part 2, TMB tissue analysis is performed. The on-study biopsy is scheduled for C3D15 ⁇ 7 days and should occur only after the tumor imaging scan in Cycle 3. It is recognized that a variety of clinical factors may make it difficult to obtain adequate specimens. Decisions not to perform biopsy on-treatment should be discussed with the Medical Monitor.
• Tumor type-relevant biomarkers Blood samples are to be collected at screening and every cycle pre-dose administration as appropriate for the tumor type. Blood sampling m; decreased to every 3rd cycle after 6 months of treatment.
• PK blood sampling Cycle 1 and Cycle 3 Day 1: blood samples are collected pre-dose, end of study drug infusion (EOI) and 1 h ( ⁇ 15 min) post-study drug administration. O 1 and Cycle 3 Day 3, blood samples are collected at one time point, any time. Cycle 1 and Cycle 3 Day 8, Day 15, and Day 22, blood samples are collected pre-dose and at EOI ⁇ Cycle 2 and every even cycle there after: blood samples are collected Day 1 only and should occur pre-dose and at EOI. Blood samples for PK will not be collected on every odd cycle after Cycle 3.
• EOI end of study drug infusion
• ADA blood sampling Blood samples are collected Day 1 and Day 15 of Cycles 1 and 2, pre-dose. Thereafter, it is collected every cycle, Day 1, pre-dose (ie, C3D1, C4D1, eti hd
• Tumor imaging should continue, where possible, for patients discontinuing treatment due to reasons other than progression of disease and not receivir O additional systemic anticancer treatments. Survival data is collected at a minimum every 3 months. It can be collected more frequently to support data cleaning or regulatory 5
• Procedures conducted as part of the participant’s routine clinical management (e.g., blood count) and obtained before signing of the ICE may be utilized for screening or baseline purposes provided the procedures met the protocol-specified criteria and were performed within the time frame defined in the SoA.
• the SoA (Tables 5-6) provides a list of assessments to be performed during the screening period (up to 28 days), the treatment period (presented as 28-day cycles), the End of Treatment/Early Termination period, IMAR follow-up and the long-term follow-up period. Optional visits are allowed during each treatment cycle if medically indicated, during which any of the study assessments may be performed.
• Tumor imaging assessment computed tomography [CT] or magnetic resonance imaging (MRI), with or without contrast; or positron emission tomography (PET)-CT; CT with contrast is preferred
• Thyroid stimulating hormone Thyroid stimulating hormone
• fT4 free T4 or thyroxine
• serum lipase amylase
• PTH parathyroid hormone
• FSH follicle-stimulating hormone
• LH luteinizing hormone
• Tumor biopsy o Biopsy can be omitted if it is deemed that the procedure is a risk to the patient. o If a biopsy is unavailable, site will make every effort to obtain an archival tumor tissue sample available as a formalin-fixed paraffin-embedded (PEPE) block. Acceptable archival samples include those obtained via a core needle biopsy or excisional surgery within the last five years.
• PEPE formalin-fixed paraffin-embedded
• Each treatment cycle has a duration of 28 days. Refer to Table 5 for Cohorts 1-6 of Part
• CT Tumor imaging assessment
• Cohorts 1-6 Treatment Procedures for Day 15 of each Cycle (CXD15 ⁇ 7 day for
• Cohorts 7 and 8 Treatment Procedures for Dav 3 of Cycle 1 and Cycle 3 (C1D3 ⁇ 7 day and C3D3 ⁇ 7 day)
• Cohorts 7 and 8 Treatment Procedures for Day 8 of each Cycle ( CXD8 ⁇ 7 day)
• Tumor type-relevant biomarkers • Tumor biopsy on C3D15 ⁇ 7 days (Cycle 3 ONLY ; can be eliminated if it is deemed too risky for the patient)
• Treatment cycles beyond Cycle 4 can be repeated as indicated in the SoA (Tables 5-6). If the patient is experiencing clinical benefit, even in the event of radiological progression, the patient can continue on treatment.
• the following procedures are done 30 days ( ⁇ 3 days) after the last dose, including patients who have discontinued treatment early.
• Tumor imaging assessment Confirmatory scan if end of study is > 8 weeks after previous scan.
• OS is assessed every 3 months for up to 2 years after the patient has the End of Treatment/Early Termination. Tumor imaging assessment continues, where possible, for patients discontinuing treatment due to reasons other than progression of disease and not receiving additional systemic anticancer treatments.
• tumor lesions/lymph nodes are categorized as measurable or non-measurable with measurable tumor lesions recorded according to the longest diameter in the plane of measurement (except for pathological lymph nodes, which are measured in the shortest axis).
• measurable lesion When more than one measurable lesion is present at screening all lesions up to a maximum of five lesions total (and a maximum of two lesions per organ) representative of all involved organs should be identified as target lesions.
• Target lesions should be selected on the basis of their size (lesions with the longest diameter). A sum of the diameters for all target lesions is calculated and reported as the baseline sum diameters.
• All other lesions (or sites of disease) including pathological lymph nodes should be identified as non-target lesions and should also be recorded at screening. Measurements are not required, and these lesions should be followed as ‘present’, ‘absent’, or ‘unequivocal progression’.
• Tumor target lesions are assessed according to the RECIST vl.l Guidelines (Eisenhauer et al., 2009) using the following disease response measures:
• CR Complete Response
• Partial Response At least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum diameters.
• Stable Disease Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on study.
• CR Disappearance of all non-target lesions and normalization of tumor marker level. All lymph nodes must be non-pathological in size ( ⁇ 10 mm short axis).
• Non-CR/Non-progressive disease Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limits.
• Non-evaluable Complete Response
• Non-PD Non-progressive Disease
• PR Partial Response
• SD Stable Disease
• NE Non-evaluable
• the disease response measures at different timepoints allow for the calculation of the following:
• DCR Disease control rate
• ORR Objective response rate
• PFS Progression-free survival
• DoR Duration of response
• OS Overall survival
• Vital signs are measured in a post-supine position after 5 minutes rest and include temperature, blood pressure (systolic and diastolic), heart rate, and respiratory rate.
• ECG 12-lead ECG is obtained as outlined in the SoA (see Tables 5-6) using an ECG machine that automatically calculates the heart rate and measures heart rate, PR interval, QRS duration, distance in time on the ECG tracing from the start of the QRS complex to the end of T-wave (QT) interval, and QTcF intervals.
• the clinical laboratory parameters are analyzed at the site’s local laboratory. Laboratory assessments completed include hematology and serum chemistry and is defined as following:
• Serum Chemistry Includes glucose, total protein, albumin, electrolytes [sodium, potassium, chloride, magnesium, phosphorus], calcium, bilirubin (total, direct), SGPT (ALT) or SGOT (AST), alkaline phosphatase, gamma glutamyl transferase (gamma GT), lactate dehydrogenase (LDH), creatinine, hemoglobin Ale (HgbAlc) (only if history of Type 1 or Type 2 diabetes mellitus), blood urea nitrogen, creatine phosphokinase (CPK) o TSH, fT4, lipase, amylase, PTH, ESH, LH, free cortisol additionally at specified visits o Fasting glucose is assessed only if clinically indicated
• Hematology Includes complete blood count, differential, platelets, hemoglobin
• Coagulation Includes prothrombin time (PT) and PIT, activated partial thromboplastin time (APTT) and INR (if on allowable anti-coagulants) C-reactive protein (CRP), and troponin
• Urinalysis Patients have urine samples collected for routine urinalysis.
• the urinalysis includes color, appearance, and dipstick for specific gravity, protein, white blood cell-esterase, glucose, ketones, urobilinogen, nitrite, white blood cell count (WBC), red blood cell count (RBC), and pH, and urine culture (if patient is clinically symptomatic).
• WOCBP should only be included after a confirmed menstrual period and a negative highly sensitive urine or serum pregnancy test.
• Pregnancy testing is performed whenever a menstrual cycle is missed or when pregnancy is otherwise suspected.
• Cycle Day 1 i.e., C2D1, C4D1, etc.
• additional PK and safety assessments are collected upon resumption of dosing; additional PK assessments may be performed during the interruption. If the dose of study drug is reduced, additional PK assessments are collected pre-administration of the reduced dose (within 2 h pre-dosing), and 2 to 4 h after starting the reduced study drug dose. Additional PK, and other blood assessments may be taken if clinically indicated. Centers that are not able to hold patients more than 2 h post-dose due to CO VID- 19 restrictions, contribute samples at EOI and 2 h post-dose only.
• Samples are tested for PD biomarkers (by flow cytometry, ELISA, IHC, or multiplex phenotyping) to evaluate their association with the observed clinical responses to G9.2-17 (IGG4) using validated assays.
• Tumor markers blood: CAI 5 -3, CA-125, carcinoembryonic antigen (CEA), CA19-9, alpha fetoprotein, neuron-specific enolase (NSE), cytokeratin-fragment-21 (CYFRA-21) to be assessed every cycle pre-dose administration as needed per tumor type. This may be decreased to every 3 cycles after 6 months of treatment, following the same schedule as tumor imaging assessments, as appropriate.
• CEA carcinoembryonic antigen
• NSE neuron-specific enolase
• CYFRA-21 cytokeratin-fragment-21
• PBMC phenotype e.g., CD3, CD4, CDS, CD45RO, forkhead-box-protein P3 (FOXP3), CD11B, CD14, CD15, CD16, CD33, CD68, human leukocyte antigen (HLA) DR, CD163, arginase 1, granzyme B, KI67, PD-1, PD LI, pan cytokeratin (PAN CK)
• HLA human leukocyte antigen
• PAN CK pan cytokeratin
• Cytokines (blood): eg, interferon gamma (IFN y), IL 10, IL 12p70, IL 13, IL ip, IL 2, IL 4, IL 6, IL 8, TNF a, MIP-lb, monocyte chemoattractant protein 1 (MCP-1), MIP-la, IL 17a, IL 5, TGF ⁇
• TLB Tumor Mutational Burden
• Exploratory biomarker changes, if any, are correlated with safety and response outcomes.
• Samples may be stored for a maximum of 2 years (or according to local regulations) following the last patient’s last visit for the study at a facility selected to enable further analysis of the effect of G9.2-17 (IGG4) on pharmacodynamic biomarkers.
• G9.2-17 G9.2-17
• Blood samples (approximately 3 mL) are collected from all participants according to the SoA (Tables 5-6) and processed to serum. Additionally, serum samples should also be collected at the end of treatment/early termination visit from patients who discontinued study intervention or were withdrawn from the study.
• Cohorts 1-6 Every 2 cycles beyond Cycle 4 on Day 1 (i.e., C6D1, C8D1 etc.):
• Serum samples are screened for antibodies binding to G9.2-17 (IgG4) (ADA) and the titer of confirmed positive samples is reported. Other analyses may be performed to verify the stability of antibodies to G9.2-17 (IgG4) and/or further characterize the immunogenicity of G9.2-17 (IgG4).
• the detection and characterization of antibodies to G9.2-17 (IgG4) is performed using a validated assay method. All samples collected for detection of antibodies to study intervention are evaluated for G9.2-17 (IgG4) serum concentration to enable interpretation of the antibody data. Antibodies may be further characterized and/or evaluated for their ability to neutralize the activity of the study intervention. Samples may be stored for a maximum of 2 years (or according to local regulations) following the last patient’s last visit for the study at a suitable facility to enable further analysis of immune responses to G9.2-17 (IgG4).
• patient demographic data is collected. These include age, gender, race, and ethnicity.
• the medical history includes oncology history, surgical/transplant history radiation therapy history, and COVID 19 history and testing.
• pancreatic adenocarcinoma For patients with previously resected pancreatic adenocarcinoma, record whether the primary tumor was localized to the head of pancreas, pancreatic body or the pancreatic tail.
• Prior and concomitant Medications Prior and concomitant medications, including vaccines and complementary treatments/supplements, are documented for each patient at each scheduled visit (Tables 5-6).
• Tumor assessments are performed using CT or MRI with or without contrast; a PET-CT is performed.
• CT with contrast is the preferred modality (MRI, PET-CT, or other imaging modalities instead of, or in addition to, the CT scan, if CT is not feasible or appropriate, given location of the disease).
• Assessment should include the chest/abdomen/pelvis at a minimum and should include other anatomic regions as indicated, based on the patient’s tumor type and/or disease history. Imaging scans must be de-identified and archived in their native format as part of the patient study file. While the type of scan obtained, as appropriate for the disease, the same method should be used for the duration of the study.
• assessments are done every 8 weeks ⁇ 7 days according to the SoA (i.e., C3D1, C5D1, C7D1, C9D1, etc.) and at the End of Treatment if not assessed within the last 4-6 weeks. Assessments may be performed more frequently if clinically indicated. For Part 2 only, if an objective response is seen on a scan, a confirmation scan is done 4 weeks (+7 days) later. After a confirmatory scan, the scheduled scans are to be resumed at a frequency of every 8 weeks ( ⁇ 7 days) from the date of the confirmatory scan.
• SoA i.e., C3D1, C5D1, C7D1, C9D1, etc.

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