Combination cancer therapy with anti-galactogenin-9 antibodies and chemotherapy agents

Abstract

Combination therapy for solid tumors includes antibodies that bind to human galactagogue-9 (anti-Gal9 antibodies, such as G9.2-17) and one or more chemotherapeutic agents (such as gemcitabine, paclitaxel, or combinations thereof).

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 881,894, filed August 1, 2019, and PCT Application No. PCT / US2020 / 031181, filed May 1, 2020, the entire contents of which are incorporated herein by reference. Background of the Invention

[0004] Galactochonin-9 is a tandem repeat lectin composed of two carbohydrate recognition domains (CRDs), first discovered and described in patients with Hodgkin's lymphoma (HL) in 1997 (Tureci et al., J. Biol. Chem. 1997, 272, 6416-6422). Three isoforms exist, which can be located intracellularly or extracellularly. Elevated galactochonin-9 levels have been observed in many different cancers, including melanoma, Hodgkin's lymphoma, hepatocellular carcinoma, pancreatic cancer, gastric cancer, colon cancer, and clear cell renal cell carcinoma (Wdowiak et al., Int. J. Mol. Sci. 2018, 19, 210). In renal cell carcinoma, patients with high galactochonin-9 expression exhibit more advanced disease progression and larger tumor size (Kawashima et al.; BJUInt. 2014;113:320-332). In melanoma, galactagogue-9 is expressed in 57% of tumors and is 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). Numerous studies have demonstrated the utility of galactagogue-9 as a prognostic biomarker, and more recently, its potential as a novel 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).

[0005] Galactochonin-9 has been described as playing important roles in many cellular processes, such as adhesion, cancer cell aggregation, apoptosis, and chemotaxis. Recent studies have shown that galactochonin-9 plays a role in supporting tumor-mediated immune regulation, for example, by negatively regulating Th1 responses, Th2 polarization, and macrophage polarization towards the M2 phenotype. This work also includes studies demonstrating that galactochonin-9 participates in the direct inactivation of T cells through interactions with T cell immunoglobulin and mucin 3 (TIM-3) receptors (Dardalhon et al., J Immunol.,2010, 185, 1383-1392; Sanchez-Fueyo et al., Nat Immunol., 2003, 4, 1093-1101).

[0006] It was also found that galactagogue-9 plays a role in differentiating polarized T cells into tumor suppressor phenotypes, as well as promoting tolerance-inducing macrophage programming and adaptive immunosuppression (Daley et al.). Nat Med., 2017, 23, 556-567. In a mouse model of pancreatic ductal adenocarcinoma (PDA), it has been shown that blocking the checkpoint interaction between galactagogue-9 and its receptor Dectin-1 on innate immune cells in the tumor microenvironment (TME) increases the antitumor immune response in the TME and slows tumor progression (Daley et al., 2017, 23, 556-567). Nat Med., 2017, 23, 556-567 It was also found that galactagogue-9 binds to CD206, a surface marker of M2 macrophages, leading to a decrease in the secretion of macrophage-derived chemokine CVL22 (MDC), which is associated with longer survival and lower risk of recurrence in lung cancer (Enninga et al., J Pathol. 2018 Aug;245(4): 468-477). Invention Overview

[0008] This disclosure is based on the unexpected discovery of a synergistic effect observed in animal models of combination therapy involving exemplary anti-galactotropic 9 antibodies (e.g., G9.2-17(IgG4)) and chemotherapeutic agents (e.g., gemcitabine and paclitaxel, such as nanoparticle albumin-bound paclitaxel or nab-paclitaxel).

[0009] Therefore, this article provides a method for treating solid tumors involving the co-use of an anti-galactotropic 9 antibody (e.g., G9.2-17 or a functional variant thereof) and one or more chemotherapeutic agents (e.g., gemcitabine, paclitaxel, or protein-bound paclitaxel such as nab-paclitaxel or abraxane). ® (or a combination thereof).

[0010] In some embodiments, the methods disclosed herein for treating solid tumors may include administering an effective amount of an antibody binding to human galactagogue-9 (anti-Gal9 antibody) to a subject in need. The anti-galactagogue-9 antibody may have the same heavy chain complementarity-determining region (CDR) and the same light chain CDR as antibody G9.2-17. The subject may be receiving anticancer therapy comprising one or more chemotherapeutic agents.

[0011] In some embodiments, the methods of treating solid tumors disclosed herein may include administering to a subject in need an effective amount of an antibody binding to human galactagogue-9 (anti-Gal9 antibody) and an effective amount of one or more chemotherapeutic agents. The anti-Gal9 antibody may have the same heavy chain complementarity-determining region (CDR) and the same light chain CDR as antibody G9.2-17.

[0012] In some implementations, the methods of treating solid tumors disclosed herein may include administering an effective amount of one or more chemotherapeutic agents to a subject in need. The subject may be receiving therapy comprising an antibody that binds to human galactagogue-9 (anti-Gal9 antibody) having the same heavy chain complementarity-determining region (CDR) and the same light chain CDR as antibody G9.2-17.

[0013] Any of the methods disclosed in this article can be used to treat metastatic solid tumors. In some examples, the solid tumor is pancreatic ductal adenocarcinoma (PDAC), such as metastatic PDAC.

[0014] In some embodiments, one or more chemotherapeutic agents involved in any of the methods disclosed herein may include antimetabolites (e.g., nucleoside analogs), microtubule inhibitors, or combinations thereof. In some examples, the nucleoside analog is gemcitabine and / or the microtubule inhibitor is paclitaxel, such as albumin-bound nanoparticle paclitaxel (e.g., Abraxane). ® ).

[0015] In some embodiments, the anti-galactoglobulin-9 antibody is administered to the subject at a dose of about 0.5 mg / kg to about 32 mg / kg (e.g., about 0.5 mg / kg to about 16 mg / kg, about 2 mg / kg to about 32 mg / kg, or about 2 mg / kg to about 16 mg / kg). In some embodiments, the anti-galactoglobulin-9 antibody is administered to the subject once weekly. In some embodiments, the anti-galactoglobulin-9 antibody is administered to the subject every 2 or 3 weeks. In some embodiments, the anti-galactoglobulin-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. In some embodiments, the antibody is administered every 2 weeks. In some embodiments, the anti-galactoglobulin-9 antibody is administered to the subject every 2 weeks at a dose selected from 2 mg / kg, 4 mg / kg, 8 mg / kg, 12 mg / kg, or 16 mg / kg. In some embodiments, the anti-galactoglobulin-9 antibody is administered for one cycle every two weeks, two cycles every two weeks, three cycles every two weeks, four cycles every two weeks, or more than four cycles every two weeks. In some embodiments, the duration of treatment is 0 to 3 months, 0 to 6 months, 3 to 6 months, 6 to 12 months, 12 to 24 months, or longer. In some embodiments, the duration of treatment is 12 to 24 months or longer. In some embodiments, the cycle is extended to a duration of 3 to 6 months, or 6 to 12 months, or 12 to 24 months or longer. In some embodiments, the cycle length is modified, for example, temporarily or permanently, to a longer duration, such as 3 weeks or 4 weeks. In any of these embodiments, the anti-galactoglobulin-9 antibody is administered to the subject once a week, once every two weeks, once every three weeks, or once every four weeks. In some embodiments, the anti-galactoglobulin-9 antibody is administered to the subject via intravenous infusion. In some embodiments, the cancer is a PDA. In some implementations, the cancer is metastatic cancer.

[0016] In some embodiments, anti-Gal9 antibody may be administered to the subject intravenously at a dose of about 0.5 mg / kg to about 32 mg / kg every 2 weeks. In some examples, anti-Gal9 antibody may be administered to the subject intravenously at a dose of about 0.5 mg / kg every 2 weeks. In some embodiments, anti-Gal9 antibody may be administered to the subject intravenously at a dose of about 2 mg / kg to about 16 mg / kg every 2 weeks. In some examples, anti-Gal9 antibody may be administered to the subject intravenously at a dose of about 2 mg / kg every 2 weeks. In some examples, anti-Gal9 antibody may be administered to the subject intravenously at a dose of about 4 mg / kg every 2 weeks. In some examples, anti-Gal9 antibody may be administered to the subject intravenously at a dose of about 8 mg / kg every 2 weeks. In some examples, anti-Gal9 antibody may be administered to the subject intravenously at a dose of about 12 mg / kg every 2 weeks. In some cases, the anti-Gal9 antibody was administered to subjects intravenously at a dose of approximately 16 mg / kg every 2 weeks. In some cases, the anti-Gal9 antibody was administered to subjects intravenously at a dose of approximately 32 mg / kg every 2 weeks.

[0017] In some implementations, the method includes a 28-day cycle, wherein the anti-Gal9 antibody is administered to the subject on days 1 and 15, and gemcitabine and paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) are administered to the subject on days 1, 8, and 15. In some examples, paclitaxel is administered at 125 mg / m². 2 Gemcitabine was administered intravenously to the subjects. In some examples, gemcitabine was administered at a dose of 1000 mg / m². 2 Administered to subjects.

[0018] In some embodiments, the anti-galactoglobulin-9 antibody comprises light chain complementarity-determining region 1 (CDR1) as shown in SEQ ID NO: 1, light chain complementarity-determining region 2 (CDR2) as shown in SEQ ID NO: 2, and light chain complementarity-determining region 3 (CDR3) as shown in SEQ ID NO: 3, and / or comprises heavy chain complementarity-determining region 1 (CDR1) as shown in SEQ ID NO: 4, heavy chain complementarity-determining region 2 (CDR2) as shown in SEQ ID NO: 5, and heavy chain complementarity-determining region 3 (CDR3) as shown in SEQ ID NO: 6.

[0019] In some embodiments, the anti-Gal9 antibody may comprise: a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO:7. H), and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 8. L In some examples, the anti-Gal9 antibody may be an IgG molecule, such as an IgG4 molecule. In a specific example, the anti-Gal9 antibody may comprise: a heavy chain containing the amino acid sequence of SEQ ID NO: 19 and a light chain containing the amino acid sequence of SEQ ID NO: 15.

[0020] In some implementations, the subject to be treated by any of the methods disclosed herein may be a human patient. In some examples, the subject has galactoglobulin-9 positive cancer cells or immune cells. Such galactoglobulin-9 positive cancer cells or immune cells can be detected in tumor organoids derived from the subject. In some examples, the subject may have elevated galactoglobulin-9 levels relative to control values. For example, the subject may have elevated serum or plasma galactoglobulin-9 levels relative to control values.

[0021] In some implementations, the subject may have already received at least one line of systemic anticancer therapy. Alternatively or additionally, the subject may not have prior therapy involving gemcitabine and / or paclitaxel. In some examples, the subject may have received prior therapy involving gemcitabine and / or paclitaxel for at least 6 months prior to administration of the anti-Gal9 antibody.

[0022] In any of the methods disclosed herein, one or more of the following characteristics are detected in a subject before, during, and / or after treatment: (a) one or more tumor markers in a blood sample from the subject, optionally including CA15-3, CA-125, CEA, CA19-9, and / or alpha-fetoprotein, and any other tumor type-specific tumor markers; (b) cytokine profile; and (c) galactagogue-9 serum / plasma levels; (d) peripheral blood mononuclear cell immunophenotyping; (e) multiple immunophenotyping of tumor tissue biopsy / resected specimens; (f) galactagogue-9 expression levels and patterns in tumor tissue biopsy / resected specimens; and (g) any other immunoscoring test, such as PDL-1 immunohistochemistry, tumor mutational burden (TMB), tumor microsatellite instability status, and panels, such as Immunoscore®-HalioDx, ImmunoSeq-Adaptive Biotechnologies, and NanoString. The nCounter® gene expression system has been developed for TIS, 18-gene signature, and PanCancer IO 360™ assay (NanoString Technologies). It can also detect other suitable biomarkers specific to the target tumor.

[0023] Any of the methods disclosed herein may also include monitoring for the occurrence of one or more adverse reactions in subjects. Exemplary adverse reactions include, but are not limited to, liver injury, hematologic toxicity, neurotoxicity, skin toxicity, gastrointestinal toxicity, or combinations thereof. When one or more adverse reactions are observed, the methods disclosed herein may further include reducing the dose of the anti-Gal9 antibody, the dose of one or more chemotherapeutic agents, or both. For example, when moderate to severe liver injury is observed in a subject, the method may further include reducing the dose of the anti-Gal9 antibody, the dose of gemcitabine, the dose of paclitaxel, or combinations thereof.

[0024] In some examples, paclitaxel administration is discontinued when a subject has an aspartate aminotransferase (AST) level greater than 10 times the upper limit of normal (ULN), a bilirubin level greater than 5 times the ULN, or both. In some embodiments, the method may further include reducing or discontinuing the administration of anti-Gal9 antibody, gemcitabine, paclitaxel, or combinations thereof when serious hematologic, neurotoxic, skin, and / or gastrointestinal toxicities are observed.

[0025] In some cases, the dosage of paclitaxel can be reduced to 100 mg / m². 2 Up to 75mg / m 2 Optionally or otherwise, the gemcitabine dose may be reduced to 800 mg / m². 2 Up to 600mg / m 2 .

[0026] The scope of this disclosure also includes pharmaceutical compositions comprising any anti-Gal9 antibody and one or more chemotherapeutic agents for treating solid tumors (e.g., PDAC), and the use of a combination of anti-Gal9 antibody and one or more chemotherapeutic agents in the preparation of a medicament for treating solid tumors.

[0027] Details of one or more embodiments of the present invention are set forth in the following description. Other features or advantages of the invention will become apparent from the following drawings and detailed description of several embodiments, as well as from the appended claims. Brief description of the attached diagram

[0029] The following figures form part of this specification and are included to further illustrate certain aspects of this disclosure, which can be better understood by referring to the figures and the detailed description of the specific embodiments described herein.

[0030] Figures 1A to 1DThis includes plots showing Kaplan-Meier survival curves and log-rank tests for orthotopic mPA6115 pancreatic cancer xenograft mouse models grouped by treatment regimen. Group 1 = untreated; Group 2 = chemotherapy mediator control, saline; Group 3 = allotype IgG1 mice; Group 4 = anti-Gal9 mAb; Group 5 = gemcitabine / Abraxane; and Group 6 = anti-Gal9 mAb and gemcitabine / Abraxane. Figure 1A Show the survival curves for all 6 groups. Figure 1B The survival curves for groups 1, 5, and 6 are shown. Figure 1C The survival curves for groups 1, 4, and 6 are shown. Figure 1D The survival curves for groups 1, 4, 5, and 6 are shown.

[0031] Figure 2 The graphs include those showing the hazard ratios (HRs) of groups 4 through 6 relative to groups 1, 2, and 3, respectively, calculated from Cox regression analysis, and their 95% confidence intervals (%95CI), where group 1 = untreated orthotopic mPA6115 mice; group 2 = orthotopic mPA6115 mice treated with chemotherapeutic agent and saline; group 3 = orthotopic mPA6115 mice treated with isotype IgG1 mice; group 4 = orthotopic mPA6115 mice treated with anti-Gal9 mAb; group 5 = orthotopic mPA6115 mice treated with gemcitabine / Abraxane; and group 6 = orthotopic mPA6115 mice treated with both anti-Gal9 mAb and gemcitabine / Abraxane.

[0032] Figure 3 The study included a graph of mean body weight measured twice weekly for each treatment group during the study period, where group 1 = untreated orthotopic mPA6115 mice; group 2 = chemotherapy-mediated control, saline-treated orthotopic mPA6115 mice; group 3 = orthotopic mPA6115 mice treated with isotype IgG1 mice; group 4 = orthotopic mPA6115 mice treated with anti-Gal9 mAb; group 5 = orthotopic mPA6115 mice treated with gemcitabine / Abraxane; and group 6 = orthotopic mPA6115 mice treated with both anti-Gal9 mAb and gemcitabine / Abraxane. Invention Details

[0034] This document provides methods for treating solid tumors (e.g., pancreatic cancer (PDA)) using a combination of an anti-galactotropic 9 antibody (e.g., G9.2-17) and a chemotherapeutic agent (e.g., gemcitabine and paclitaxel, such as protein-bound paclitaxel, such as nanoparticle albumin-conjugated paclitaxel, e.g., Abraxane®). In some embodiments, the solid tumor is metastatic. In some embodiments, the methods disclosed herein provide specific dosages and / or dosing schedules. In some cases, the methods disclosed herein are targeted at specific patient populations, such as patients who have received prior treatment and have shown disease progression during prior treatment, or patients who are resistant to prior treatment (de novo or acquired).

[0035] Galactochonin-9 is a tandem repeat lectin, a β-galactoside-binding protein, which has been shown to play a role in regulating cell-cell and cell-matrix interactions. It has been found to be strongly overexpressed in Hodgkin's disease tissues and other pathological conditions. In some cases, it has also been found to circulate in the tumor microenvironment (TME).

[0036] Galactagogue-9 interacts with Dectin-1, an innate immune receptor that is highly expressed on macrophages in PDA and on cancer cells (Daley et al.). Nat Med 2017;23(5):556-6). Regardless of the source of galactagogue-9, disruption of its interaction with Dectin-1 has been shown to lead to CD4+. + and CD8 + Cellular reprogramming is an essential mediator of anti-tumor immunity. Therefore, galactagogue-9 can serve as a valuable therapeutic target for blocking Dectin-1-mediated signaling. Consequently, in some embodiments, the anti-galactagogue-9 antibody described herein disrupts the interaction between galactagogue-9 and Dectin-1.

[0037] Galactagogue-9 also interacts with TIM-3, a type I cell surface glycoprotein expressed on the surface of leukemia stem cells in all types of acute myeloid leukemia (except M3 (acute promyelocytic leukemia)), but not in normal human hematopoietic stem cells (HSCs). TIM-3 signaling linked by galactagogue-9 has been found to have multiple effects on immune cells, including inducing Th1 cell apoptosis (Zhu et al.). Nat Immunol. (Kuchroo et al., 2005, 6: 1245-1252) and stimulate the secretion of tumor necrosis factor-α (TNF-α), leading to the maturation of monocytes into dendritic cells, which causes inflammation due to innate immunity (Kuchroo et al., 2005, 6: 1245-1252). Nat Rev Immunol., 2008, 8: 577-580). Further galactagogue-9 / TIM-3 signaling has been found to co-activate NF-κB and β-catenin signaling, both pathways that promote LSC self-renewal (Kikushige et al., 2008, 8: 577-580). Cell Stem Cell, 2015, 17(3):341-352). Anti-galactoglobulin-9 antibodies that interfere with the binding of galactoglobulin-9 / TIM-3 may have therapeutic effects, particularly for leukemia and other hematologic malignancies. Therefore, in some embodiments, the anti-galactoglobulin-9 antibodies described herein disrupt the interaction between galactoglobulin-9 and TIM-3.

[0038] Furthermore, galactagogue-9 interacts with CD206 to promote tumor survival (Enninga et al., J Pathol. 2018 Aug;245(4):468-477), CD206 is a mannose receptor highly expressed on M2 polarized macrophages. Tumor-associated macrophages expressing CD206 are mediators of tumor immunosuppression, angiogenesis, metastasis, and recurrence (see, for example, Scodeller et al., 2018 Aug;245(4):468-477). Sci Rep . 2017 Nov 7;7(1):14655, and references therein). Specifically, M1 (also known as classical activated macrophages) are triggered by Th1-related cytokines and bacterial products, express high levels of IL-12, and have tumor-killing effects. In contrast, M2 (so-called alternative activated macrophages) are activated by Th2-related factors, express high levels of anti-inflammatory cytokines such as IL-10, and promote tumor progression (Biswas and Mantovani; Nat Immunol. 2010 Oct; 11(10):889-96). The pro-tumor effects of M2 include promoting angiogenesis, promoting invasion and metastasis, and protecting tumor cells from chemotherapy-induced apoptosis (Hu et al., 2010 Oct; 11(10):889-96). Tumour Biol . 2015 Dec; 36(12): 9119–9126, and references therein). It is believed that tumor-associated macrophages possess an M2-like phenotype and have pro-tumorigenic effects. It has been shown that galactagogue-9 can mediate the differentiation of bone marrow cells toward the M2 phenotype (Enninga et al., 2015 Dec; 36(12): 9119–9126, and references therein). Melanoma Res(2016 Oct;26(5):429-41). Gastrin-9 binding to CD206 may lead to TAM reprogramming to the M2 phenotype, similar to what has been shown with Dectin. Not wishing to be bound by theory, blocking the interaction between galactoglobulin-9 and CD206 could provide a mechanism through which anti-galactoglobulin-9 antibodies (e.g., G9.2-17 antibodies) could be therapeutically beneficial. Therefore, in some embodiments, the anti-galactoglobulin-9 antibodies described herein disrupt the interaction between galactoglobulin-9 and CD206.

[0039] It was also shown that galactagogue-9 interacts with protein disulfide isomerase (PDI) and 4-1BB (Bi S, et al.). Proc Natl Acad Sci USA . 2011;108(26):10650-5; Madireddi et al. J Exp Med. 2014;211(7):1433-48).

[0040] Anti-galactoglobulin-9 antibodies can be used as therapeutic agents to treat diseases associated with galactoglobulin-9 (e.g., diseases in which galactoglobulin-9 signaling plays a role). Without being bound by theory, anti-galactoglobulin-9 antibodies can block galactoglobulin-9-mediated signaling pathways. For example, the antibodies may interfere with the interaction between galactoglobulin-9 and its binding partners (e.g., Dectin-1, TIM-3, or CD206), thereby blocking signaling triggered by galactoglobulin-9 / ligand interactions. Optionally or additionally, anti-galactoglobulin-9 antibodies can also exert their therapeutic effect by inducing blocking and / or cytotoxicity, such as targeting ADCC, CDC, or ADCP in pathological cells expressing galactoglobulin-9. Pathological cells are those that directly or indirectly contribute to the initiation and / or development of disease.

[0041] The anti-galactoglobulin-9 antibody disclosed herein can inhibit galactoglobulin-9-mediated signaling (e.g., galactoglobulin-9 / Dectin-1 or galactoglobulin-9 / Tim-3 mediated signaling pathways) or eliminate pathological cells expressing galactoglobulin-9 through methods such as ADCC. Therefore, the anti-galactoglobulin-9 antibody described herein can be used to inhibit any galactoglobulin-9 signaling and / or eliminate galactoglobulin-9-positive pathological cells, thereby benefiting the treatment of galactoglobulin-9-related diseases. See, for example, WO2019 / 084553, PCT / US2020 / 024767 and PCT / US2020 / 031181, the respective disclosures of which are incorporated herein by reference for the purposes and subject matter herein.

[0042] As reported herein, combination therapy with representative anti-Gal9 antibodies (G9.2-17) and chemotherapeutic agents (gemcitabine and nab-paclitaxel) successfully prolonged survival in the animal models disclosed herein. A synergistic effect on survival time was observed between representative anti-Gal9 antibodies and gemcitabine and nab-paclitaxel in the animal models. These results suggest that the antitumor approaches disclosed herein, involving combinations of anti-galactotropic 9 antibodies and chemotherapeutic agents (such as those disclosed herein), will achieve superior therapeutic efficacy against target solid tumors compared to antibody or chemotherapy alone.

[0043] Therefore, this article describes the therapeutic use of anti-galactoglobulin-9 antibodies and chemotherapeutic agents for the treatment of certain solid tumors disclosed herein.

[0044] Antibodies that bind to galactagogue-9

[0045] This disclosure provides anti-galactoglobulin-9 antibody G9.2-17 and its functional variants for use in the treatment methods disclosed herein.

[0046] Antibodies (the plural form is used interchangeably) are immunoglobulin molecules capable of specifically binding to targets, such as carbohydrates, polynucleotides, lipids, peptides, etc., through at least one antigen recognition site located in the variable region of an immunoglobulin molecule. As used herein, the term "antibody," such as anti-galactoglobulin-9 antibody, includes not only complete (e.g., full-length) polyclonal or monoclonal antibodies, but also their antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv), single chains (scFv), their mutants, fusion proteins containing antibody portions, humanized antibodies, chimeric antibodies, biantibodies, nanobodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified conformation of an immunoglobulin molecule containing an antigen recognition site of desired specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies, such as anti-galactoglobulin-9 antibody, include any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or their subclasses), and antibodies need not be of any particular type. Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant domains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are designated as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well-known.

[0047] Typical antibody molecules contain a heavy chain variable region (V). H ) and light chain variable region (V LThese typically participate in antigen binding. H and V L The region can be further subdivided into high-variability regions, also known as "complementary determinant regions" ("CDRs"), within which are interspersed more conservative regions, known as "frame regions" ("FRs"). Each V H and V L Typically, it consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the frame region and CDRs can be precisely identified using methods known in the art, such as the Kabat definition, Chothia definition, AbM definition, EU definition, “Contact” numbering scheme, “IMGT” numbering scheme, “AHo” numbering scheme, and / or the Contact definition, all of which are well known in the art. See, for example, Kabat, EA, et al. (1991). Sequences of Proteins of Immunological Interest, Fifth Edition , US Department of Health and Human Services, NIHPublication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948; Edelman et al., Proc Natl Acad Sci USA . 1969 May;63(1):78-85; and Almagro, J. Mol. Recognit 17:132-143 (2004); MacCallum et al., J. Mol. Biol .262:732-745 (1996), Lefranc MP et al., Dev Comp Immunol , 2003 January; 27(1):55-77; and Honegger A and Pluckthun A, J Mol Biol, 2001 Jun. 8; 309(3):657-70. See also hgmp.mrc.ac.uk and bioinf.org.uk / abs).

[0048] In some embodiments, the anti-galactoglobulin-9 antibody described herein is a full-length antibody comprising two heavy chains and two light chains, each including a variable domain and a constant domain. Alternatively, the anti-galactoglobulin-9 antibody may be an antigen-binding fragment of a full-length antibody. Examples of binding fragments included in the term "antigen-binding fragment" of a full-length antibody include (i) a Fab fragment, consisting of V... L V H C L and C H 1) a monovalent segment composed of structural domains; (ii) an F(ab')2 segment, comprising a divalent segment of two Fab segments connected by disulfide bonds in the hinge region; (iii) a segment composed of V H and C H (iv) Fd fragment composed of 1 domain; (iv) Fv fragment composed of V of the antibody single arm. L and V H Domain composition; (v)dAb fragment (Ward et al., (1989) Nature 341:544-546), by V H The structural domains consist of (vi) separate complementary determinant regions (CDRs) that preserve functionality. Furthermore, although the two structural domains V of the Fv segment... L and V H Encoded by different genes, but which can be linked together via synthetic adapters using recombination methods, allowing them to form a single protein chain, where V L and V H Block pairing forms a monovalent molecule called a single-chain Fv (scFv). See, for example, Bird et al. (1988). Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883.

[0049] Any antibody described herein, such as anti-galactoglobulin-9 antibody, can be monoclonal or polyclonal. "Monoclonal antibody" refers to a homogeneous group of antibodies, while "polyclonal antibody" refers to a heterogeneous group of antibodies. These terms do not limit the source of the antibody or its preparation method.

[0050] Reference antibody G9.2-17 refers to an antibody capable of binding to human galactoglobulin-9, comprising the heavy chain variable region of SEQ ID NO: 7 and the light chain variable region of SEQ ID NO: 8, both of which are provided below. In some embodiments, the anti-galactoglobulin-9 antibody used in the methods disclosed herein is antibody G9.2-17. In some embodiments, the anti-galactoglobulin-9 antibody used in the methods disclosed herein is an antibody having the same heavy chain complementarity-determining region (CDR) and / or the same light chain complementarity-determining region as reference antibody G9.2-17. Having the same V H and / or V L Two antibodies with a CDR mean that their CDRs are identical when determined by the same method (e.g., the Kabat method, Chothia method, AbM method, Contact method, or IMGT method known in the art; see, for example, bioinf.org.uk / abs / ).

[0051] The heavy and light chain CDRs of reference antibody G9.2-17 are provided in Table 1 below (determined using the Kabat method):

[0052] Table 1. CDRs of heavy and light chains in G9.2-17

[0053]

[0054] In some examples, the anti-galactoglobulin-9 antibody used in the methods disclosed herein may comprise (according to the Kabat protocol) a heavy chain complementarity-determining region 1 (CDR1) as shown in SEQ ID NO: 4, a heavy chain complementarity-determining region 2 (CDR2) as shown in SEQ ID NO: 5, and a heavy chain complementarity-determining region 3 (CDR3) as shown in SEQ ID NO: 6, and / or may comprise a light chain complementarity-determining region 1 (CDR1) as shown in SEQ ID NO: 1, a light chain complementarity-determining region 2 (CDR2) as shown in SEQ ID NO: 2, and a light chain complementarity-determining region 3 (CDR3) as shown in SEQ ID NO: 3. The anti-galactoglobulin-9 antibody, including the reference antibody G9.2-17, may be in any form disclosed herein, such as a full-length antibody or a Fab. As used herein, the term “G9.2-17(Ig4)” refers to the G9.2-17 antibody as an IgG4 molecule. Similarly, the term “G9.2-17(Fab)” refers to the G9.2-17 antibody as a Fab molecule.

[0055] In some embodiments, the anti-galactoglobulin-9 antibody or its binding portion comprises heavy and light chain variable regions, wherein the amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment thereof) sequence identity with the amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 1, 2, and 3. In some embodiments, the anti-galactoglobulin-9 antibody or its binding portion comprises heavy and light chain variable regions, wherein the amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments thereof) sequence identity with the amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 4, 5, and 6.

[0056] Other galactagogue-9 antibodies, such as those binding to the CRD1 and / or CRD2 regions of galactagogue-9, are described in jointly owned, jointly pending U.S. Patent Application 16 / 173,970 and jointly owned, jointly pending international Patent Applications PCT / US18 / 58028 and PCT / US2020 / 024767, the contents of which are incorporated herein by reference in their entirety.

[0057] In some embodiments, the anti-galactophlebin-9 antibody disclosed herein comprises the corresponding V of the reference antibody G9.2-17. L The CDR has at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and any increments thereof) sequence identity compared to the reference antibody G9.2-17, either individually or overall. Alternatively or additionally, in some embodiments, the anti-hemegruin 9 antibody comprises the corresponding V of the reference antibody G9.2-17. H CDRs have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments thereof) sequence identity with each other individually or in whole.

[0058] The "percentage of identity" between two amino acid sequences was determined using the following algorithm: Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, improved from Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. This algorithm was incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecule of this invention. In the case of gaps between two sequences, Gapped BLAST as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997 can be used. When using the BLAST and Gapped BLAST programs, the default parameters of each program (such as XBLAST and NBLAST) can be used.

[0059] In other embodiments, the anti-galactogenin-9 antibody described herein comprises: V containing HC CDR1, HC CDR2, and HCCDR3. H These antibodies, relative to the reference antibody G9.2-17, generally contain up to 8 amino acid residue variations (8, 7, 6, 5, 4, 3, 2, or 1 variation, including additions, deletions, and / or substitutions) of HC CDR1, HC CDR2, and HC CDR3. Alternatively or additionally, in some embodiments, the anti-galactogenin-9 antibodies described herein comprise: V containing LC CDR1, LC CDR2, and LC CDR3. H They contain up to 8 amino acid residue variations (8, 7, 6, 5, 4, 3, 2 or 1 variation, including addition, deletion and / or substitution) in total relative to the reference antibody G9.2-17 LC CDR1, LC CDR2 and LC CDR3.

[0060] In one example, amino acid residue variation is a conserved amino acid residue substitution. As used herein, “conserved amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein to which the substitution is made. Variants can be prepared according to methods of altering polypeptide sequences known to those skilled in the art, for example, as compiled in references such as *Molecular Cloning: A Laboratory Manual*, J. Sambrook et al., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or *Current Protocols in Molecular Biology*, FM Ausubel et al., John Wiley & Sons, Inc., New York. Conserved substitutions of amino acids include substitutions between amino acids within the following group: (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.

[0061] In some embodiments, the disclosed anti-galactoglobulin-9 antibody having the heavy chain CDR disclosed herein comprises germline V H The frame region of the subclass of the fragment. This phylogenetic V HRegions are well known in the art. See, for example, the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php. Examples include the IGHV1 subfamily (e.g., IGHV1-2, IGHV1-3, IGHV1-8, IGHV1-18, IGHV1-24, IGHV1-45, IGHV1-46, IGHV1-58, and IGHV1-69), the IGHV2 subfamily (e.g., IGHV2-5, IGHV2-26, and IGHV2-70), and the IGHV3 subfamily (e.g., IGHV3-7, IGHV3-9, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-20, IGHV3-21, IGHV3-23, IGHV3-30, I...). The 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, IGHV4-31, IGHV4-34, IGHV4-39, IGHV4-59, IGHV4-61 and IGHV4-B), the IGHV subfamily (e.g., IGHV5-51 or IGHV6-1), and the IGHV7 subfamily (e.g., IGHV7-4-1).

[0062] Alternatively or additionally, in some embodiments, an anti-galactocyanin-9 antibody having the light chain CDR disclosed herein comprises germline V The frame region of the fragment. Examples include the IGKV1 frame (e.g., IGKV1-05, IGKV1-12, IGKV1-27, IGKV1-33, or IGKV1-39), the IGKV2 frame (e.g., IGKV2-28), the IGKV3 frame (e.g., IGKV3-11, IGKV3-15, or IGKV3-20), and the IGKV4 frame (e.g., IGKV4-1). In other cases, anti-galactoglobulin-9 antibodies contain a light chain variable region, which contains components derived from germline V. The frame of the clip. Examples include IG. 1. Framework (e.g., IG) V1-36, IG V1-40, IG V1-44, IG V1-47, IG V1-51), IG 2. Framework (e.g., IG) V2-8, IG V2-11, IG V2-14, IG V2-18, IG V2-23), IG 3-framework (e.g., IG) V3-1, IG V3-9, IG V3-10, IG V3-12, IG V3-16, IG V3-19, IG V3-21, IG V3-25, IG V3-27), IG 4-framework (e.g., IG) V4-3, IG V4-60, IG V4-69), IG 5 frames (e.g., IG) V5-39, IG V5-45), IG 6 frames (e.g., IG) V6-57), IG 7 frames (e.g., IG) V7-43, IG V7-46), IG 8 frames (e.g., IG) V8-61), IG 9 frames (e.g., IG) V9-49), or IG 10 frames (e.g., IG) V10-54).

[0063] In some embodiments, the anti-galactophlebin-9 antibody used in the methods disclosed herein may have the same heavy chain variable region (V) as the reference antibody G9.2-17. H ) and / or the same light chain variable region (V L The antibody, V H and V L The amino acid sequence of the region is as follows:

[0064] V H :

[0065] EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSS (SEQ ID NO: 7)

[0066] V L :

[0067] DIQMTQSPSSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSTDPITFGQGTKVEIKR (SEQ ID NO: 8)

[0068] In some embodiments, the anti-galactoglobulin-9 antibody has at least 80% sequence identity with the heavy chain variable region of SEQ ID NO: 7 (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity). Alternatively or additionally, the anti-galactoglobulin-9 antibody has at least 80% sequence identity with the light chain variable region of SEQ ID NO: 8 (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity).

[0069] In some cases, the anti-galactoglobulin-9 antibodies disclosed herein are functional variants of the reference antibody G9.2-17. These functional variants may be structurally similar to the reference antibody (e.g., in one or more heavy and / or light chain CDRs of G9.2-17 as disclosed herein, or in sequence identity with respect to the heavy and / or light chain CDRs of G9.2-17, or in the VH and / or VL of G9.2-17 as disclosed herein), and have substantially similar binding affinity for human anti-galactoglobulin-9 (e.g., having KD values ​​of the same order of magnitude).

[0070] In some embodiments, the anti-galactoglobulin-9 antibody, as described herein, can bind to galactoglobulin-9 and inhibit its activity by at least 20% (e.g., 31%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or higher, including any increments thereof). The epigenetic inhibition constant (Ki) app or K i,appThis provides a measure of inhibitory efficacy, which is related to the concentration of inhibitor required to reduce enzyme activity, but is independent of enzyme concentration. The inhibitory activity of the anti-galactoglobulin-9 antibody described herein can be determined using conventional methods known in the art.

[0071] Ki of antibodies app The value can be determined by measuring the inhibitory effect of different concentrations of antibody on the degree of reaction (e.g., enzyme activity); the pseudo-first-order rate constant ( v The change in Ki is fitted as a function of inhibitor concentration to the improved Morrison equation (Equation 1) to produce an estimate of the apparent Ki value. For competitive inhibitors, Ki app Available from Ki app The y-intercept was obtained from the linear regression analysis of the substrate concentration map.

[0072] (Equation 1)

[0073] in A equal v o / E In the absence of inhibitors ( I The initial rate of the enzyme-catalyzed reaction under the condition of ) v o Divide by the total enzyme concentration (E). In some embodiments, the anti-galactoglobulin-9 antibody described herein is effective against the Ki of the target antigen or epitope. app Values ​​were 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. In some implementations, the anti-galactoglobulin-9 antibody exhibited a lower Ki value against the first target (e.g., galactoglobulin-9 CRD2) relative to the second target (e.g., CRD1 of galactoglobulin-9). app Ki app The difference (e.g., for specificity or other comparisons) 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 In some examples, the anti-galactoglobulin-9 antibody inhibits the first antigen (e.g., the first conformation of the first protein or its mimicry) more than the second antigen (e.g., a second conformation of the same first protein or a mimicry of it; or a second protein). In some embodiments, any anti-galactoglobulin-9 antibody undergoes further affinity maturation to reduce the Ki of the antibody against the target antigen or its epitope. app .

[0074] In some embodiments, the anti-galactoglobulin-9 antibody inhibits Dectin-1 signaling, for example, in tumor-infiltrating immune cells such as macrophages. In some embodiments, the anti-galactoglobulin-9 antibody inhibits Dectin-1 signaling triggered by galactoglobulin-9 by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher, including any increments thereof). This inhibitory activity can be determined by conventional methods, such as conventional assays. Optionally or additionally, the anti-galactoglobulin-9 antibody inhibits T-cell immunoglobulin mucin 3 (TIM-3) signaling initiated by galactoglobulin-9. In some embodiments, the anti-galactoglobulin-9 antibody inhibits T-cell immunoglobulin mucin 3 (TIM-3) signaling, such as in tumor-infiltrating immune cells, for example, in some embodiments, by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher, including any increments thereof). This inhibitory activity can be determined by conventional methods, such as conventional assays.

[0075] In some embodiments, the anti-galactoglobulin-9 antibody inhibits CD206 signaling, for example, in tumor-infiltrating immune cells. In some embodiments, the anti-galactoglobulin-9 antibody inhibits at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher, including any increments thereof) of CD206 triggered by galactoglobulin-9. This inhibitory activity can be determined by conventional methods, such as conventional assays. In some embodiments, the anti-galactoglobulin-9 antibody blocks or prevents the binding of galactoglobulin-9 to CD206 by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher, including any increments thereof). This inhibitory activity can be determined by conventional methods, such as conventional assays.

[0076] In some embodiments, the anti-galactotropic antibody induces cytotoxicity, such as ADCC, in target cells expressing galactotropic 9, for example, where the target cells are cancer cells or immunosuppressive immune cells. In some embodiments, the anti-galactotropic antibody induces at least 30% apoptosis in immune cells (e.g., T cells) or cancer cells (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater, including any increments thereof). This inhibitory activity can be determined by conventional methods, such as conventional assays. In some embodiments, any anti-galactotropic antibody described herein induces cytotoxicity, such as complement-dependent cytotoxicity (CDC) against target cells expressing galactotropic 9.

[0077] Antibody-dependent cell-mediated phagocytosis (ADCP) is an important mechanism by which antibodies mediate some or all of their effects through phagocytosis. In this case, antibodies mediate the uptake of specific antigens by antigen-presenting cells. ADCP can be mediated by monocytes, macrophages, neutrophils, and dendritic cells via FcγRIIa, FcγRI, and FcγRIIIa, with FcγRIIa (CD32a) on macrophages being the main pathway.

[0078] In some embodiments, the anti-galactoglobulin-9 antibody induces phagocytosis (ADCP) of target cells, such as cancer cells expressing galactoglobulin-9 or immunosuppressive immune cells. In some embodiments, the anti-galactoglobulin-9 antibody increases the phagocytosis of target cells (e.g., cancer cells or immunosuppressive immune cells) by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher, including any increments thereof).

[0079] In some embodiments, the anti-galactotropic antibody described herein induces cytotoxicity against target cells, such as cancer cells or immunosuppressive immune cells, such as complement-dependent cytotoxicity (CDC). In some embodiments, the anti-galactotropic antibody increases the CDC against target cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher, including any increments thereof).

[0080] In some embodiments, the anti-galactoglobulin-9 antibody induces T cell activation, such as in tumor-infiltrating T cells, i.e., directly or indirectly inhibits galactoglobulin-9-mediated T cell activation. In some embodiments, the anti-galactoglobulin-9 antibody promotes T cell activation by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher, including any increments thereof). T cell activation can be determined by conventional methods, such as assays (e.g., measuring CD44, TNFα, IFNγ, and / or PD-1). In some embodiments, the anti-galactoglobulin-9 antibody promotes CD4+ cell activation by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher, including any increments thereof). In a non-limiting example, the anti-galactoglobulin antibody induces CD44 expression in CD4+ cells. In some embodiments, the anti-galactoglobulin-9 antibody increases CD44 expression in CD4+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher, including any increments therein). In a non-limiting example, the anti-galactoglobulin antibody induces IFNγ expression in CD4+ cells. In some embodiments, the anti-galactoglobulin-9 antibody increases IFNγ expression in CD4+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher, including any increments therein). In a non-limiting example, the anti-galactoglobulin antibody induces TNFα expression in CD4+ cells. In some embodiments, the anti-galactoglobulin-9 antibody increases TNFα expression in CD4+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher, including any increments therein).

[0081] In some embodiments, the anti-galactoglobulin-9 antibody promotes CD8+ cell activation by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher, including any increments therein). In a non-limiting example, the anti-galactoglobulin antibody induces CD44 expression in CD8+ cells. In some embodiments, the anti-galactoglobulin-9 antibody increases CD44 expression in CD8+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher, including any increments therein). In a non-limiting example, the anti-galactoglobulin antibody induces IFNγ expression in CD8+ cells. In some embodiments, the anti-galactoglobulin-9 antibody increases IFNγ expression in CD8+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher, including any increments therein). In a non-limiting example, the anti-galactoglobulin antibody induces TNFα expression in CD8+ cells. In some embodiments, the anti-galactoglobulin-9 antibody increases TNFα expression in CD8+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher, including any increment therein).

[0082] In some embodiments, the anti-galactophage-9 antibody described herein has a suitable binding affinity for the target antigen (e.g., galactophage-9) or its antigenic epitope. As used herein, "binding affinity" refers to the apparent binding constant or K0. A K A It is the dissociation constant (K) D The reciprocal of ). The anti-galactogenin-9 antibody described herein may have at least 10 against the target antigen or epitope. -5 10 -6 10 -7 10 -8 10 -9 10 -10 M or lower binding affinity (K D Increased binding affinity corresponds to decreased K. D Binding affinity (or binding specificity) can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using fluorescence assays). Exemplary conditions for assessing binding affinity are in HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20).

[0083] These techniques can be used to measure the concentration of bound proteins as a function of target protein concentration. Under certain conditions, the fractional concentration of bound proteins ([bound] / [total]) is often correlated with the total target protein concentration ([target]), as shown in the following formula:

[0084] [Combined] / [Total] = [Target] / (Kd+[Target])

[0085] However, it is not always necessary to accurately measure K. A Because sometimes obtaining a quantitative measurement of affinity is sufficient, for example, the affinity determined using methods such as ELISA or FACS analysis relative to K. A The affinity is directly proportional to the in vivo affinity, and therefore can be used for comparisons, such as determining whether a higher affinity is, for example, 2 times higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, for example, by activity in functional assays such as in vitro or in vivo assays. In some cases, in vitro binding assays indicate in vivo activity. In other cases, in vitro binding assays do not necessarily indicate in vivo activity. In some cases, tight binding is beneficial, but in others, tight binding is not expected in vivo, and binding to antibodies with lower affinity is more desirable.

[0086] In some embodiments, the heavy chain of any anti-galactoglobulin-9 antibody described herein further comprises a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or combinations thereof). The heavy chain constant region can be from any suitable source, such as human, mouse, rat, or rabbit. In a specific example, the heavy chain constant region is derived from human IgG (γ heavy chain) of any of the IgG subfamily described herein.

[0087] In some embodiments, the heavy chain constant region of the antibody described herein comprises a single domain (e.g., CH1, CH2, or CH3) or any combination of single domains of the constant region (e.g., SEQ ID NO: 10, 12-14, and 21). In some embodiments, the light chain constant region of the antibody described herein comprises a single domain (e.g., CL) of the constant region. Exemplary light and heavy chain sequences are listed below. The hIgG1 LALA sequence includes two mutations, L234A and L235A (EU number), which inhibit FcgR binding, and a P329G mutation (EU number) to eliminate complement C1q binding, thereby eliminating all immune effector functions. The hIgG4 Fab arm exchange mutation sequence includes a mutation (S228P; EU number) that inhibits Fab arm exchange. The IL2 signaling sequence (MYRMQLLSCIALSLALVTNS; SEQ ID NO: 9) may be located at the N-terminus of the variable region. It is used in the expression vector and is cleaved during secretion, therefore it is not present in the mature antibody molecule. Mature proteins (post-secretionary) begin with "EVQ" for heavy chains and "DIM" for light chains. The following provides an example amino acid sequence of the heavy chain constant region:

[0088] hIgG1 heavy chain constant region (SEQ ID NO: 10)

[0089] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0090] hIgG1 LALA heavy chain constant region (SEQ ID NO: 12)

[0091] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL G APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0092] hIgG4 heavy chain constant region (SEQ ID NO: 13)

[0093] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0094] hIgG4 heavy chain constant region (SEQ ID NO: 20)

[0095] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0096] hIgG4 mut heavy chain constant region (SEQ ID NO: 14)

[0097] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP P CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0098] hIgG4 mut heavy chain constant region (SEQ ID NO: 21)

[0099] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0100] In some implementations, anti-galactocyanin-9 antibodies having any of the above heavy chain constant regions are paired with light chains having the following light chain constant regions:

[0101] Light chain constant region (SEQ ID NO: 11)

[0102] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0103] An example full-length anti-galactoglobulin-9 antibody is provided as follows:

[0104] G9.2-17 hIgG1 heavy chain (SEQ ID NO: 16)

[0105] EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0106] G9.2-17 hIgG1 LALA heavy chain (SEQ ID NO: 17)

[0107] EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL GAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0108] G9.2 - 17 hIgG4 heavy chain (SEQ ID NO: 18)

[0109] EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0110] G9.2 - 17 hIgG4 heavy chain (SEQ ID NO: 22)

[0111] EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0112] G9.2-17 hIgG4 Fab-arm exchange mut heavy chain (SEQ ID NO: 19)

[0113] EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0114] G9.2-17 hIgG4 Fab-arm exchange mut heavy chain (SEQ ID NO: 23)

[0115] EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP P CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0116] Any of the above heavy chains can be paired with the light chain shown below (SEQ ID NO: 15):

[0117] DIQMTQSPSSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSTDPITFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0118] In some embodiments, the anti-galactoglobulin-9 antibody comprises a heavy chain IgG1 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments thereof) sequence identity with SEQ ID NO: 10. In one embodiment, the constant region of the anti-galactoglobulin-9 antibody comprises the heavy chain IgG1 constant region contained in SEQ ID NO: 13. In one embodiment, the constant region of the anti-galactoglobulin-9 antibody comprises the heavy chain IgG4 constant region constituted by SEQ ID NO: 10.

[0119] In one embodiment, the anti-galactoglobulin-9 antibody comprises a heavy chain IgG4 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments thereof) sequence identity with SEQ ID NO: 13. In one embodiment, the anti-galactoglobulin-9 antibody comprises the heavy chain IgG4 constant region containing SEQ ID NO: 13. In one embodiment, the anti-galactoglobulin-9 antibody comprises the heavy chain IgG4 constant region consisting of SEQ ID NO: 13.

[0120] In some embodiments, the constant region is derived from human IgG4. In one embodiment, the anti-galactoglobulin-9 antibody comprises a heavy chain IgG4 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments thereof) sequence identity with SEQ ID NO: 20. In one embodiment, the anti-galactoglobulin-9 antibody comprises the heavy chain IgG4 constant region containing SEQ ID NO: 20. In one embodiment, the anti-galactoglobulin-9 antibody comprises the heavy chain IgG4 constant region consisting of SEQ ID NO: 20.

[0121] In any of these embodiments, the anti-galactoglobulin-9 antibody comprises a light chain constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments thereof) sequence identity with SEQ ID NO: 11. In some embodiments, the anti-galactoglobulin-9 antibody comprises the light chain constant region containing SEQ ID NO: 11. In some embodiments, the anti-galactoglobulin-9 antibody comprises a light chain constant region consisting of SEQ ID NO: 11.

[0122] In some embodiments, IgG is a mutant with minimal Fc receptor binding. In one example, the constant region is derived from human IgG1 LALA. In one embodiment, the anti-galactoglobulin-9 antibody comprises a heavy chain IgG1 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment thereof) sequence identity with SEQ ID NO: 12. In one embodiment, the anti-galactoglobulin-9 antibody comprises the heavy chain IgG1 constant region containing SEQ ID NO: 12. In one embodiment, the anti-galactoglobulin-9 antibody comprises the heavy chain IgG1 constant region consisting of SEQ ID NO: 12.

[0123] In some embodiments, the anti-galactoglobulin-9 antibody comprises a modified constant region. In some embodiments, the anti-galactoglobulin-9 antibody comprises an immune-inert modified constant region, for example, that does not trigger complement-mediated cleavage or stimulate antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC activity can be evaluated using the method disclosed in U.S. Patent No. 5,500,362. In other embodiments, the constant region is as follows: Eur. J. Immunol. Modifications are made as described in (1999) 29:2613-2624; PCT application number PCT / GB99 / 01441; and / or UK patent application number 9809951.8. In some embodiments, the IgG4 constant region is a mutant with reduced heavy chain exchange. In some embodiments, the constant region is derived from the human IgG4 Fab arm exchange mutant S228P.

[0124] In one embodiment, the constant region of the anti-galactoglobulin-9 antibody comprises a heavy chain IgG4 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments thereof) sequence identity with SEQ ID NO: 14. In one embodiment, the constant region of the anti-galactoglobulin-9 antibody comprises a heavy chain IgG4 constant region containing SEQ ID NO: 14. In one embodiment, the constant region of the anti-galactoglobulin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO: 14.

[0125] In one embodiment, the anti-galactoglobulin-9 antibody comprises a heavy chain IgG4 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments thereof) sequence identity with SEQ ID NO: 21. In one embodiment, the anti-galactoglobulin-9 antibody comprises the heavy chain IgG4 constant region containing SEQ ID NO: 21. In one embodiment, the anti-galactoglobulin-9 antibody comprises the heavy chain IgG4 constant region consisting of SEQ ID NO: 21.

[0126] In some embodiments, the anti-galactoglobulin-9 antibody has a chain corresponding to the light chain of SEQ ID NO: 15; and the exemplary heavy chain amino acid sequence corresponds to SEQ ID NO: 10 (hIgG1); 12 (hIgG1 LALA); 13 (hIgG4); 20 (hIgG4); 14 (hIgG4 mut); and 21 (hIgG4 mut).

[0127] In some embodiments, the anti-galactoglobulin-9 antibody has a light chain comprising, substantially composed of, or consisting of SEQ ID NO: 15. In some embodiments, the anti-galactoglobulin-9 antibody has a heavy chain comprising any one of sequences selected from SEQ ID NO: 16-19, 22, and 23, substantially composed of, or consisting of, any one of sequences selected from SEQ ID NO: 16-19, 22, and 23. In some embodiments, the anti-galactoglobulin-9 antibody has a light chain comprising, substantially composed of, or consisting of SEQ ID NO: 15, and a heavy chain comprising any one of sequences selected from SEQ ID NO: 16-19, substantially composed of, or consisting of, any one of sequences selected from SEQ ID NO: 16-19. In some embodiments, the anti-galactoglobulin-9 antibody has a light chain comprising SEQ ID NO: 15 and a heavy chain comprising any sequence selected from SEQ ID NO: 16-19, 22, and 23. In some embodiments, the anti-galactoglobulin-9 antibody has a light chain consisting essentially of SEQ ID NO: 15 and a heavy chain consisting essentially of any sequence selected from SEQ ID NO: 16-19, 22, and 23. In some embodiments, the anti-galactoglobulin-9 antibody has a light chain consisting essentially of SEQ ID NO: 15 and a heavy chain consisting essentially of any sequence selected from SEQ ID NO: 16-19, 22, and 23. In one specific embodiment, the anti-galactoglobulin-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-galactoglobulin-9 antibody has a light chain consisting essentially of SEQ ID NO: 15 and a heavy chain consisting essentially of SEQ ID NO: 20.

[0128] In one embodiment, the anti-galactoglobulin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments thereof) sequence identity with SEQ ID NO: 16. In one embodiment, the anti-galactoglobulin-9 antibody comprises the heavy chain sequence comprising SEQ ID NO: 16. In one embodiment, the anti-galactoglobulin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 16.

[0129] In one embodiment, the anti-galactoglobulin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments thereof) sequence identity with SEQ ID NO: 17. In one embodiment, the anti-galactoglobulin-9 antibody comprises the heavy chain sequence comprising SEQ ID NO: 17. In one embodiment, the anti-galactoglobulin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 17.

[0130] In one embodiment, the anti-galactoglobulin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments thereof) sequence identity with SEQ ID NO: 18. In one embodiment, the anti-galactoglobulin-9 antibody comprises the heavy chain sequence containing SEQ ID NO: 18. In one embodiment, the anti-galactoglobulin-9 antibody comprises the heavy chain sequence consisting of SEQ ID NO: 18.

[0131] In one embodiment, the anti-galactoglobulin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments thereof) sequence identity with SEQ ID NO: 22. In one embodiment, the anti-galactoglobulin-9 antibody comprises the heavy chain sequence containing SEQ ID NO: 22. In one embodiment, the anti-galactoglobulin-9 antibody comprises the heavy chain sequence consisting of SEQ ID NO: 22.

[0132] In one embodiment, the anti-galactoglobulin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments thereof) sequence identity with SEQ ID NO: 19. In one embodiment, the anti-galactoglobulin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 19. In one embodiment, the anti-galactoglobulin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 19.

[0133] In one embodiment, the anti-galactoglobulin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments thereof) sequence identity with SEQ ID NO: 23. In one embodiment, the anti-galactoglobulin-9 antibody comprises the heavy chain sequence containing SEQ ID NO: 23. In one embodiment, the anti-galactoglobulin-9 antibody comprises the heavy chain sequence consisting of SEQ ID NO: 23.

[0134] In any of these embodiments, the anti-galactoglobulin-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 therewith) sequence identity with SEQ ID NO: 15. In some embodiments, the anti-galactoglobulin-9 antibody comprises a light chain sequence containing SEQ ID NO: 15. In some embodiments, the anti-galactoglobulin-9 antibody comprises a light chain sequence consisting of SEQ ID NO: 15.

[0135] In a specific example, the anti-galactoglobulin-9 antibody used in the treatment methods disclosed herein has the heavy chain of SEQ ID NO: 19 and the light chain of SEQ ID NO: 15. In some embodiments, the anti-galactoglobulin-9 antibody used in the treatment methods disclosed herein is G9.2-17 IgG4.

[0136] Preparation of anti-galactophlebin-9 antibody

[0137] The antibodies described herein capable of binding galactagogue-9 can be prepared by any method known in the art, including but not limited to recombinant techniques. An example is provided below.

[0138] The nucleic acids encoding the heavy and light chains of the anti-galactoglobulin-9 antibody described herein can be cloned into the same expression vector, with each nucleotide sequence operatively linked to a suitable promoter. In one example, each nucleotide sequence encoding the heavy and light chains is operatively linked to a different promoter. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operatively linked to a single promoter, such that both the heavy and light chains are expressed by the same promoter. If necessary, an internal ribosome entry site (IRES) can be inserted between the heavy and light chain coding sequences.

[0139] In some examples, the nucleotide sequences encoding the two chains of an antibody are cloned into two vectors, which can be introduced into the same or different cells. When the two chains are expressed in different cells, each of them can be isolated from the host cell expressing them, and the separated heavy and light chains can be mixed and incubated under suitable conditions that allow for antibody formation.

[0140] Typically, a nucleic acid sequence encoding one or all strands of an antibody can be cloned into a suitable expression vector and operatively ligated to a suitable promoter using methods known in the art. For example, the nucleotide sequence and the vector can be contacted with a restriction enzyme under suitable conditions to generate complementary ends on each molecule, which can then pair with each other and be linked together by a ligase. Alternatively, synthetic nucleic acid adapters can be ligated to the ends of a gene. These synthetic adapters contain nucleic acid sequences corresponding to specific restriction sites in the vector. The choice of expression vector / promoter will depend on the type of host cell used to produce the antibody.

[0141] Various promoters can be used to express the antibodies described herein, including but not limited to the cytomegalovirus (CMV) immediate early promoter and viral LTRs such as those for Rouss sarcoma (CRS). Rous sarcoma The promoters include viral LTR, HIV-LTR, HTLV-1 LTR, simian virus 40 (SV40) early promoter, Escherichia coli lac UV5 promoter, and herpes simplex virus TK promoter.

[0142] Adjustable promoters can also be used. Such adjustable promoters include those that use lac repressors from *E. coli* as transcriptional regulators to regulate transcription from mammalian cell promoters carrying lac operator genes [Brown, M. et al., ...]. Cell [49:603-612 (1987)], using those promoters of tetracycline repressors (tetR) [Gossen, M., and Bujard, H., , 49:603-612 (1987)], Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al. Human Gene Therapy , 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA [92:6522-6526 (1995)]. Other systems include FK506 dimer, VP16 or p65 using estradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech, and Ariad.

[0143] A regulated promoter comprising an operon repressor can be used. In one embodiment, a lac repressor derived from *E. coli* can act as a transcriptional regulator to control transcription from a mammalian cell promoter carrying the lac operator gene (M. Brown et al.). Cell , 49:603-612 (1987); Gossen and Bujard (1992); M. Gossen et al., Natl. Acad. Sci. USA , 89:5547-5551 (1992)), combining a tetracycline repressor (tetR) with a transcription activator (VP 16) to generate the tetR-mammalian cell transcription activator fusion protein tTa (tetR-VP 16), using a minimal promoter with tetO derived from the major immediate early promoter of human cytomegalovirus (hCMV) to form a tetR-tet operator gene system to control gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. When the tetracycline operator gene is correctly located downstream of the TATA element of the CMVIE promoter, the tetracycline repressor (tetR) alone, rather than the tetR-mammalian cell transcription factor fusion derivative, can act as an effective trans-regulator to regulate gene expression in mammalian cells (Yao et al., 89:5547-5551 (1992)). Human Gene Therapy , 10(16):1392-1399 (2003)). A particular advantage of this tetracycline-inducible switch is that it does not require the use of tetracycline repressors—mammalian cell transactivators or repressor fusion proteins—which may be cellularly toxic in some cases (Gossen et al., 10(16):1392-1399 (2003)). Natl. Acad. Sci. USA , 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA , 92:6522-6526 (1995)), in order to achieve its controllable effect.

[0144] In addition, the vector may contain some or all of the following: optional marker genes, such as the neomycin gene for selecting stable or transient transfectants in mammalian cells; enhancer / promoter sequences from human CMV immediate early genes for high-level transcription; transcription termination and RNA processing signals from SV40 for stabilizing mRNA; SV40 multitumor origin of replication and ColE1 for appropriate episome replication; internal ribosome binding site (IRES), multifunctional multiple cloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.

[0145] Examples of polyadenylation signals that can be used to implement the methods described herein include, but are not limited to, human collagen I polyadenylation signals, human collagen II polyadenylation signals, and SV40 polyadenylation signals.

[0146] One or more vectors (e.g., expression vectors) containing nucleic acids encoding any antibody can be introduced into suitable host cells to produce antibodies. Host cells can be cultured under conditions suitable for expressing the antibody or any of its polypeptide chains. Such antibodies or their polypeptide chains can be recovered from cultured cells (e.g., from cells or culture supernatant) using conventional methods such as affinity purification. If necessary, the antibody polypeptide chain can be incubated under suitable conditions for a suitable period of time to produce antibodies.

[0147] In some embodiments, the methods for preparing the antibodies described herein involve a recombinant expression vector encoding the heavy and light chains of an anti-galactophage-9 antibody, which is also described herein. The recombinant expression vector can be introduced into suitable host cells (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Positive transformant host cells can be selected and cultured under suitable conditions that allow expression of the two polypeptide chains that form the antibody; the antibody can be recovered from the cells or from the culture medium. If necessary, the two chains recovered from the host cells can be incubated under conditions suitable for antibody formation.

[0148] In one example, two recombinant expression vectors are provided: one encoding a heavy chain of an anti-galactocyanin-9 antibody, and the other encoding a light chain of an anti-galactocyanin-9 antibody. Both recombinant expression vectors can be introduced into suitable host cells (e.g., dhfr-CHO cells) via conventional methods such as calcium phosphate-mediated transfection. Alternatively, each expression vector can be introduced into a suitable host cell. Positive transformants can be selected and cultured under suitable conditions that allow for antibody polypeptide chain expression. When both expression vectors are introduced into the same host cell, the antibody produced can be recovered from the host cell or from the culture medium. If desired, the polypeptide chain can be recovered from the host cell or culture medium and then incubated under suitable conditions that allow for antibody formation. When the two expression vectors are introduced into different host cells, each can be recovered from the respective host cell or from the respective culture medium. The two polypeptide chains can then be incubated under suitable conditions to form antibodies.

[0149] Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture host cells, and recover antibodies from the culture medium. For example, some antibodies can be separated using protein A or protein G conjugated matrix via affinity chromatography.

[0150] Any nucleic acid encoding the heavy chain, light chain, or both of the anti-galactoglobulin-9 antibody as described herein, a vector (e.g., an expression vector) containing such a vector; and a host cell containing such a vector within the scope of this disclosure.

[0151] The anti-galactoglobulin-9 antibody thus prepared can be characterized using methods known in the art, thereby detecting and / or measuring the reduction, improvement, or neutralization of galactoglobulin-9 biological activity. For example, in some embodiments, ELISA-type assays are suitable for qualitative or quantitative measurement of the inhibition of Dectin-1 or TIM-3 signaling by galactoglobulin-9.

[0152] The bioactivity of anti-galactoglobulin-9 antibodies can be verified by incubating the candidate antibody with Dectin-1 and galactoglobulin-9 and monitoring any one or more of the following characteristics: (a) binding between Dectin-1 and galactoglobulin-9 and inhibition of binding-mediated signal transduction; (b) prevention, improvement, or treatment of any aspect of solid tumors; (c) blocking or reducing Dectin-1 activation; (d) inhibition (reduction) of the synthesis, production, or release of galactoglobulin-9. Alternatively, TIM-3 can be used to verify the bioactivity of anti-galactoglobulin-9 antibodies using the above protocol. Alternatively, CD206 can be used to verify the bioactivity of anti-galactoglobulin-9 antibodies using the above protocol.

[0153] In some implementations, bioactivity or efficacy is assessed in subjects, for example, by measuring the ratio of peripheral and intratumoral T cells, T cell activation, or by macrophage phenotype analysis.

[0154] Other assays for determining the bioactivity of anti-galactoglobulin-9 antibodies include measuring CD8+ and CD4+ (conventional) T cell activation (in vitro or in vivo assays, for example, by measuring levels of inflammatory cytokines such as IFNγ, TNFα, CD44, ICOS granzyme B, perforin, IL2 (upregulated); CD26L and IL-10 (downregulated)); and measuring macrophage reprogramming (in vitro or in vivo), such as from the M2 to M1 phenotype (e.g., increased MHCII, decreased CD206, increased TNF-α and iNOS). Alternatively, ADCC levels may be assessed, for example, in vitro assays as described herein.

[0155] Pharmaceutical Composition

[0156] Anti-galactoglobulin-9 antibodies, as described herein, as well as carriers encoding nucleic acids or nucleic acid sequences, or host cells containing such carriers, can be mixed with pharmaceutically acceptable excipients to form pharmaceutical compositions for treating a target disease. "Acceptable" means that the carrier must be compatible with (and preferably, capable of stabilizing) the active ingredient of the composition and harmless to the subject being treated. Pharmaceutically acceptable excipients (carriers) include buffers well known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Areiams and Wilkins, Ed. KE Hoover.

[0157] The pharmaceutical compositions used in this method may comprise pharmaceutically acceptable carriers, excipients, or stabilizers in lyophilized or aqueous forms (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Areiams and Wilkins, Ed. KE Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and contain buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); and low molecular weight (less than about 10). (1 residue) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextran; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; counterions that form salts, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as TWEEN. TM PLURONICS TMOr polyethylene glycol (PEG). In some examples, the pharmaceutical compositions described herein comprise liposomes containing antibodies (or encoding nucleic acids), which can be prepared by methods known in the art, such as Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and as described in U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced cycle time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be produced by reverse-phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter defining the pore size to produce liposomes with the desired diameter.

[0158] In some embodiments, anti-galactogen-9 antibodies or encoding nucleic acids are encapsulated in microcapsules, such as those prepared by coagulation techniques or interfacial polymerization, for example, hydroxymethyl cellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in crude emulsions. Such techniques are known in the art; see, for example, Remington, *The Science and Practice of Pharmacy*, 20th Ed. Mack Publishing (2000).

[0159] In other examples, the pharmaceutical compositions described herein may be formulated into sustained-release forms. Suitable examples of sustained-release preparations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, in the form of a molded article, such as a film or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (US Patent No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamic acid ester, non-degradable ethylene-vinyl acetate, and degradable lactic-glycolic acid copolymers such as LUPRON DEPOT. TM (Injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose isobutyrate acetate and poly-D-(-)-3-hydroxybutyric acid.

[0160] Pharmaceutical compositions intended for internal administration must be sterile. This can be easily achieved through filtration, such as via a sterile filter membrane. Therapeutic antibody compositions are typically placed in containers with sterile access openings, such as intravenous bags or vials with stoppers that can be punctured by a hypodermic needle.

[0161] The pharmaceutical compositions described herein may be in unit dosage forms, such as tablets, pills, capsules, powders, granules, solutions, suspensions, or suppositories for oral, parenteral, or rectal administration, or for administration by inhalation or blowing.

[0162] To prepare solid compositions such as tablets, the main active ingredient can be mixed with a pharmaceutical carrier, such as conventional tableting ingredients like corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gum, and other pharmaceutical diluents such as water, to form a solid pre-formulation composition containing a homogeneous mixture of the compound of the present invention or a non-toxic, pharmaceutically acceptable salt thereof. When these pre-formulation compositions are referred to as homogeneous, it means that the active ingredient is uniformly dispersed throughout the composition, allowing the composition to be easily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. The solid pre-formulation composition is then subdivided into unit dosage forms of the type described above, containing 0.1 to approximately 500 mg of the active ingredient of the present invention. Tablets or pills of the new composition can be coated or otherwise compounded to provide a dosage form with sustained-action advantages. For example, tablets or pills may contain an internal dose and an external dose component, the latter encapsulated on top of the former. These two components can be separated by an enteric coating layer, which resists breakdown in the stomach and allows the internal component to enter the duodenum intact or with delayed release. A variety of materials can be used for such enteric coatings or coatings, including various polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate. Suitable surfactants include, in particular, nonionic agents such as polyoxyethylene sorbitan anhydride (e.g., Tween). TM 20, 40, 60, 80 or 85) and other sorbitan anhydrides (e.g., Span TM (20, 40, 60, 80, or 85). Compositions containing surfactants conveniently contain 0.05% to 5%, and may be 0.1% to 2.5%, of the surfactant. It is understood that other ingredients, such as mannitol or other pharmaceutically acceptable mediators, may be added if desired.

[0163] Suitable emulsions can be prepared using commercially available fat emulsions, such as Intralipid. TM Liposyn TM Infonutrol TM Lipofundin TM and Lipiphysan TMThe active ingredient may be soluble in the premixed emulsion composition, or alternatively, it may be soluble in oils (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and mixed with phospholipids (e.g., lecithin, soybean lecithin, or soybean phospholipid) and water to form an emulsion. It is understood that other ingredients (e.g., glycerol or glucose) may be added to adjust the emulsion tension. Suitable emulsions typically contain up to 20% oil, such as 5% to 20%. Fat emulsions may contain 0.1 to 1.0 μm, particularly 0.1 to 0.5 μm, fat droplets and have a pH range of 5.5 to 8.0.

[0164] The emulsion composition can be made by combining antibodies with Intralipids TM Those prepared by mixing with its components (soybean oil, lecithin, glycerin and water).

[0165] Pharmaceutical compositions for inhalation or inhalation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the composition is administered via oral or nasal inhalation to produce local or systemic effects.

[0166] The composition, preferably in a sterile, pharmaceutically acceptable solvent, can be nebulized using a gas. The nebulized solution can be inhaled directly from a nebulizer, or the nebulizer can be connected to a mask, tent, or intermittent positive pressure ventilation machine. The solution, suspension, or powder composition can be administered from a device that delivers the formulation in a suitable manner, preferably orally or nasally.

[0167] Combination cancer therapy

[0168] This disclosure provides a method for using any anti-galactagogue antibody such as G9.2-17 (e.g., G9.2-17(IgG4)) in combination with one or more chemotherapeutic agents such as gemcitabine and / or paclitaxel (e.g., Abraxane®) to treat solid tumors such as PDAC, colorectal cancer (CRC), hepatocellular carcinoma (HCC), or cholangiocarcinoma (CAA).

[0169] Unbound by theory, it is believed that anti-galactotropic 9 antibodies reprogram the immune response against tumor cells through their inhibition of Dectin-1, via, for example, by inhibiting the infiltration of β-galactotropic enzymes into the tumor microenvironment. T cell activity and / or enhanced immune surveillance against tumor cells, for example, by activating CD4+ and / or CD8+ T cells. Therefore, the combined use of anti-galactoside-9 antibodies with one or more chemotherapeutic agents as described herein is expected to significantly enhance antitumor efficacy.

[0170] Pancreatic ductal adenocarcinoma (PDA) is a devastating disease with few long-term survivors (Yadav et al., Gastroenterology (Guerra et al., 2013, 144, 1252-1261). Inflammation is crucial in the progression of PDA because, in the absence of accompanying inflammation, oncogenic mutations alone are insufficient to lead to tumorigenesis (Guerra et al., 2013, 144, 1252-1261). Cancer Cell 2007, 11, 291-302). Innate and adaptive immunity synergistically promote tumor progression in PDA. In particular, specific innate immune subsets within the tumor microenvironment (TME) readily culture adaptive immune effector cells into tumor-permissive phenotypes. Antigen-presenting cell (APC) populations, including M2-polarized tumor-associated macrophages (TAMs) and myeloid dendritic cells (DCs), induce the generation of immunosuppressive Th2 cells that favor tumor-protective Th1 cells (Ochi et al., 2007, 11, 291-302). J of Exp Med., 2012, 209, 1671-1687; Zhu et al., Cancer Res., 2014, 74, 5057-5069). Similarly, myeloid-derived suppressor cells (MDSCs) have been shown to eliminate anti-tumor CD8 in PDA. + Cytotoxic T lymphocyte (CTL) responses promote the progression of metastasis (Connolly et al., J Leuk Biol., 2010, 87, 713-725; Pylayeva-Gupta et al., Cancer Cell 2012, 21, 836-847; Bayne et al., Cancer Cell 2012, 21, 822-835).

[0171] Colorectal cancer (CRC), also known as bowel cancer, colon cancer, or rectal cancer, is any cancer that affects the colon and rectum. CRC is known to be driven by genetic alterations in tumor cells and is also influenced by tumor-host interactions. Recent reports have shown a direct correlation between the density of certain T lymphocyte subsets and favorable clinical outcomes in CRC, supporting the major role of T cell-mediated immunity in suppressing CRC tumor progression.

[0172] Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. HCC most frequently occurs in people with chronic liver diseases, such as cirrhosis caused by hepatitis B or hepatitis C infection. HCC is often accompanied by cirrhosis with extensive lymphocytic infiltration due to chronic viral infection. Many studies have shown that tumor-invasive effector CD8+ T cells and T helper 17 (Th17) cells are associated with improved survival after surgical resection of the tumor. However, tumor-invasive effector T cells cannot control tumor growth and metastasis (Pang et al., Cancer Immunol Immunother 2009;58:877-886).

[0173] Cholangiocarcinoma is a group of cancers that originate in the bile ducts. Cholangiocarcinomas are typically classified according to their location relative to the liver. For example, intrahepatic cholangiocarcinomas, which account for less than 10% of all cholangiocarcinoma cases, begin in small bile ducts within the liver. In another example, hilar cholangiocarcinomas (also known as Klatskin tumors), which account for more than half of all cholangiocarcinoma cases, begin at the porta hepatis, where two major bile ducts connect and exit the liver. Others are classified as distal cholangiocarcinomas, which begin in bile ducts outside the liver.

[0174] In some aspects, this disclosure provides methods for treating solid tumors, such as those disclosed herein. In some embodiments, this disclosure provides methods for reducing, improving, or eliminating one or more symptoms associated with solid tumors. The treatment methods disclosed herein involve combination therapy of an anti-Gal9 antibody, such as G9.2-17, and one or more chemotherapeutic agents. In some examples, an effective amount of an anti-Gal9 antibody is given to a subject with a solid tumor (e.g., PDAC) who is receiving treatment involving one or more chemotherapeutic agents. In some examples, an effective amount of one or more chemotherapeutic agents is given to a subject with a solid tumor (e.g., PDAC) who is receiving treatment involving an anti-Gal9 antibody. In other examples, an effective amount of an anti-Gal9 antibody and an effective amount of one or more chemotherapeutic agents are given to a subject simultaneously or sequentially.

[0175] In some embodiments, the methods of this disclosure increase antitumor activity (e.g., reduction in cell proliferation, tumor growth, tumor volume and / or tumor burden or load, or reduction in 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 higher compared to pre-treatment or control levels in the subject. In some embodiments, the reduction is measured by comparing cell proliferation, tumor growth, and / or tumor volume in the subject before and after administration of the pharmaceutical composition. In some embodiments, the method of treating or improving cancer in the subject allows for improvement of one or more symptoms of cancer by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher. In some embodiments, cancer cells and / or biomarkers in the subject are measured in biological samples, such as blood, serum, plasma, urine, peritoneal fluid, and / or biopsies from tissues or organs, before, during, and after administration of the pharmaceutical composition. In some embodiments, the method includes administering the composition of the invention to reduce the tumor volume, size, load, or burden in a subject to an undetectable size, or to a reduction of about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the pre-treatment tumor volume, size, load, or burden in the subject. In other embodiments, the method includes administering the composition of the invention to reduce the cell proliferation rate or tumor growth rate in a subject to an undetectable rate, or to a reduction of about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the pre-treatment rate. In other embodiments, the method includes administering the composition of the invention to reduce the development, number, or size of metastatic lesions in a subject to an undetectable rate, or to a rate less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the pre-treatment rate.

[0176] The term "about" or "approximately" refers to a specific value within an acceptable range of error as determined by a person skilled in the art, depending in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" may mean within an acceptable standard deviation. Alternatively, "about" may mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and even more preferably up to ±1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude of the value, preferably within twice that. Where a specific value is described in this application and claims, the term "about" is implied unless otherwise stated and herein means within an acceptable range of error for the specific value.

[0177] As used herein, the term “treatment” means the application or administration of a composition comprising one or more active agents to a subject having a target disease or condition, symptoms of the disease or condition, or susceptibility to the disease or condition, with the aim of curing, restoring, alleviating, reducing, altering, remedying, improving, refining, or influencing the condition, symptoms of the disease or condition, or susceptibility to the disease or condition.

[0178] Relieving a target disease / symptom involves delaying the development or progression of the disease, or reducing its severity or prolonging survival. Disease relief or prolonged survival does not necessarily require a curative outcome. As used herein, “delaying” the development of a target disease or symptom means delaying, hindering, slowing, stabilizing, and / or postponing the progression of the disease. This delay can vary in length depending on the patient’s medical history and / or the individual’s treatment. Methods of “delaying” or mitigating disease development or delaying the onset of the disease include reducing the likelihood of developing one or more symptoms of the disease within a given timeframe and / or reducing the severity of symptoms within a given timeframe, compared to not using such methods. This comparison is typically based on clinical studies using a sufficient number of participants to yield statistically significant results.

[0179] The term "development" or "progression" of a disease refers to the initial presentation and / or subsequent progression of the disease. The development of a disease can be detected and assessed using standard clinical techniques well known in the art. However, development also refers to progression that may be undetectable. For the purposes of this disclosure, development or progression refers to the biological process of symptoms. "Development" includes occurrence, relapse, and onset. As used herein, an "onset" or "occurrence" of a target disease or condition includes an initial onset and / or relapse.

[0180] In some embodiments, the antibody described herein, such as G9.2-17, for example in its IgG4 form, is administered to the subject in need of treatment in an amount sufficient to inhibit the activity of galactagogue-9 (and / or Dectin-1 or TIM-3 or CD206) in tumor immunosuppressive immune cells by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher) in vivo. In other embodiments, the antibody described herein, such as G9.2-17, is administered in an amount that effectively reduces the activity level of galactagogue-9 (and / or Dectin-1 or TIM-3 or CD206) in tumor immunosuppressive immune cells by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher) (compared to the level in the subject before treatment or in a control subject). In some implementations, the antibody described herein, such as G9.2-17, is administered to the subject in need of treatment in an amount sufficient to promote at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher) of M1-like programming in TAM in vivo (compared to levels in pre-treatment or control subjects).

[0181] Depending on the type or location of the disease to be treated, the pharmaceutical composition may be administered to the subject using conventional methods known to those skilled in the art. In some embodiments, the anti-galactogenin-9 antibody may be administered to the subject via intravenous infusion.

[0182] Injectable compositions may contain various carriers such as vegetable oils, dimethyl lactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies can be administered via infusion, thereby infusing a pharmaceutical preparation containing antibodies and physiologically acceptable excipients. Physiologically acceptable excipients may include, for example, 5% glucose, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular preparations, such as sterile formulations in the form of suitable soluble salts of antibodies, can be dissolved and administered in pharmaceutical excipients (e.g., water for injection, 0.9% saline, or 5% glucose solution).

[0183] In some embodiments, a method is provided for the simultaneous administration of an anti-galactoglobulin-9 antibody with one or more chemotherapeutic agents. In some embodiments, the anti-galactoglobulin-9 antibody is administered before or after one or more chemotherapeutic agents. In some embodiments, one or more chemotherapeutic agents are administered systemically. In some embodiments, one or more chemotherapeutic agents are administered locally. In some embodiments, one or more chemotherapeutic agents are administered via intravenous administration, such as a bolus injection or by continuous infusion over a period of time, via intramuscular, intraperitoneal, intraspinal, subcutaneous, intraarticular, intra-articular, intrasynovial, intrathecal, intratumoral, oral, inhalation, or local routes. In one embodiment, one or more chemotherapeutic agents are administered to the subject via intravenous infusion.

[0184] An effective amount of the pharmaceutical composition described herein may be administered systemically or locally to a subject requiring treatment (e.g., a human) via a suitable route. In some embodiments, the anti-galactogenin-9 antibody is administered intravenously, such as by bolus injection or by continuous infusion over a period of time, via intramuscular, intraperitoneal, intraspinal, subcutaneous, intraarticular, intra-articular, intrasynovial, intrathecal, intratumoral, oral, inhalation, or local routes. In one embodiment, the anti-galactogenin-9 antibody is administered to the subject via intravenous infusion.

[0185] As used herein, “effective amount” means the amount of each active agent required, alone or in combination with one or more other active agents, to confer a therapeutic effect on a subject. In some embodiments, the therapeutic effect is a reduction in galactagogue-9 activity and / or amount / expression, a reduction in Dectin-1 signaling, a reduction in TIM-3 signaling, a reduction in CD206 signaling, or an increase in antitumor immune responses in the tumor microenvironment. Non-limiting examples of increased antitumor responses include increased levels of effector T cell activation, or a TAM phenotype transition from M2 to M1. In some cases, the antitumor response includes an increased ADCC response. Determining whether a given amount of antibody achieves a therapeutic effect will be apparent to those skilled in the art. As will be appreciated by those skilled in the art, the effective amount varies with the specific condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex, and weight, the duration of treatment, the nature of any concurrent therapy, the specific route of administration, and similar factors within the knowledge and expertise of a medical practitioner. These factors are well known to those skilled in the art and can be resolved using only routine laboratory methods. It is generally preferred to use the maximum dose of a single component or its combination, that is, the highest safe dose based on reasonable medical judgment.

[0186] Empirical considerations, such as half-life, often help determine dosage. For example, antibodies compatible with the human immune system, such as humanized antibodies or intact human antibodies, are used in some cases to prolong the antibody's half-life and prevent the antibody from being attacked by the host immune system. The frequency of administration can be determined and adjusted during treatment and is generally, but not necessarily, based on the treatment and / or suppression and / or improvement and / or delay of the target disease / symptom. Alternatively, a sustained-release formulation of the antibody may be suitable. Various formulations and devices for achieving sustained release are known in the art.

[0187] In one example, the antibody dosage, as described herein, is determined empirically in individuals who have received one or more antibody administrations. Individuals are given escalating doses of the antagonist. To assess the efficacy of the antagonist, indicators of disease / symptom can be tracked.

[0188] Any anti-galactoglobulin-9 antibody described herein may be used in any of the methods described herein. In some embodiments, the anti-galactoglobulin-9 antibody is G9.2-17. The G9.2-17 antibody may be an IgG4 molecule (G9.2-17 (IgG4)) as disclosed herein. In a specific example, the anti-galactoglobulin-9 antibody (G9.2-17) used herein has the heavy chain of SEQ ID NO: 19 and the light chain of SEQ ID NO: 15. The anti-Gal9 antibody may be formulated as disclosed herein and administered to a subject in need of treatment via a suitable route, such as intravenous infusion.

[0189] In some cases, anti-galactoglobulin-9 antibodies (e.g., G9.2-17) disclosed herein may be administered to subjects at appropriate doses, such as from about 0.5 to about 32 mg / kg. Examples include 0.5 mg / kg to 1 mg / kg, 1 mg / kg to 2 mg / kg, 2 mg / kg to 3 mg / kg, 3 mg / kg to 4 mg / kg, 4 mg / kg to 8 mg / kg, 8 mg / kg to 12 mg / kg, 12 mg / kg to 16 mg / kg, 16 mg / kg to 20 mg / kg, 20 mg / kg to 24 mg / kg, 24 mg / kg to 28 mg / kg, or 28 mg / kg to 32 mg / kg (e.g., 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, etc.). The dosage ranges from 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, or 32 mg / kg, or any escalating dose within these ranges. In some embodiments, the antibody is administered at a dose of approximately 0.5 mg / kg. mg / kg to 1 mg / kg, about 1 mg / kg to 2 mg / kg, about 2 mg / kg to 4 mg / kg, about 4 mg / kg to 8 mg / kg, about 8 mg / kg to 12 mg / kg, about 12 mg / kg to 16 mg / kg, about 16 mg / kg to 20 mg / kg, about 20 mg / kg to 24 mg / kg, about 24 mg / kg to 28 mg / kg, or about 28 mg / kg to 32 mg / kg (e.g., about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, 7 mg / kg, about 8 mg / kg). / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, about 20 mg / kg, about 21 mg / kg, about 22 mg / kg, about 23 mg / kg, about 24 mg / kg, about 25 mg / kg, about 26 mg / kg, about 27 mg / kg, about 28 mg / kg, about 29 mg / kg, about 30 mg / kg, about 31 mg / kg or about 32 mg / kg) or any escalating dose within these ranges.

[0190] In some embodiments, galactoglobulin-9 antibody is administered at 2 mg / kg. In some embodiments, galactoglobulin-9 antibody is administered at 4 mg / kg. In some embodiments, galactoglobulin-9 antibody is administered at 8 mg / kg. In some embodiments, galactoglobulin-9 antibody is administered at 12 mg / kg. In some embodiments, galactoglobulin-9 antibody is administered at 16 mg / kg. In some cases, multiple doses of anti-galactoglobulin-9 antibody may be administered to the subject at appropriate intervals or cycles, such as once weekly, every 2 to 4 weeks (e.g., every 2, 3, or 4 weeks). Treatment may continue for an appropriate duration, such as up to 3 months, up to 6 months, or up to 12 months, or up to 24 months or longer.

[0191] In some examples, anti-galactoglobulin-9 antibody was administered intravenously at a dose of approximately 3 mg / kg every 2 weeks to human patients with the solid tumors disclosed herein (e.g., PDA). In other examples, anti-galactoglobulin-9 antibody was administered intravenously at a dose of approximately 15 mg / kg every 2 weeks to human patients with the target solid tumor.

[0192] In some examples, anti-Gal9 antibodies (e.g., G9.2-17 in IgG4 form) of about 2 mg / kg to 16 mg / kg can be administered to subjects in need of treatment every 2 weeks. In some examples, anti-Gal9 antibodies (e.g., G9.2-17 in the form of IgG4 disclosed herein, having the heavy chain of SEQ ID NO: 19 and the light chain of SEQ ID NO: 15) were administered to subjects via intravenous injection at the following doses every 2 weeks: about 0.5 mg / kg, 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, or about 20 mg / kg or any increment thereof.

[0193] In some examples, anti-Gal9 antibody (e.g., G9.2-17 in the IgG4 form disclosed herein, having the heavy chain of SEQ ID NO: 19 and the light chain of SEQ ID NO: 15) was administered to subjects intravenously at a dose of about 2 mg / kg every 2 weeks. In some examples, anti-Gal9 antibody was administered to subjects intravenously at a dose of about 4 mg / kg every 2 weeks. In some examples, anti-Gal9 antibody was administered to subjects intravenously at a dose of about 8 mg / kg every 2 weeks. In some examples, anti-Gal9 antibody was administered to subjects intravenously at a dose of about 12 mg / kg every 2 weeks. In some examples, anti-Gal9 antibody was administered to subjects intravenously at a dose of about 16 mg / kg every 2 weeks.

[0194] In some examples, anti-Gal9 antibodies (e.g., G9.2-17 in IgG4 form disclosed herein, having the heavy chain of SEQ ID NO: 19 and the light chain of SEQ ID NO: 15) were administered to subjects once weekly via intravenous injection at the following doses: 0.5 mg / kg to 1 mg / kg, about 1 mg / kg to 2 mg / kg, about 3 mg / kg to 4 mg / kg, about 4 mg / kg to 8 mg / kg, about 8 mg / kg to 12 mg / kg, about 12 mg / kg to 16 mg / kg, about 16 mg / kg to 20 mg / kg, about 20 mg / kg to 24 mg / kg, about 24 mg / kg to 28 mg / kg, or about 28 mg / kg to 32 mg / kg (e.g., about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 ... (mg / kg, 7mg / kg, about 8mg / kg, about 9mg / kg, about 10mg / kg, about 11mg / kg, about 12mg / kg, about 13mg / kg, about 14mg / kg, about 15mg / kg, about 16mg / kg, about 17mg / kg, about 18mg / kg, about 19mg / kg, about 20mg / kg, about 21mg / kg, about 22mg / kg, about 23mg / kg, about 24mg / kg, about 25mg / kg, about 26mg / kg, about 27mg / kg, about 28mg / kg, about 29mg / kg, about 30mg / kg, about 31mg / kg, or about 32mg / kg) or any increment thereof.

[0195] In some examples, anti-Gal9 antibodies (e.g., G9.2-17 in IgG4 form disclosed herein, having the heavy chain of SEQ ID NO: 19 and the light chain of SEQ ID NO: 15) were administered to subjects once weekly via intravenous injection at the following doses: 0.5 mg / kg to 1 mg / kg, 1 mg / kg to 2 mg / kg, 3 mg / kg to 4 mg / kg, 4 mg / kg to 8 mg / kg, 8 mg / kg to 12 mg / kg, 12 mg / kg to 16 mg / kg, 16 mg / kg to 20 mg / kg, 20 mg / kg to 24 mg / kg, 24 mg / kg to 28 mg / kg, or 28 mg / kg to 32 mg / kg (e.g., 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, etc.). (kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, or 32 mg / kg) or any increment within these ranges or any increase in dose within these ranges.

[0196] In some examples, anti-Gal9 antibodies (e.g., G9.2-17 in the IgG4 form disclosed herein, having the heavy chain of SEQ ID NO: 19 and the light chain of SEQ ID NO: 15) are administered to subjects once weekly via intravenous injection at the following doses: 0.5 mg / kg, 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, or about 20 mg / kg or any increment thereof.

[0197] In some implementations, the interval or cycle is 1 week. In other implementations, the interval or cycle is 2 weeks. In some implementations, the regimen is 1 cycle every 2 weeks, 2 cycles every 2 weeks, 3 cycles every 2 weeks, 4 cycles every 2 weeks, or more than 4 cycles every 2 weeks. In some implementations, the treatment is 1 to 3 months every 2 weeks, 3 to 6 months every 2 weeks, 6 to 12 months every 2 weeks, or 12 to 24 months or longer every 2 weeks.

[0198] In specific implementations, the interval or cycle is 3 weeks. In some implementations, the regimen is one cycle every 3 weeks, two cycles every 3 weeks, three cycles every 3 weeks, four cycles every 3 weeks, or more than four cycles every 3 weeks. In some implementations, treatment is administered every 3 weeks for 1 to 3 months, every 3 weeks for 3 to 6 months, every 3 weeks for 6 to 12 months, or every 3 weeks for 12 to 24 months or longer.

[0199] In specific implementations, the interval or cycle is 4 weeks or more. In some implementations, the regimen is one cycle every 4 weeks or more, two cycles every 4 weeks or more, three cycles every 4 weeks or more, four cycles every 4 weeks or more, or more than four cycles every 4 weeks or more. In some implementations, treatment is every 4 weeks or more for 1 to 3 months, every 4 weeks or more for 3 to 6 months, every 4 weeks or more for 6 to 12 months, or every 4 weeks or more for 12 to 24 months or longer. In some implementations, treatment is a combination of treatments at different times, such as a combination of 2 weeks, 3 weeks, 4 weeks, or more than 4 weeks. In some implementations, the treatment interval is adjusted based on the patient's response to treatment. In some implementations, the dose is adjusted based on the patient's response to treatment. In some implementations, the dose varies between treatment intervals. In some implementations, treatment may be temporarily discontinued. In some implementations, anti-galactotropic-9 therapy is temporarily discontinued. In some implementations, chemotherapy is temporarily discontinued. In some implementations, both are temporarily discontinued. In any of these embodiments, the anti-Gal9 antibody may be G9.2-17 in the form of IgG4 as disclosed herein, having a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 15.

[0200] One or more chemotherapeutic agents may contain antimetabolites, microtubule inhibitors, or combinations thereof. Antimetabolites include, for example, folate antagonists (e.g., methotrexate) and nucleotide analogues such as pyrimidine antagonists (e.g., 5-fluorouracil, foxuridine, cytarabine, capecitabine, and gemcitabine), purine antagonists (e.g., 6-mercaptopurine and 6-thioguanine), and adenosine deaminase inhibitors (e.g., cladribine, fludarabine, and pentostatin).

[0201] In some examples, the antimetabolite used in the methods disclosed herein is gemcitabine, which can be administered via intravenous infusion. The amount of gemcitabine administered to the subject depends on many factors, including height and weight, general health or other health problems, and the type of cancer to be treated, within the knowledge of a medical practitioner, based on guidance provided by the Food and Drug Administration (e.g., see the drug label of the approved gemcitabine product). In some examples, it can be administered via intravenous infusion at a dose of 1000 mg / m². 2 Gemcitabine is administered to the subject at a dose that is optionally administered once weekly over 30 minutes for up to 7 weeks, followed by a one-week rest period. Subsequent cycles may consist of weekly infusions for three consecutive weeks over four weeks. If one or more adverse reactions occur, the dose of gemcitabine may be reduced or treatment may be discontinued. Further details on managing adverse reactions associated with gemcitabine treatment are provided in Example 2 below.

[0202] Microtubule inhibitors are a class of compounds that inhibit the formation of cellular microtubules, thereby blocking cell proliferation. In some examples, microtubule inhibitors are stabilizers that promote microtubule polymerization. Examples include taxane and epothilone. In other examples, microtubule inhibitors are destabilizers that promote microtubule depolymerization. Examples include vinca alkaloids. In some examples, the microtubule inhibitor used in the methods disclosed herein is paclitaxel. In some cases, paclitaxel is in its free form. In other cases, paclitaxel is conjugated to proteins such as albumin. In a specific example, paclitaxel is Abraxane®, a nanoparticle albumin-bound paclitaxel.

[0203] The amount of paclitaxel (e.g., protein-bound paclitaxel, such as nab-paclitaxel) administered to a subject depends on many factors, including height and weight, overall health or other health problems, and the type of cancer to be treated, in accordance with guidelines provided by the Food and Drug Administration (e.g., see the drug label of approved paclitaxel products) within the knowledge of a medical practitioner. For example, when nanoparticle albumin-bound paclitaxel (nab-paclitaxel, such as Abraxane®), it can be administered at 260 mg / m² over 30 minutes every 3 weeks. 2The paclitaxel is administered to the subject via intravenous injection. If serious adverse reactions (e.g., neutropenia or severe sensory neuropathy) are observed, the dose of paclitaxel may be reduced. In some cases, the dose of nab-paclitaxel may be reduced to 180 mg / m². 2 When combined with anti-Gal9 antibody, the dosage of paclitaxel can be 125 mg / m². 2 If necessary, the dosage of paclitaxel can be reduced to 100 mg / m². 2 Or 75mg / m 2 Further details on managing adverse reactions related to paclitaxel are provided in Example 2 below.

[0204] In some specific examples, anti-Gal9 antibodies (e.g., G9.2-17 in IgG4 form), gemcitabine, and paclitaxel (e.g., nanoparticle albumin-conjugated paclitaxel or Abraxane®) may be administered to subjects requiring treatment according to the treatment and dosing regimens provided in Example 2 below. For example, treatment may comprise one or more cycles, each consisting of 28 days. In each cycle, anti-Gal9 antibodies (e.g., G9.2-17 (IgG4)) are administered to subjects (e.g., human patients with PDAC) via intravenous infusion at doses ranging from about 2 mg / kg to 16 mg / kg (e.g., about 2 mg / kg, about 4 mg / kg, about 8 mg / kg, about 12 mg / kg, or about 16 mg / kg) every two weeks (e.g., on day 1 and day 15). Using FDA-approved dosage and dosing regimens, gemcitabine and paclitaxel (e.g., protein-bound paclitaxel, such as Abraxane®) can be administered to subjects once weekly for 3 weeks, followed by a 1-week treatment-free period (e.g., on days 1, 8, and 15 of a 28-day cycle). For example, gemcitabine can be administered at 1000 mg / m² in each cycle. 2 Paclitaxel can be administered to subjects once a week via intravenous injection at a dose of 125 mg / m². 2 Administered to subjects once weekly. If necessary, the gemcitabine dose may be reduced to 800 mg / m². 2 Or 600mg / m 2 Optionally or additionally, the dose of paclitaxel may be reduced to 100 mg / m². 2 Or 75mg / m 2 .

[0205] In some embodiments, the method described herein for treating solid tumors (e.g., PDA) comprises one or more 28-day treatment cycles, wherein an anti-Gal9 antibody is administered to the subject via intravenous infusion on days 1 and 15 (i.e., every 2 weeks (q2w)) at a dose of about 0.5 mg / kg to about 32 mg / kg, and gemcitabine and paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) are administered to the subject on days 1, 8, and 15. In some examples, paclitaxel is administered at 125 mg / m². 2 Gemcitabine is administered intravenously (e.g., intravenous injection) to the subject. In some examples, gemcitabine is administered at 1000 mg / m². 2 Administered intravenously (e.g., intravenous injection) to the subject. If necessary, the gemcitabine dose may be reduced to 800 mg / m². 2 Or 600mg / m 2 Optionally or additionally, the dose of paclitaxel may be reduced to 100 mg / m². 2 Or 75mg / m 2 .

[0206] In some embodiments, the method described herein for treating solid tumors (e.g., PDA) comprises one or more 28-day treatment cycles, wherein the anti-Gal9 antibody is administered to the subject via intravenous infusion on day 1 and day 15 (i.e., every 2 weeks (q2w)) at doses including: about 0.5 mg / kg to 1 mg / kg, about 1 mg / kg to 2 mg / kg, about 3 mg / kg to 4 mg / kg, about 4 mg / kg to 8 mg / kg, about 8 mg / kg to 12 mg / kg, about 12 mg / kg to 16 mg / kg, about 16 mg / kg to 20 mg / kg, about 20 mg / kg to 24 mg / kg, about 24 mg / kg to 28 mg / kg, or about 28 mg / kg to 32 mg / kg (e.g., about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg). Approximately 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg, approximately 10 mg / kg, approximately 11 mg / kg, approximately 12 mg / kg, approximately 13 mg / kg, approximately 14 mg / kg, approximately 15 mg / kg, approximately 16 mg / kg, approximately 17 mg / kg, approximately 18 mg / kg, approximately 19 mg / kg, approximately 20 mg / kg, approximately 21 mg / kg, approximately 22 mg / kg, approximately 23 mg / kg, approximately 24 mg / kg, approximately 25 mg / kg, approximately 26 mg / kg, approximately 27 mg / kg, approximately 28 mg / kg, approximately 29 mg / kg, approximately 30 mg / kg, approximately 31 mg / kg, or approximately 32 mg / kg), as well as gemcitabine and paclitaxel (e.g., albumin-bound paclitaxel) were administered to subjects on days 1, 8, and 15. In some examples, paclitaxel was used at 125 mg / m². 2 Gemcitabine is administered intravenously (e.g., intravenous injection) to the subject. In some examples, gemcitabine is administered at 1000 mg / m². 2 Administered intravenously (e.g., intravenous injection) to the subject. If necessary, the gemcitabine dose may be reduced to 800 mg / m². 2 Or 600mg / m 2 Optionally or additionally, the dose of paclitaxel may be reduced to 100 mg / m². 2 Or 75mg / m 2 .

[0207] In some embodiments, the method of treating solid tumors (e.g., PDA) described herein comprises one or more 28-day treatment cycles, wherein the anti-Gal9 antibody is administered to the subject via intravenous infusion on day 1 and day 15 (i.e., every 2 weeks (q2w)) at doses including: 0.5 mg / kg to 1 mg / kg, 1 mg / kg to 2 mg / kg, 3 mg / kg to 4 mg / kg, 4 mg / kg to 8 mg / kg, 8 mg / kg to 12 mg / kg, 12 mg / kg to 16 mg / kg, 16 mg / kg to 20 mg / kg, 20 mg / kg to 24 mg / kg, 24 mg / kg to 28 mg / kg, or 28 mg / kg to 32 mg / kg (e.g., 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 11 mg / kg, 12 mg / kg, 12 mg / kg to 16 mg / kg, 16 mg / kg to 20 mg / kg, 20 mg / kg to 24 mg / kg, 24 mg / kg to 28 mg / kg, or 28 mg / kg to 32 mg / kg (e.g., 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 12 mg / kg, 12 mg / kg, 12 mg / kg, 12 mg / kg, 12 mg / kg, 12 mg / 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, or 32 mg / kg) or any increased doses within these ranges, and gemcitabine and paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) were administered to subjects on days 1, 8, and 15. In some examples, paclitaxel was administered at 125 mg / kg. 2 Gemcitabine is administered intravenously (e.g., intravenous injection) to the subject. In some examples, gemcitabine is administered at 1000 mg / m². 2 Administered intravenously (e.g., intravenous injection) to the subject. If necessary, the gemcitabine dose may be reduced to 800 mg / m². 2 Or 600mg / m 2 Optionally or additionally, the dose of paclitaxel may be reduced to 100 mg / m². 2 Or 75mg / m 2 .

[0208] In some embodiments, the method described herein for treating solid tumors (e.g., PDA) comprises one or more 28-day treatment cycles, wherein an anti-Gal9 antibody is administered to the subject via intravenous infusion on days 1 and 15 (i.e., every 2 weeks (q2w)) at a dose of about 2 mg / kg to about 16 mg / kg, and gemcitabine and paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) are administered to the subject on days 1, 8, and 15. In some examples, paclitaxel is administered at 125 mg / m². 2 Gemcitabine is administered intravenously (e.g., intravenous injection) to the subject. In some examples, gemcitabine is administered at 1000 mg / m². 2 Administered intravenously (e.g., intravenous injection) to the subject. If necessary, the gemcitabine dose may be reduced to 800 mg / m². 2 Or 600mg / m 2 Optionally or additionally, the dose of paclitaxel may be reduced to 100 mg / m². 2 Or 75mg / m 2 .

[0209] In some embodiments, the method described herein for treating solid tumors (e.g., PDA) comprises one or more 28-day treatment cycles, wherein the anti-Gal9 antibody is administered to the subject via intravenous infusion on day 1 and day 15 (i.e., every 2 weeks (q2w)) at the following doses: about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, or about 20 mg / kg or any increment thereof, and gemcitabine and paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) are administered to the subject on day 1, day 8, and day 15. In some examples, paclitaxel was used at 125 mg / m². 2 Gemcitabine is administered intravenously (e.g., intravenous injection) to the subject. In some examples, gemcitabine is administered at 1000 mg / m². 2 Administered intravenously (e.g., intravenous injection) to the subject. If necessary, the gemcitabine dose may be reduced to 800 mg / m². 2 Or 600mg / m 2 Optionally or otherwise, the dose of paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) can be reduced to 100 mg / m². 2 Or 75mg / m 2 .

[0210] In some implementations, the method includes one or more 28-day treatment cycles, wherein

[0211] (1) Anti-Gal9 antibody was administered to subjects via intravenous infusion on days 1 and 15 (i.e., every 2 weeks (q2w)) at the following doses: approximately 0.5 mg / kg to 1 mg / kg, approximately 1 mg / kg to 2 mg / kg, approximately 3 mg / kg to 4 mg / kg, approximately 4 mg / kg to 8 mg / kg, approximately 8 mg / kg to 12 mg / kg, approximately 12 mg / kg to 16 mg / kg, approximately 16 mg / kg to 20 mg / kg, approximately 20 mg / kg to 24 mg / kg, approximately 24 mg / kg to 28 mg / kg, or approximately 28 mg / kg to 32 mg / kg (e.g., approximately 0.5 mg / kg, approximately 1 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 ...1 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 (approximately 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, or 32 mg / kg) or any increment thereof.

[0212] (2) Gemcitabine was administered at a dose of 1000 mg / m² on days 1, 8, and 15. 2 The dose was administered to the subject intravenously (e.g., intravenous injection).

[0213] (3) Paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) was administered at 125 mg / m² on days 1, 8, and 15. 2 The dose is administered intravenously (e.g., intravenous injection) to the subject.

[0214] In some implementations, the method includes one or more 28-day treatment cycles, wherein

[0215] (1) Anti-Gal9 antibody was administered to subjects via intravenous infusion on days 1 and 15 (i.e., every 2 weeks (q2w)) at the following doses: 0.5 mg / kg to 1 mg / kg, 1 mg / kg to 2 mg / kg, 3 mg / kg to 4 mg / kg, 4 mg / kg to 8 mg / kg, 8 mg / kg to 12 mg / kg, 12 mg / kg to 16 mg / kg, 16 mg / kg to 20 mg / kg, 20 mg / kg to 24 mg / kg, 24 mg / kg to 28 mg / kg, or 28 mg / kg to 32 mg / kg (e.g., 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg to 20 mg / kg, 20 mg / kg to 24 mg / kg, 24 mg / kg to 28 mg / kg, or 28 mg / kg to 32 mg / kg). (mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, or 32 mg / kg) or any increment thereof,

[0216] (2) Gemcitabine was administered at a dose of 1000 mg / m² on days 1, 8, and 15. 2 The dose is administered intravenously (e.g., intravenous injection) to the subject.

[0217] (3) Paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) was administered at 125 mg / m² on days 1, 8, and 15. 2 The dose is administered intravenously (e.g., intravenous injection) to the subject.

[0218] In some implementations, the method includes one or more 28-day treatment cycles, wherein

[0219] (1) Anti-Gal9 antibody was administered to subjects via intravenous infusion on day 1 and day 15 (i.e., every 2 weeks (q2w)) at the following doses: approximately 0.5 mg / kg, approximately 1 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, approximately 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg, approximately 10 mg / kg, approximately 11 mg / kg, approximately 12 mg / kg, approximately 13 mg / kg, approximately 14 mg / kg. / kg, about 15mg / kg, about 16mg / kg, about 17mg / kg, about 18mg / kg, about 19mg / kg, about 20mg / kg, about 21mg / kg, about 22mg / kg, about 23mg / kg, about 24mg / kg, about 25mg / kg, about 26mg / kg, about 27mg / kg, about 28mg / kg, about 29mg / kg, about 30mg / kg, about 31mg / kg, or about 32mg / kg) or any increment thereof,

[0220] (2) Gemcitabine was administered at a dose of 1000 mg / m² on days 1, 8, and 15. 2 The dose was administered to the subject intravenously (e.g., intravenous injection).

[0221] (3) Paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) was administered at 125 mg / m² on days 1, 8, and 15. 2 The dose is administered intravenously (e.g., intravenous injection) to the subject.

[0222] In some implementations, the method includes one or more 28-day treatment cycles, wherein

[0223] (1) Anti-Gal9 antibody was administered to subjects via intravenous infusion on days 1 and 15 (i.e., every 2 weeks (q2w)) at the following doses: approximately 1 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, approximately 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg, approximately 10 mg / kg, approximately 11 mg / kg, approximately 12 mg / kg, approximately 13 mg / kg, approximately 14 mg / kg, approximately 15 mg / kg, approximately 16 mg / kg, approximately 17 mg / kg, approximately 18 mg / kg, approximately 19 mg / kg, or approximately 20 mg / kg or any increment thereof.

[0224] (2) Gemcitabine was administered at a dose of 1000 mg / m² on days 1, 8, and 15. 2 The dose was administered to the subject intravenously (e.g., intravenous injection).

[0225] (3) Paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) was administered at 125 mg / m² on days 1, 8, and 15. 2 The dose is administered intravenously (e.g., intravenous injection) to the subject.

[0226] In some implementations, the method includes one or more 28-day treatment cycles, wherein

[0227] (1) Anti-Gal9 antibody was administered to subjects via intravenous infusion at a dose of approximately 2 mg / kg on days 1 and 15 (i.e., every 2 weeks (q2w)).

[0228] (2) Gemcitabine was administered at a dose of 1000 mg / m² on days 1, 8, and 15. 2 The dose was administered to the subject intravenously (e.g., intravenous injection).

[0229] (3) Paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) was administered at 125 mg / m² on days 1, 8, and 15. 2 The dose is administered intravenously (e.g., intravenous injection) to the subject.

[0230] In some implementations, the method includes one or more 28-day treatment cycles, wherein

[0231] (1) Anti-Gal9 antibody was administered to subjects via intravenous infusion at a dose of approximately 4 mg / kg on days 1 and 15 (i.e., every 2 weeks (q2w)).

[0232] (2) Gemcitabine was administered at a dose of 1000 mg / m² on days 1, 8, and 15. 2 The dose was administered to the subject intravenously (e.g., intravenous injection).

[0233] (3) Paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) was administered at 125 mg / m² on days 1, 8, and 15. 2 The dose is administered intravenously (e.g., intravenous injection) to the subject.

[0234] In some implementations, the method includes one or more 28-day treatment cycles, wherein

[0235] (1) Anti-Gal9 antibody was administered to subjects via intravenous infusion at a dose of approximately 8 mg / kg on days 1 and 15 (i.e., every 2 weeks (q2w)).

[0236] (2) Gemcitabine was administered at a dose of 1000 mg / m² on days 1, 8, and 15. 2 The dose was administered to the subject intravenously (e.g., intravenous injection).

[0237] (3) Paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) was administered at 125 mg / m² on days 1, 8, and 15. 2 The dose is administered intravenously (e.g., intravenous injection) to the subject.

[0238] In some implementations, the method includes one or more 28-day treatment cycles, wherein

[0239] (1) Anti-Gal9 antibody was administered to subjects via intravenous infusion at a dose of approximately 12 mg / kg on days 1 and 15 (i.e., every 2 weeks (q2w)).

[0240] (2) Gemcitabine was administered at a dose of 1000 mg / m² on days 1, 8, and 15. 2 The dose was administered to the subject intravenously (e.g., intravenous injection).

[0241] (3) Paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) was administered at 125 mg / m² on days 1, 8, and 15. 2 The dose is administered intravenously (e.g., intravenous injection) to the subject.

[0242] In some implementations, the method includes one or more 28-day treatment cycles, wherein

[0243] (1) Anti-Gal9 antibody was administered to subjects via intravenous infusion at a dose of approximately 16 mg / kg on days 1 and 15 (i.e., every 2 weeks (q2w)).

[0244] (2) Gemcitabine was administered at a dose of 1000 mg / m² on days 1, 8, and 15. 2 The dose was administered to the subject intravenously (e.g., intravenous injection).

[0245] (3) Paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) was administered at 125 mg / m² on days 1, 8, and 15. 2 The dose is administered intravenously (e.g., intravenous injection) to the subject.

[0246] In some implementations, the method includes one or more 28-day treatment cycles, wherein

[0247] (1) Anti-Gal9 antibody was administered to subjects via intravenous infusion at a dose of approximately 32 mg / kg on days 1 and 15 (i.e., every 2 weeks (q2w)).

[0248] (2) Gemcitabine was administered at a dose of 1000 mg / m² on days 1, 8, and 15. 2 The dose was administered to the subject intravenously (e.g., intravenous injection).

[0249] (3) Paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) was administered at 125 mg / m² on days 1, 8, and 15. 2 The dose is administered intravenously (e.g., intravenous injection) to the subject.

[0250] In any of the above-described administration methods, the gemcitabine dose may be reduced to 800 mg / m² when necessary. 2 Or 600mg / m 2 And optionally or additionally, the dose of paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) can be reduced to 100 mg / m². 2 Or 75mg / m 2 .

[0251] In some implementations, the method includes one or more 28-day treatment cycles, wherein

[0252] (1) Anti-Gal9 antibody was administered to subjects via intravenous infusion on days 1 and 15 (i.e., every 2 weeks (q2w)) at the following doses: approximately 0.5 mg / kg to 1 mg / kg, approximately 1 mg / kg to 2 mg / kg, approximately 3 mg / kg to 4 mg / kg, approximately 4 mg / kg to 8 mg / kg, approximately 8 mg / kg to 12 mg / kg, approximately 12 mg / kg to 16 mg / kg, approximately 16 mg / kg to 20 mg / kg, approximately 20 mg / kg to 24 mg / kg, approximately 24 mg / kg to 28 mg / kg, or approximately 28 mg / kg to 32 mg / kg (e.g., approximately 0.5 mg / kg, approximately 1 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 ...1 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 mg / kg, approximately 1 (approximately 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, or 32 mg / kg) or any increment thereof.

[0253] (2) Gemcitabine was administered at a dose of 800 mg / m² on days 1, 8, and 15. 2 600mg / m 2 Or 1000mg / m 2The dose was administered to the subject intravenously (e.g., intravenous injection).

[0254] (3) Paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) was administered at 100 mg / m² on days 1, 8, and 15. 2 75mg / m 2 Or 125mg / m 2 The dose is administered intravenously (e.g., intravenous injection) to the subject.

[0255] In some implementations, the method includes one or more 28-day treatment cycles, wherein

[0256] (1) Anti-Gal9 antibody was administered to subjects via intravenous infusion on days 1 and 15 (i.e., every 2 weeks (q2w)) at the following doses: 0.5 mg / kg to 1 mg / kg, 1 mg / kg to 2 mg / kg, 3 mg / kg to 4 mg / kg, 4 mg / kg to 8 mg / kg, 8 mg / kg to 12 mg / kg, 12 mg / kg to 16 mg / kg, 16 mg / kg to 20 mg / kg, 20 mg / kg to 24 mg / kg, 24 mg / kg to 28 mg / kg, or 28 mg / kg to 32 mg / kg (e.g., 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 20 mg / kg, 20 mg / kg to 24 mg / kg, 24 mg / kg to 28 mg / kg, or 28 mg / kg to 32 mg / kg). 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, or 32 mg / kg) or any increments within these ranges or any increment thereof,

[0257] (2) Gemcitabine was administered at a dose of 800 mg / m² on days 1, 8, and 15. 2 600mg / m 2 Or 1000mg / m 2 The dose was administered to the subject intravenously (e.g., intravenous injection).

[0258] (3) Paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) was administered at 100 mg / m² on days 1, 8, and 15. 2 75mg / m 2Or 125mg / m 2 The dose is administered intravenously (e.g., intravenous injection) to the subject.

[0259] In some implementations, the method includes one or more 28-day treatment cycles, wherein

[0260] (1) Anti-Gal9 antibody was administered to subjects via intravenous infusion on days 1 and 15 (i.e., every 2 weeks (q2w)) at the following doses: approximately 1 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, approximately 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg, approximately 10 mg / kg, approximately 11 mg / kg, approximately 12 mg / kg, approximately 13 mg / kg, approximately 14 mg / kg, approximately 15 mg / kg, approximately 16 mg / kg, approximately 17 mg / kg, approximately 18 mg / kg, approximately 19 mg / kg, or approximately 20 mg / kg or any increment thereof.

[0261] (2) Gemcitabine was administered at a dose of 800 mg / m² on days 1, 8, and 15. 2 600mg / m 2 Or 1000mg / m 2 The dose was administered to the subject intravenously (e.g., intravenous injection).

[0262] (3) Paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) was administered at 100 mg / m² on days 1, 8, and 15. 2 75mg / m 2 Or 125mg / m 2 The dose is administered intravenously (e.g., intravenous injection) to the subject.

[0263] In any of the above-mentioned methods of administration, the treatment period can last from 12 to 24 months.

[0264] In any of the method embodiments described herein, the anti-galactagogue-9 antibody may be administered (alone or in combination with one or more chemotherapeutic agents such as gemcitabine and nab-paclitaxel, for example, at the doses described herein) as follows: once weekly, once every two weeks for one cycle, once every two weeks for two cycles, once every two weeks for three cycles, once every two weeks for four cycles, or once every two weeks for more than four cycles. In some embodiments, treatment is for 1 to 3 months, 3 to 6 months, 6 to 12 months, 12 to 24 months, or longer. In some embodiments, treatment is once every two weeks for 1 to 3 months, once every two weeks for 3 to 6 months, once every two weeks for 6 to 12 months, or once every two weeks for 12 to 24 months, or longer.

[0265] In some implementations, the method described herein for treating solid tumors (e.g., PDA) comprises one or more 28-day treatment cycles, wherein the anti-Gal9 antibody is administered to the subject via intravenous infusion on days 1, 7, 15, and 21 (i.e., once weekly (q1w)) at the following doses:

[0266] Approximately 0.5 mg / kg to 1 mg / kg, approximately 1 mg / kg to 2 mg / kg, approximately 3 mg / kg to 4 mg / kg, approximately 4 mg / kg to 8 mg / kg, approximately 8 mg / kg to 12 mg / kg, approximately 12 mg / kg to 16 mg / kg, approximately 16 mg / kg to 20 mg / kg, approximately 20 mg / kg to 24 mg / kg, approximately 24 mg / kg to 28 mg / kg, or approximately 28 mg / kg to 32 mg / kg (e.g., approximately 0.5 mg / kg, approximately 1 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg, approximately 10 mg / kg). Approximately 11 mg / kg, approximately 12 mg / kg, approximately 13 mg / kg, approximately 14 mg / kg, approximately 15 mg / kg, approximately 16 mg / kg, approximately 17 mg / kg, approximately 18 mg / kg, approximately 19 mg / kg, approximately 20 mg / kg, approximately 21 mg / kg, approximately 22 mg / kg, approximately 23 mg / kg, approximately 24 mg / kg, approximately 25 mg / kg, approximately 26 mg / kg, approximately 27 mg / kg, approximately 28 mg / kg, approximately 29 mg / kg, approximately 30 mg / kg, approximately 31 mg / kg, or approximately 32 mg / kg) or any increments thereof, and gemcitabine and paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) were administered to subjects on days 1, 8, and 15. In some examples, paclitaxel was administered at 125 mg / kg. 2 Gemcitabine is administered intravenously (e.g., intravenous injection) to the subject. In some examples, gemcitabine is administered at 1000 mg / m². 2 Administered intravenously (e.g., intravenous injection) to the subject. If necessary, the gemcitabine dose may be reduced to 800 mg / m². 2 Or 600mg / m 2 Optionally or otherwise, the dose of paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) can be reduced to 100 mg / m². 2 Or 75mg / m 2 .

[0267] In some embodiments, the method described herein for treating solid tumors (e.g., PDA) comprises one or more 28-day treatment cycles, wherein the anti-Gal9 antibody is administered to the subject via intravenous infusion on days 1, 7, 15, and 21 (i.e., once weekly (q1w)) at the following doses: 0.5 mg / kg to 1 mg / kg, 1 mg / kg to 2 mg / kg, 3 mg / kg to 4 mg / kg, 4 mg / kg to 8 mg / kg, 8 mg / kg to 12 mg / kg, 12 mg / kg to 16 mg / kg, 16 mg / kg to 20 mg / kg, 20 mg / kg to 24 mg / kg, 24 mg / kg to 28 mg / kg, or 28 mg / kg to 32 mg / kg (e.g., 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 9 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 ... 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, or 32 mg / kg) or any increments within these ranges, and gemcitabine and paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) were administered to subjects on days 1, 8, and 15. In some examples, paclitaxel was administered at 125 mg / kg. 2 Gemcitabine is administered intravenously (e.g., intravenous injection) to the subject. In some examples, gemcitabine is administered at 1000 mg / m². 2 Administered intravenously (e.g., intravenous injection) to the subject. If necessary, the gemcitabine dose may be reduced to 800 mg / m². 2 Or 600mg / m 2 Optionally or otherwise, the dose of paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) can be reduced to 100 mg / m². 2 Or 75mg / m 2 .

[0268] In some implementations, the method of treating solid tumors (e.g., PDA) described herein comprises one or more 28-day treatment cycles, wherein anti-Gal9 antibody is administered to the subject via intravenous infusion on days 1, 7, 15, and 21 (i.e., once weekly (q1w)) at the following doses: about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, or about 20 mg / kg or any increment thereof, and gemcitabine and paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) are administered to the subject on days 1, 8, and 15. In some examples, paclitaxel was used at 125 mg / m². 2 Gemcitabine is administered intravenously (e.g., intravenous injection) to the subject. In some examples, gemcitabine is administered at 1000 mg / m². 2 Gemcitabine is administered intravenously (e.g., intravenous injection) to the subject. If necessary, the dose of gemcitabine may be reduced to 800 mg / m². 2 Or 600mg / m 2 Optionally or otherwise, the dose of paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) can be reduced to 100 mg / m². 2 Or 75mg / m 2 .

[0269] In some cases, Gal-9 antibody therapy can be initiated concurrently with chemotherapy (e.g., gemcitabine and nab-paclitaxel). Alternatively, Gal-9 antibody therapy can be initiated after a chemotherapy regimen (e.g., gemcitabine and nab-paclitaxel) has already begun. In some cases, Gal-9 antibody therapy is administered concurrently with chemotherapy (e.g., gemcitabine and nab-paclitaxel), followed by discontinuation of chemotherapy. In some cases where chemotherapy has been discontinued, the anti-Gal-9 antibody therapy regimen may be continued.

[0270] In any of the above embodiments, the interval or cycle may be once a week. In any of the above embodiments, the interval or cycle may be once every two weeks. In some embodiments, the regimen may be once every two weeks for one cycle, once every two weeks for two cycles, once every two weeks for three cycles, once every two weeks for four cycles, or once every two weeks for more than four cycles. In some embodiments, treatment may be once every two weeks for one to three months, once every two weeks for three to six months, once every two weeks for six to twelve months, or once every two weeks for twelve to twenty-four months or longer.

[0271] In any of the above embodiments, the interval or cycle may be 3 weeks. In some embodiments, the regimen may be one cycle every 3 weeks, two cycles every 3 weeks, three cycles every 3 weeks, four cycles every 3 weeks, or more than four cycles every 3 weeks. In some embodiments, treatment may be every 3 weeks for 1 to 3 months, every 3 weeks for 3 to 6 months, every 3 weeks for 6 to 12 months, or every 3 weeks for 12 to 24 months or longer.

[0272] In any of the above embodiments, the interval or cycle may be 4 weeks or more. In some embodiments, the regimen is one cycle every 4 weeks or more, two cycles every 4 weeks or more, three cycles every 4 weeks or more, four cycles every 4 weeks or more, or more than four cycles every 4 weeks or more. In some embodiments, treatment may be every 4 weeks or more for 1 to 3 months, every 4 weeks or more for 3 to 6 months, every 4 weeks or more for 6 to 12 months, or every 4 weeks or more for 12 to 24 months or longer. In some embodiments, treatment may be a combination of treatments at different times, such as a combination of 2 weeks, 3 weeks, 4 weeks, or more than 4 weeks. In some embodiments, the treatment interval may be adjusted based on the patient's response to treatment. In some embodiments, the dose may be adjusted based on the patient's response to treatment. In some embodiments, the dose may vary between treatment intervals. In some embodiments, treatment may be temporarily discontinued. In some embodiments, antigalactotropic-9 therapy may be temporarily discontinued. In some embodiments, chemotherapy may be temporarily discontinued. In some embodiments, both may be temporarily discontinued. In any of these embodiments, the anti-Gal9 antibody may be G9.2-17 in the form of IgG4 as disclosed herein, having a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 15.

[0273] Treatment response can also be characterized by the following: one or more immunophenotypes in the blood and tumor, cytokine profile (serum), soluble galactagogue-9 levels in the blood (serum or plasma), galactagogue-9 tumor tissue expression levels and immunohistochemical expression patterns (tumor, stroma, immune cells), tumor mutational burden (TMB), PDL-1 expression (e.g., by immunohistochemistry), mismatch repair status, or disease-related tumor markers (e.g., measured at 3, 6, or 12 months or later). Non-limiting examples of such tumor markers include Ca15-3, CA-125, CEA, CA19-9, and alpha-fetoprotein. These parameters can be compared to baseline levels before treatment initiation or to the control group described herein.

[0274] In any of the methods disclosed herein, one or more of the following characteristics of a subject may be detected before, during, and / or after treatment: (a) one or more tumor markers in a blood sample from the subject, optionally including one or more tumor markers including CA15-3, CA-125, CEA, CA19-9, and / or alpha-fetoprotein, and any other tumor type-specific tumor markers; (b) cytokine profile; and (c) galactagogue-9 serum / plasma levels; (d) peripheral blood mononuclear cell immunophenotyping; (e) multiple immunophenotyping of tumor tissue biopsy / resected specimens; (f) galactagogue-9 expression levels and patterns in tumor tissue biopsy / resected specimens; and (g) any other immunoscoring test, such as: PDL-1 immunohistochemistry, tumor mutational burden (TMB), tumor microsatellite instability status, and the following series such as: Immunoscore®-HalioDx, ImmunoSeq-Adaptive Biotechnologies, and NanoString. TIS, 18-gene signature, and PanCancer IO 360™ assays (NanoString Technologies) have been developed on the nCounter® gene expression system. Other suitable biomarkers specific to target tumors such as PDAC can also be used.

[0275] In some implementations, the methods described herein (where the Gal-9 antibody is administered in conjunction with chemotherapy (e.g., gemcitabine and nab-paclitaxel)) modulate the levels of immune cells and immune cell markers in the blood or tumor. 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. For example, a set of phenotypic and functional PBMC immune markers can be evaluated at baseline before treatment initiation and at different time points during treatment. Table A lists non-limiting examples of markers that can be used in these evaluation methods. Flow cytometry (FC) is a rapid and highly informative selection technique for analyzing cell phenotypes and functions and plays an important role in immunophenotyping surveillance. It allows for the characterization of many cell subpopulations, including rare subpopulations, in complex mixtures (e.g., blood) and represents a method for rapidly acquiring large amounts of data. The advantages of FC are its speed, high sensitivity, and high specificity. Standardized antibody series and procedures can be used to analyze and classify immune cell subtypes. Multiplex IHC is a powerful research tool that provides objective quantitative data describing the number and location of immune subsets in the tumor immune background and allows for the evaluation of multiple biomarkers on a single tissue slide. Computer algorithms can be used to quantify IHC-based biomarker levels from whole-slide images of patient biopsies, combining chromogenic IHC methods and staining with digital pathology approaches.

[0276] Table A. Phenotypic biomarkers for PBMC

[0277]

[0278] Therefore, in some embodiments, the method of combining anti-gal9 antibodies with chemotherapy as described herein can modulate immune activation markers, such as those in Table A. These markers can be compared to baseline levels prior to the start of treatment or to a control group receiving chemotherapy alone (e.g., at certain intervals, such as 3 months, 6 months, or 12 months). In some embodiments, the cytokine profile is modulated.

[0279] In some embodiments, this disclosure provides methods for modulating the immune response of a subject. The immune response may be a T-cell-mediated and / or B-cell-mediated immune response, which is influenced by the regulation of immune cell activity, such as T-cell activation. In one embodiment of this disclosure, the immune response is T-cell-mediated. As used herein, the term “modulation” means alteration or modification and includes both upregulation and downregulation. For example, “modulating an immune response” means altering or modifying the state of one or more immune response parameters. Exemplary parameters of a T-cell-mediated immune response include T-cell levels (e.g., an increase or decrease in effector T cells) and T-cell activation levels (e.g., an increase or decrease in the production of certain cytokines). Exemplary parameters of a B-cell-mediated immune response include an increase in B-cell levels, B-cell activation, and B-cell-mediated antibody production.

[0280] When an immune response is modulated, some immune response parameters may decrease while others may increase. For example, in some cases, modulating the immune response leads to an increase (or upregulation) in one or more immune response parameters and a decrease (or downregulation) in one or more other immune response parameters, resulting in an overall increase in the immune response, such as an overall increase in the inflammatory immune response. In another example, modulating the immune response leads to an increase (or upregulation) in one or more immune response parameters and a decrease (or downregulation) in one or more other immune response parameters, resulting in an overall decrease in the immune response, such as an overall decrease in the inflammatory response.

[0281] In some implementations, the method of combining anti-gal9 antibody with chemotherapy described herein can modulate the level of soluble galactagogue-9 in the blood (serum or plasma) of a subject, or modulate the expression level and pattern of galactagogue-9 in tumor tissue (by immunohistochemistry (tumor, stroma, immune cells)) (e.g., measured at 3 months, 6 months, or 12 months or later). The galactagogue-9 level in the subject can be compared to baseline levels before the start of treatment, or it can be compared to a control group, such as one receiving chemotherapy alone.

[0282] In some implementations, the methods described herein can reduce the levels of one or more soluble galactagogue-9 in the blood (serum or plasma), or reduce the expression levels and patterns of galactagogue-9 in tumor tissues (by immunohistochemistry (tumor, stroma, immune cells)) (e.g., measured at 3 months, 6 months, or 12 months or later).

[0283] In some implementations, the method of combining anti-gal9 antibody administration with chemotherapy described herein can modulate one or more disease-related tumor markers (increase or decrease) (e.g., as measured at 3 months, 6 months, or 12 months or later). Non-limiting examples of such tumor markers include Ca15-3, CA-125, CEA, CA19-9, and alpha-fetoprotein. These parameters can be compared to baseline levels before the start of treatment or to a control group, such as one receiving chemotherapy alone.

[0284] In some embodiments, the method of combining the anti-gal-9 antibody described herein with chemotherapy (e.g., gemcitabine and nab-paclitaxel) may improve overall response (e.g., at 3, 6, or 12 months) compared to baseline levels before treatment initiation or to a control group receiving chemotherapy alone. In some embodiments, the method described herein may result in a complete response, partial response, or disease stabilization (e.g., as measured at 3, 6, or 12 months, according to RECIST or iRECIST criteria). In some embodiments, the method may increase the likelihood of a complete response, partial response, or disease stabilization (e.g., as measured at 3, 6, or 12 months), for example, compared to a control group receiving chemotherapy alone. In some embodiments, treatment may result in longer survival or a greater likelihood of survival, for example at a time, such as 6 or 12 months or at a later time point.

[0285] In any of the methods described herein, partial response, stable disease, complete response, partial response, stable disease, progressive disease, and disease progression (e.g., measured at 3 months, 6 months, or 12 months or later) may be assessed according to RECIST criteria or iRECIST criteria.

[0286] In some implementations, the combination of the anti-gal-9 antibody and chemotherapy (e.g., gemcitabine and nab-paclitaxel) described herein, compared to the control group (e.g., chemotherapy alone), may increase time before disease progression or increase progression-free survival (e.g., as measured at 6 months). In some implementations, treatment may result in a greater likelihood of progression-free survival compared to the control group (e.g., as measured at time points 3 months, 6 months, or 12 months or later after treatment initiation).

[0287] In some implementations, the combination of the anti-gal-9 antibody and chemotherapy (e.g., gemcitabine and nab-paclitaxel) provided herein, compared with the control group (e.g., chemotherapy alone), improved the duration and depth of response according to RECIST 1.1 criteria (e.g., measured at time points of 3 months, 6 months, or 12 months or later after the start of treatment).

[0288] In some implementations, the method of combining anti-gal-9 antibody with chemotherapy (e.g., gemcitabine and nab-paclitaxel) provided herein can improve quality of life and / or symptom control (e.g., as measured using the ECOG scale at 1 month, 3 months, 6 months, or 12 months or later) compared to baseline before treatment or compared to the control group.

[0289] Subjects with a target solid tumor (e.g., PDAC) as disclosed herein can be identified through routine medical examinations, such as laboratory tests, organ function tests, genetic testing, interventional procedures (biopsy, surgery), and any and all relevant imaging modalities. In some embodiments, the subject to be treated by the methods described herein is a human cancer patient who has received or is receiving anticancer therapies such as chemotherapy, radiation therapy, immunotherapy, or surgery. In some embodiments, the subject has received prior immunomodulatory antitumor agents. Non-limiting examples of such immunomodulatory agents include, but are not limited to, anti-PD1, anti-PD-L1, anti-CTLA-4, anti-OX40, anti-CD137, etc. In some embodiments, the subject shows disease progression during treatment. In other embodiments, the subject is resistant to treatment (congenital or acquired). In some embodiments, such subjects show advanced malignancy (e.g., unresectable or metastatic). Alternatively or additionally, in some embodiments, the subject has no standard treatment options available or is unsuitable for standard treatment options, which refer to therapies commonly used in the clinical setting for treating the corresponding solid tumor.

[0290] In some cases, the subject may be a human patient with a refractory disease (e.g., refractory PDAC). As used herein, “refractory” means a tumor that is unresponsive to treatment or resistant to treatment. In some cases, the subject may be a human patient with a recurrent disease such as recurrent PDAC. As used herein, “recurrent” or “relapsed” means that the tumor has relapsed or progressed after a period of treatment improvement (e.g., partial or complete response).

[0291] In some implementations, human patients to be treated by the methods disclosed herein may meet one or more of the inclusion and exclusion criteria disclosed in Example 2 below. For example, a human patient may be 18 years of age or older and have histologically confirmed unresectable metastatic cancer (e.g., adenocarcinoma and squamous cell carcinoma). The patient may have a disease measurable according to RECIST v. 1.1. In some cases, a human patient may have a recently archived tumor sample (e.g., obtained within 5 years) available for biomarker analysis (e.g., galactagogue-9 tumor tissue expression assessable by IHC). In some cases, a human patient is a PDAC patient in a metastatic cancer setting who has received at least one systemic therapy. Such patients may be those who have not received a gemcitabine-containing regimen or those who have discontinued treatment with a gemcitabine-containing regimen for at least 6 months. The patient may have an Eastern Cooperative Oncology Group (ECOG) performance status of 0–1 and / or a Karnofsky score > 70. The patient may also have adequate hematological and end-organ function, e.g., a neutrophil count ≥ 1 x 10⁻⁶. 9 / L, platelet count ≥100 x10 9 / L, for HCC ≥50 x 10 in Part 1 9 / L; hemoglobin ≥8.5 g / dL without transfusion in the previous week, creatinine ≤1.5 x ULN, AST (SGOT) ≤3 x ULN (≤5 x ULN in the presence of HCC or liver metastasis), ALT (SGPT) ≤3 x ULN (≤5 x ULN in the presence of HCC or liver metastasis), bilirubin ≤1.5 x ULN (bilirubin ≤3.0 x ULN in patients with known Gilbert's disease), albumin ≥3.0 g / dL, INR and PTT ≤1.5 x ULN; and / or amylase and lipase ≤1.5 x ULN. In some cases, human patients do not show evidence of active infection or infection requiring parenteral antibiotics and have not had a serious infection within 4 weeks prior to the start of treatment. Pancreatic fistula, bile fistula, or intestinal fistula is permissible, provided they are controlled by appropriate non-infectious and open (patent) drainage.

[0292] Optionally or otherwise, human patients receiving any of the treatments disclosed herein may not have: (i) metastatic cancer of unknown primary origin; (ii) clinically significant uncontrolled active bleeding, any bleeding predisposition (e.g., active peptic ulcer disease); (iii) radiotherapy within 4 weeks of the first dose; (iv) fungal tumor mass or locally advanced PDAC; (v) ≥CTCAE grade 3 toxicity due to prior cancer treatment (excluding alopecia and vitiligo); (v) history of secondary malignancy; (vi) evidence of severe or uncontrolled systemic disease, congestive heart failure >NYHA grade 2, or myocardial infarction (MI) within 6 months; (vii) severe unhealed wound, active ulcer, or untreated fracture; (viii) uncontrolled pleural effusion, pericardial effusion, or ascites requiring repeated drainage procedures; (ix) severe hypersensitivity, allergy, or other allergic reactions to chimeric or humanized antibodies or fusion proteins. History of hypersensitivity response; (x) Major vascular disease within 6 months after treatment (e.g., aortic aneurysm requiring surgical repair or recent arterial thrombosis), history of pulmonary embolism, stroke or transient ischemic attack within 3 months prior to treatment, and / or history of abdominal fistula or gastrointestinal perforation within 6 months prior to treatment; (xi) Active autoimmune disease (except type I diabetes, hypothyroidism requiring only hormone replacement, vitiligo, psoriasis or alopecia); (xii) Requirement of systemic immunosuppressive therapy; (xii) Tumor-related pain (> grade 3) unresponsive to extensive analgesic interventions (oral and / or patch); (xiii) Hypercalcemia uncontrolled despite bisphosphonate use; (xiv) Receiving organ transplantation.

[0293] In some cases, the subject is a human patient with elevated galactagogue-9 levels relative to a control level. Galactagogue-9 levels can be plasma or serum levels of galactagogue-9 in a human patient. In other examples, galactagogue-9 levels can be cell surface galactagogue-9 levels, such as galactagogue-9 levels on cancer cells. In one example, galactagogue-9 levels can be surface galactagogue-9 levels expressed on cancer cells in a patient-derived organoid tumor spheroid (PDOT), which can be prepared by methods disclosed, for example, in the examples below. A control level can refer to the galactagogue-9 level in a matched sample from a subject of the same species (e.g., a human) without a solid tumor. In some examples, a control level represents the galactagogue-9 level in a healthy subject.

[0294] To identify such subjects, suitable biological samples can be obtained from subjects suspected of having solid tumors using conventional methods (e.g., ELISA or FACS), and the biological samples can be analyzed to determine the level of galactagogue-9 contained therein (e.g., free, cell surface expressed, or total). In some embodiments, organoid cultures, such as those described herein, are prepared and used to assess galactagogue-9 levels in subjects. Single cells obtained from certain portions as part of the organoid preparation process are also suitable for assessing galactagogue-9 levels in subjects. In some cases, assays for measuring levels of free form or cell surface expressed galactagogue-9 involve the use of antibodies that specifically bind to galactagogue-9 (e.g., those specifically binding to human galactagogue-9). Any anti-galactagogue-9 antibody known in the art can be suitably tested in any of the above assays and then used in a conventional manner for such assays. In some embodiments, antibodies described herein (e.g., G9.2-17 antibody) can be used in such assays. In some embodiments, antibodies are described in co-pending U.S. Patent Application No. 16 / 173,970 and co-owned co-pending International Patent Application PCT / US18 / 58028, the relevant disclosures of which are incorporated herein by reference for the purposes and subject matter herein. In some examples, the anti-galactoglobulin-9 antibody is a Fab molecule. Assays for determining galactoglobulin-9 levels as disclosed herein are also within the scope of this disclosure.

[0295] Kits for combination therapy of solid tumors

[0296] This disclosure also provides kits for treating or alleviating solid tumors such as PDA, CRC, HCC, or cholangiocarcinoma, as well as other solid tumors described herein. Such kits may include one or more containers containing an anti-galactoglobulin-9 antibody, such as any of those described herein (e.g., G9.2-17(IgG4)), and optionally one or more chemotherapeutic agents (e.g., gemcitabine and / or paclitaxel) to be used in conjunction with the anti-galactoglobulin-9 antibody, also described herein.

[0297] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include instructions for administering an anti-galactoglobulin-9 antibody and one or more chemotherapeutic agents to treat, delay the onset of, or alleviate target diseases as described herein. In some embodiments, the kit may also include instructions for selecting suitable individuals for treatment based on identifying whether an individual has the target disease (e.g., using diagnostic methods as described herein). In other embodiments, the instructions include instructions for administering the antibody to individuals at risk of having the target disease.

[0298] Instructions for use with anti-galactoside-9 antibodies and one or more chemotherapeutic agents typically include information on the dosage, dosing regimen, and route of administration for the intended treatment. Containers may be unit doses, bulk packaging (e.g., multi-dose packs), or subunit doses. Instructions provided with kits of this invention are typically written instructions on a label or packaging insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions carried on a disk or optical storage disc) are also acceptable.

[0299] The label or packaging insert indicates that the composition is used to treat, delay onset, and / or alleviate solid tumors. In some embodiments, instructions for carrying out any of the methods described herein are provided.

[0300] The kit of the present invention is packaged in a suitable manner. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. Packaging for use in combination with specific devices, such as inhalers, nasal delivery devices (e.g., nebulizers), or infusion devices such as micropumps, is also contemplated. In some embodiments, the kit has a sterile inlet (e.g., the container may be an intravenous solution bag or vial with a stopper that can be punctured by a hypodermic needle). In some embodiments, the container also has a sterile inlet (e.g., the container is an intravenous solution bag or vial with a stopper that can be punctured by a hypodermic needle). At least one active agent in the composition is an anti-galactogenin-9 antibody as described herein.

[0301] The kit may optionally include additional components, such as buffer solutions and explanatory information. Typically, the kit includes a container and a label or packaging insert on or attached to the container. In some embodiments, the invention provides an article of manufacture comprising the contents of the kit described above.

[0302] General Technology

[0303] Unless otherwise stated, the practice of this invention employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art. These techniques are well explained in the literature, for example: *Molecular Cloning: A Laboratory Manual, 2nd Edition* (Sambrook et al., 1989), Cold Spring Harbor Press; *Oligonucleotide Synthesis* (MJ Gait, ed., 1984); *Methods in Molecular Biology*, Humana Press; *Cell Biology: A Laboratory Notebook* (JE Cellis, ed., 1998), Academic Press; *Animal Cell Culture* (RIFreshney, ed., 1987); *Introduction to Cell and Tissue Culture* (JP Mather and PE Roberts, 1998), Plenum Press; *Cell and Tissue Culture: Laboratory Procedures* (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8), J. Wiley and Sons; *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (DM Weir and CC Blackwell, eds.); *Gene Transfer Vectors for Mammalian Cells* (JM Miller and MP Calos, ed., 1987); Current Protocols in Molecular Biology (FM Ausubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis, et al., eds., 1994); Current Protocols in Immunology (JEColigan et al., Ed., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., Ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999)); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995).

[0304] Without further elaboration, it is believed that those skilled in the art can fully utilize the invention based on the above description. Therefore, the following specific embodiments should be interpreted as merely illustrative and not as limiting the remainder of this disclosure in any way. All publications cited herein for the purposes or subject matter are incorporated herein by reference.

[0305] Example

[0306] Although this disclosure has been described with reference to specific embodiments thereof, those skilled in the art will understand that various changes and equivalents may be made without departing from the true spirit and scope of this disclosure. Furthermore, numerous modifications may be made to adapt particular circumstances, materials, material composition, processes, process steps, or procedures to the purpose, spirit, and scope of this disclosure. All such modifications are intended to fall within the scope of this disclosure.

[0307] Example 1 In vivo study of anti-galactophlebin-9 antibody in combination with chemotherapy agents for cancer treatment in a mouse model of pancreatic cancer.

[0308] Preclinical evaluation of the anti-galactagogue-9 IgG4 fully human antibody (G9.2-17(IgG4)) for the treatment of refractory solid tumors in a mouse model of pancreatic cancer—as a single agent or in combination with other systemic chemotherapy regimens.

[0309] The specific animal used was the orthotopic mPA6115 pancreatic cancer xenograft model from female C57BL / 6 mice. To generate this model, the tumor was first derived from mPA6115 mice, a mouse allograft model of pancreatic ductal adenocarcinoma (PDAC) that retains morphological similarity to human PDAC. mPA6115 mice were stained with the conditional mutation Kras (Kras... LSL-G12D / WT Constitutive deletion of Trp53 (P53KO / KO) and Cre driven by the Pdx1 gene promoter, and developed into a severe PDAC tumor at 8 weeks of age.

[0310] At this point, mPA6115 mice with palpable tumors were euthanized, and their pancreatic tumors were collected. The collected tumor tissue was cut into small pieces (~2 mm). 3 These seed tumors were then subcutaneously (SC) transplanted into syngeneic recipient C57BL / 6 mice. These seed tumors were maintained subcutaneously in C57BL / 6 mice until the seed tumor volume reached 700–1000 mm². 3 Once the seed tumor reaches the desired size, it is collected and cut into pieces approximately 2 mm in diameter. 3 The tumor was then washed with cold Roswell Park Memorial Institute (RPMI) 1640 medium (serum-free) to remove adjacent non-tumor tissue. The tumor blocks were then placed in cold RPMI 1640 medium until in situ implantation. On the same day as the seed tumors were collected, pancreatic in situ implantation was performed on 6–7 week old female C57BL / 6 mice. Specifically, after the animals were fully anesthetized, a small longitudinal incision was made in the left lower thoracic cavity to expose the spleen and the pancreas below it. One seed tumor block was sutured into the pancreas of each mouse using 6-0 sutures. The tissue surrounding the tumor block was then sutured with 6-0 sutures, wrapping the tumor block with pancreatic tissue. The abdomen was then sutured with 4-0 sutures. After tumor implantation, the animals were kept in warm cages and subsequently returned to the animal room after complete recovery from anesthesia.

[0311] On the day of implantation, the mice were randomly divided into 6 groups based on their body weight, with the latter group selected based on a "matched distribution" method (StudyDirector). TM The software (version 3.1.399.19) was used for randomization. The randomization date is designated as day 0. Three days post-implantation, animals began the dosing regimen according to their group number. The dosing regimen for each group is provided in Table 2 below.

[0312] Table 2. Study Dosing Schedule

[0313]

[0314] IP = intraperitoneal; IV = intravenous; QW = once a week; Q4D = once every 4 days

[0315] For these studies, anti-galactagogue-9 mouse IgG1 was used. This antibody, called anti-Gal9 mAb, is the mouse IgG1 version of the human G9.2-17 antibody. It binds to the same carbohydrate-binding domain 2 (CRD2) of galactagogue-9 as G9.2-17 and has the same VH and VL regions as G9.2-17. Therefore, the data obtained using anti-Gal9 mAb are correlated with the human efficacy of G9.2-17. In addition to treating mice with anti-Gal9 mAb alone (group 4), the implanted mice in groups 5 and 6 also received standard care chemotherapy (gemcitabine / abraxane regimen) or a combination of anti-Gal9 mAb and chemotherapy.

[0316] Following orthotopic pancreatic implantation, morbidity and mortality were assessed daily in groups 1–7. During routine monitoring, tumor growth and any effects of treatment on behavior were examined, such as activity level, food and water consumption, weight gain / loss, dull eyes / fur, and any other abnormalities. StudyDirector was used. TM After randomization with the software (version 3.1.399.19), body weight and tumor volume were measured twice weekly. Measurements were collected and monitored as described from day 0 to day 66 (when the last mouse died). Blood, plasma, spleen, and tumor were collected at the end of each mouse's life. Table 3 below shows the mean lifespan of mice by experimental group. The longest survival among all control groups (groups 1, 2, and 3) was 33 days, while the last mouse in group 4 (anti-galactagogue-9 IgG1), group 5 (gemcitabine / abraxane), and group 6 (combination therapy) died on days 55, 41, and 66, respectively.

[0317] Table 3. Average lifespan of mice in each group

[0318]

[0319] The primary survival endpoint in animals with transplanted orthotopic KPC tumors was assessed using the Kaplan-Meier method by estimating survival curves for each group separately, and statistical comparisons were performed using the log-rank test. Specifically, Kaplan-Meier survival curves / log-rank test (SPSS 18) were used. The Kaplan-Meier survival curves and log-rank test are shown below. Figures 1A to 1DAs shown in Table 4, the results of the log-rank test are presented.

[0320] Table 4. Log-rank test

[0321]

[0322] p<0.05; p<0.01; p < 0.001; ns, p ≥ 0.05

[0323] Cox regression analysis (using the Coxph function of the survival R package) was used to calculate the hazard ratios (HRs) of groups 4–6 relative to groups 1, 2, and 3, and their 95% confidence intervals (%95CI). We also used Cox regression analysis to calculate the hazard ratios (HRs) of groups 5 and 6 relative to group 4, and their 95% confidence intervals (%95CI). Finally, we used Cox regression analysis to calculate the hazard ratio (HR) of group 6 relative to group 5, and its 95% confidence interval (%95CI). The results of the Cox regression analysis are shown below. Figure 2 As shown in Table 5.

[0324] Table 5. Cox Regression Analysis

[0325]

[0326] p<0.05; p<0.01; p < 0.001; ns, p ≥ 0.05

[0327] For Cox regression analysis using group 1 as a reference, the hazard ratios of groups 4 and 6 were significantly lower than those of group 1, while the hazard ratios of groups 2 and 3 were not significantly different from those of group 1. In Cox regression analysis using group 2 as a reference, the hazard ratio of group 6 was significantly lower than that of group 2; however, the hazard ratio of group 3 was not significantly different from that of group 2. For Cox regression analysis using group 3 as a reference, the hazard ratios of groups 4, 5, and 6 were not significantly different from those of group 3. In Cox regression analysis using group 4 as a reference, the hazard ratios of groups 5 and 6 were not significantly different from those of group 4. Finally, Cox regression analysis using group 5 as a reference showed that the hazard ratio of group 6 was not significantly different from that of group 5.

[0328] These data indicate that the combination of anti-galactoglobulin-9 antibody and gemcitabine / abraxane was well-tolerated and could be administered for extended periods (up to 16 doses of anti-galactoglobulin-9 IgG1 antibody (mouse IgG1 version) and up to 10 doses of gemcitabine / abraxane), and provided a survival benefit compared to untreated animals (Group 6 vs. Group 1: Cox analysis, HR = 0.336, HR (95% CI) = (0.14, 0.806), p = 0.015; and p = 0.051, log-rank test for mean survival). Anti-galactoglobulin-9 IgG1 alone provided a survival benefit compared to untreated animals (Group 4 vs. Group 1: Cox analysis, HR = 0.348, HR (95% CI) = (0.146, 0.83), p = 0.017).

[0329] When the study ended on day 66, with the mice dying, no tumor was found in the pancreas of the last mouse in group 6. Based on historical data, the take rate of the in situ mPA6115 model was 100% in the vector group. Therefore, the last mouse in group 6 was a complete responder to the anti-galactotropic-9 / gemcitabine / abraxane combination regimen.

[0330] The body weight of mice implanted with orthotopic KPC tumors was measured twice a week after implantation / randomization (day 0) until all mice were euthanized or died. Figure 3 Showing the use of StudyDirector TM Body weight measurements collected during the study period were taken using software (version 3.1.399.19). The last mouse in group 4 was euthanized on day 55 when it was dying; its tumor weight was 2544.6 mg (TV = 1877.07 mm). 3 From day 51 to day 55, compared to body weight on day 1 of treatment, only the last mouse in group 4 showed a weight change ranging from 12.36% to -2.25%. This weight loss is likely related to conditions caused by tumor growth.

[0331] Overall, the data in this embodiment confirm the safety and efficacy of the anti-galactoglobulin-9 regimen and the anti-galactoglobulin-9 / gemcitabine / abraxane combination regimen in the mPA6115 orthotopic pancreatic cancer xenograft model.

[0332] Example 2 A phase 1a / 1b open-label, multicenter study of the safety, pharmacokinetics, and antitumor activity of G9.2-17 (IgG4) alone and in combination with chemotherapy in patients with metastatic solid tumors.

[0333] Galactochonin-9 is a molecule overexpressed in many solid tumors, including pancreatic cancer, colorectal cancer, and hepatocellular carcinoma. Furthermore, galactochonin-9 is expressed on tumor-associated macrophages and intratumoral immunosuppressive γδ T cells, thus acting as a potent mediator of cancer-associated immunosuppression. As described in this article, monoclonal antibodies targeting galactochonin-9 (e.g., G9.2-17) have been developed. Data show that G9.2-17 halted pancreatic tumor growth by 50% in an orthotopic kappa PC model and more than doubled the survival of KPC animals. Moreover, in animal studies, anti-galactochonin-9 antibodies have shown signals of synergistic therapy with chemotherapeutic agents.

[0334] The goal of this phase I / II multicenter study was to determine the safety, tolerability, maximum tolerated dose (MTD), and objective tumor response after 12 to 24 months of treatment in subjects with metastatic solid tumors (e.g., pancreatic cancer (PDA), colorectal cancer (CRC), hepatocellular carcinoma (HCC), or cholangiocarcinoma (CCA)). The study also assessed progression-free survival (PFS), duration of response (according to RESIST), disease stabilization, proportion of surviving subjects, and pharmacokinetic (PK) and pharmacodynamic (PD) parameters. Subjects underwent biopsies before and after treatment, and PET-CT imaging every 8 weeks before and during the study. In addition, immunological endpoints were assessed, including peripheral and intratumoral T cell ratios, T cell activation, macrophage phenotype analysis, serum cytokine profiling, tumor immunohistochemistry, and serum galactagogue-9 levels. The study was conducted under the master study protocol and lasted 12 to 24 months.

[0335] Subject, disease, and all clinical and safety data are presented descriptively as mean, median, or proportion, with appropriate measures of variance (e.g., 95% confidence interval range). Waterfall and Swimmers plots were used to graphically represent ORR and duration of response for each disease site in each study group, as described below. Exploratory correlation analyses were also performed to identify potential biomarkers that may be associated with ORR. All statistical analyses were performed using SAS, version 9.2 (SAS, Cary, NC).

[0336] (A) Research Design

[0337] Based on data from the KPC004 mouse model, the currently estimated minimum expected pharmacologically active dose (PAD) is 2 mg / kg, with 50 mcg / mouse (2 mg / kg; human equivalent dose HED = 0.16 mg / kg) determined as the active dose. Alternative models are effective within the dose range of 200 or 400 mcg / mouse (8–16 mg / kg; HED = 0.65–1.3 mg / kg).

[0338] Table 7 below shows the recommended clinical starting dose levels, which are based on the results of repeated-dose toxicity studies conforming to GLP at the recommended dose levels of 100 and 200 mg / kg in G9.2-17. The estimated starting dose uses 1 / 10 of the No Observed Adverse Effect Level (NOAEL) or 1 / 6 of the Highest Non-Serious Toxicity Dose (HNSTD) as the starting point, and then this dose (mg / kg) is converted to the HED (mg / kg).

[0339] Table 7 Recommended starting clinical dose

[0340]

[0341] This study included monotherapy with G9.2-17 (IgG4) and combination therapy including G9.2-17 and gemcitabine / Abraxane (paclitaxel protein-bound particles for injectable suspension; albumin-bound). The study was divided into two parts: Part 1 (Phase 1a) and Part 2 (Phase 1b).

[0342] Part 1

[0343] Part 1 of this study was a dose-exploration study using the Continuous Reassessment (CRM) method (O'Quigley et al., 1990), a model-based design that uses past trial data to inform how to adjust the dose of G9.2-17 for the next patient cohort. Two patients were given G9.2-17 individually, with a maximum available sample size of 24. Patients received five dose levels every two weeks until disease progression, unacceptable toxicity, or dose-limiting toxicity (DLT) occurred and the patient withdrew from the study. The dose levels were:

[0344] Dosage level 1 = 2 mg / kg;

[0345] Dosage level 2 = 4 mg / kg;

[0346] Dosage level 3 = 8 mg / kg;

[0347] Dosage level 4 = 12 mg / kg; and

[0348] Dosage level 5 = 16 mg / kg.

[0349] The dosing regimen is intravenous (IV) administration every 2 weeks (Q2W). A dose reduction of up to 25% may be used if necessary.

[0350] As a safety precaution, new patients were only added and treated after the first patient in each cohort had been treated with G9.2-17 and at least 7 days had elapsed since the initial treatment, with each dose increase. Part 1 was completed after 6 consecutive patients had received the same dose and that dose had been determined to be the optimal biological dose (OBD).

[0351] Part 2

[0352] Part 2 of this study was a Simon two-stage optimized design (six groups: pancreatic ductal adenocarcinoma (PDA), CRC, and cholangiocarcinoma). This study investigated the use of G9.2-17 alone (the study monotherapy group) and in combination with gemcitabine / Abraxane. The dose of the anti-galactotropic antibody used was lower than the levels of toxicity observed in Part 1.

[0353] The optimized two-stage design was used to test the null hypothesis of an ORR ≤ 5% within the single-drug groups compared to an ORR ≥ 15% for the alternatives. After drug testing in 23 patients in the first stage, the corresponding experimental group was terminated if ≤ 1 patient responded. If the trial continued to the second part of the Simon optimized design, a total of 56 patients were enrolled in each single-drug group. If the total number of responding patients was ≤ 5, the study drug in that group was rejected. If ≥ 6 patients had an ORR at 3 months, the expanded cohort of that group was activated. The above method applies to the study single-drug groups.

[0354] Treatment with G9.2-17 and gemcitabine / abraxane

[0355] This study evaluated the combination therapy of G9.2-17 and gemcitabine / Abraxane in patients with metastatic PDAC. The primary objective of this study was progression-free survival (PFS) at 6 months. Secondary objectives included improving objective response rate (ORR), disease control rate (DCR) at 6 and 12 months, patient survival at 6 and 12 months, time to response, duration and depth of response (according to RECIST 1.1 criteria), safety, and tolerability. In the combination group, G9.2-17 was initiated at a dose lower than the OBD identified in Part 1 (e.g., the RP2D dose level identified in Part 1). The dose of gemcitabine / Abraxane followed the FDA-approved label dosage and may be adjusted based on specific side effects of the treatment regimen (if any) (e.g., 2 weeks of administration followed by 1 week of rest). If DLT occurred in 3 or more patients, the dose of G9.2-17 was progressively reduced, not exceeding dose 3, unless the low dose continued to provide clinical benefit.

[0356] In a cohort of patients with metastatic PDAC, the primary efficacy endpoint was progression-free survival (PFS) at 6 months. In a first-line metastatic setting using gemcitabine / abraxane, a 6-month PFS rate of 50% has been reported (Von Hoff et al., 2013). In the first phase, the trial was terminated if 6 or fewer patients showed PFS ≥ 6 months after testing the G9.2-17 / chemotherapy combination in the first 11 patients. In the second phase of the trial, a total of 25 patients were studied. The study group was rejected if the total number of responders with PFS ≥ 6 months was ≤ 16.

[0357] Cohort expansion was implemented upon detection of early efficacy signals. Once a promising efficacy signal attributable to tumor type was identified in one of the five trial groups, an expanded cohort was initiated to validate the finding. The sample size for each expanded group was determined based on the point estimates identified in Part 2 and in conjunction with a predetermined level of precision for the ORR / patient survival around the 95% confidence interval (95% CI).

[0358] Part 3

[0359] Part 3 includes an expanded cohort of early efficacy signals that have been detected. If a promising efficacy signal attributable to the tumor type is identified in an experimental group, an expanded cohort is initiated to validate the finding. The sample size for each expanded group is determined based on the point estimates identified in Part 2 and in conjunction with a predetermined level of precision for the 95% confidence interval (95% CI) around the ORR.

[0360] The study period was 12 to 24 months.

[0361] (B) Patient Group

[0362] Patients with recurrent / refractory metastatic cancer, regardless of tumor type, are eligible for dose-exploration studies using the Continuous Reassessment Method (CRM) described by O'Quigley (1990). An extension in PDAC is envisioned, where the mode of action and / or early efficacy signals are obtained in Part 1.

[0363] The inclusion and exclusion criteria for patients are the same for Part 1 and Part 2.

[0364] Patient inclusion criteria:

[0365] 1. Written informed consent form

[0366] 2. Age ≥ 18 years, male or non-pregnant female

[0367] 3. Histologically confirmed unresectable metastatic carcinoma (adenocarcinoma and squamous cell carcinoma)

[0368] 4. Able to adhere to the research plan

[0369] 5. Life expectancy > 3 months

[0370] 6. Recently archived tumor samples (obtained within 5 years) that can be used for biomarker analysis.

[0371] 7. The patient is able and willing to undergo biopsies before, during, and after treatment.

[0372] 8. Diseases measurable according to RECIST v1.1. Note that the biopsy lesion should not be a target lesion.

[0373] 9. Expected survival > 3 months

[0374] 10. For Part 1 There are no available standard care options, or the patient refuses the available and designated standard care therapy, or does not meet the criteria for available and designated standard care therapy. For Part 2 PDAC extended cohort – Patients in a metastatic cancer setting who have received at least one line of systemic therapy and have not received a gemcitabine-containing regimen or have been off-treatment with a gemcitabine-containing regimen for at least 6 months. CCR and CCA extended cohorts – Patients in a metastatic setting who have received at least one prior therapy.

[0375] 11. COVID-19 vaccination is permitted before or during the study period. Information regarding the timing and type of vaccination must be recorded.

[0376] 12. Eastern Cooperative Oncology Group (ECOG) performance status 0-1 / Karnofsky score >70.

[0377] 13. Part 1 of the study allowed patients with high microsatellite instability (MSI-H) and microsatellite stability (MSS).

[0378] 14. Adequate hematological and end-organ function, defined as a neutrophil count ≥1 x 102 9 / L, platelet count ≥100x10 9 / L, for HCC ≥50x10 in Part 1 9 / L; Hemoglobin ≥ 8.5 g / dL in the previous week without transfusion, creatinine ≤ 1.5 x ULN, AST (SGOT) ≤ 3 x ULN (≤ 5 x ULN in the presence of HCC or liver metastasis), ALT (SGPT) ≤ 3 x ULN (≤ 5 x ULN in the presence of HCC or liver metastasis), bilirubin ≤ 1.5 x ULN (bilirubin ≤ 3.0 x ULN in patients with known Gilbert's disease), albumin ≥ 3.0 g / dL, INR and PTT ≤ 1.5 x ULN; amylase and lipase ≤ 1.5 x ULN

[0379] 15. There was no evidence of active infection or infection requiring parenteral antibiotics within 4 weeks prior to the start of the study, and there was no serious infection.

[0380] 16. Women of childbearing age must undergo a negative pregnancy test before entering the study.

[0381] 17. Women of childbearing age and men with fertile partners must be willing to use highly effective contraception during the trial and for three months after the completion of treatment.

[0382] 18. Four (4) weeks or five half-lives (whichever is shorter) from the last dose of anticancer treatment prior to the first administration of G9.2-17.

[0383] 19. For bone metastases that have been stable for at least 6 months prior to C1D1, continued use of bisphosphonates (zoledronic acid) or denosumab is permitted.

[0384] 20. For the CCR and CCA extended cohorts, at least one prior therapy in a metastatic setting is required.

[0385] twenty one. For Part 1 Hepatocellular carcinoma progresses on at least one prior systemic therapy (including sorafenib, lenvatinib, nivolumab, atezolizumab, and bevacizumab), or is intolerant to or refuses sorafenib treatment after progression on standard therapy (including surgery and / or local regional therapy), or standard therapy is deemed ineffective, intolerable, or inappropriate, or no effective standard therapy is available.

[0386] 22. Biliary or gastric outlet obstruction is permissible, provided that drainage is effectively achieved through endoscopy, surgery, or interventional procedures.

[0387] 23. Pancreatic fistula, bile fistula or intestinal fistula is permissible, provided they are controlled with appropriate, non-infectious and open drainage tubes (openness needs to be confirmed before the start of the study if there are any drainage tubes or stents in situ).

[0388] Patient exclusion criteria:

[0389] 1. Patients diagnosed with metastatic cancer of unknown primary origin.

[0390] 2. The patient is unwilling or unable to comply with the protocol requirements.

[0391] 3. Previous or current addiction to illegal drugs.

[0392] 4. Clinically significant, uncontrolled active bleeding, and any patient with a bleeding predisposition (e.g., active peptic ulcer disease). Prophylactic or therapeutic use of anticoagulants is permitted.

[0393] 5. Breastfeeding women.

[0394] 6. Received any other investigational drug or participated in any other clinical trial involving the treatment of solid tumors with other investigational drugs within 4 weeks prior to study dosing or within 5 half-lives of the drug administration (whichever is shorter).

[0395] 7. Radiotherapy within 4 weeks after the first dose of the study drug, except for palliative radiotherapy to a limited area, such as for treating bone pain or focal pain in tumor masses.

[0396] 8. Patients with mushroom-shaped tumor masses.

[0397] 9. Patients with locally advanced PDAC.

[0398] 10. Due to prior cancer treatment ≥ CTCAE Grade 3 toxicity (excluding alopecia and vitiligo). Grade 4 immune-mediated toxicity from prior checkpoint inhibitor use. Grade 2 or 3 pneumonia or any other Grade 3 checkpoint inhibitor-related toxicity leading to discontinuation of immunotherapy. Low-grade (< Grade 3) toxicities are permissible, such as neuropathy, manageable electrolyte abnormalities, and lymphopenia caused by prior treatment.

[0399] 11. History of secondary malignant tumors, except for those treated for curative purposes for more than 5 years prior with no recurrence or a low probability of recurrence (e.g., non-melanoma skin cancer, cervical carcinoma in situ, prostate cancer, or superficial bladder cancer).

[0400] 12. Severe or uncontrolled systemic disease, congestive heart failure > New York Heart Association (NYHA) Class 2, evidence of myocardial infarction (MI) within 6 months, or given that the investigator considers the patient unsuitable for participation in the trial based on laboratory findings.

[0401] 13. Any medical condition that the investigator believes seriously impairs patient safety or undermines the interpretation of the G9.2-17 toxicity assessment.

[0402] 14. Severe unhealed wounds, active ulcers, or untreated fractures

[0403] 15. Uncontrolled pleural effusion, pericardial effusion, or ascites requiring repeated drainage procedures.

[0404] 16. History of severe allergy, anaphylactic reaction, or other hypersensitivity reaction to chimeric or humanized antibodies or fusion proteins.

[0405] 17. Major vascular disease (e.g., aortic aneurysm requiring surgical repair or recent arterial thrombosis) occurring within 6 months of day 1 of cycle 1.

[0406] 18. History of pulmonary embolism, stroke, or transient ischemic attack within 3 months prior to day 1 of cycle 1.

[0407] 19. History of abdominal fistula or gastrointestinal perforation within 6 months prior to day 1 of cycle 1.

[0408] 20. Active autoimmune diseases (excluding type 1 diabetes, hypothyroidism requiring only hormone replacement, vitiligo, psoriasis, or alopecia)

[0409] 21. Systemic immunosuppressive therapy is required, including but not limited to cyclophosphamide, azathioprine, methotrexate, thalidomide, and anti-tumor necrosis factor [anti-TNF] agents. Patients who have received or are currently receiving acute, low-dose systemic immunosuppressants (e.g., dexamethasone 4 mg) are eligible. Replacement therapies (e.g., thyroid hormone, insulin, or physiological corticosteroid replacement therapy for adrenal or pituitary insufficiency [e.g., prednisone equivalent ≤10 mg / day]) are not considered forms of systemic therapy. Inhaled corticosteroids and mineralocorticoids (e.g., fludrocortisone), topical steroids, intranasal steroids, intra-articular steroids, and ophthalmic steroids are permitted.

[0410] 23. Tumor-related pain (> grade 3) that is unresponsive to extensive analgesic interventions (oral and / or patch).

[0411] 24. Uncontrolled hypercalcemia despite the use of bisphosphonates.

[0412] 25. Any other disease, metabolic disorder, physical examination results, or clinical laboratory results that reasonably suggest a disease or condition that precludes the use of investigational drugs or may affect the interpretation of the results or place the patient at high risk of treatment complications.

[0413] 26. Received an organ transplant.

[0414] 27. Dialysis is in progress.

[0415] 28. For Part 1, continued hormone androgen deprivation therapy is permitted for subjects with metastatic castration-resistant pancreatic cancer.

[0416] Additional exclusion criteria for subjects with hepatobiliary carcinoma (HCC):

[0417] 1. Any ablation therapy (radiofrequency ablation or percutaneous ethanol injection) for HCC within 6 weeks prior to entering the trial.

[0418] 2. Hepatic encephalopathy or severe hepatic adenoma

[0419] 3. Child-Pugh score ≥ 7

[0420] 4. Metastatic hepatocellular carcinoma that has progressed while receiving at least one prior systemic therapy (including sorafenib), or that is intolerant to or refuses sorafenib treatment after progression with standard therapy (including surgery and / or local regional therapy), or that standard therapy is deemed ineffective, intolerable, or inappropriate, or that no effective standard therapy is available.

[0421] 5. Biliary or gastric outlet obstruction is permissible, provided that drainage is effectively achieved through endoscopic, surgical, or interventional methods.

[0422] 6. Pancreatic fistula, bile fistula, or intestinal fistula are permissible, provided they are controlled with appropriate, non-infectious, and open drainage tubes (openness needs to be confirmed before the start of the study if there are any drainage tubes or stents in situ).

[0423] The patient should discontinue treatment if one or more of the following conditions occur:

[0424] Pregnant

[0425] Unmanageable toxicity

[0426] Symptom exacerbation attributed to disease progression, determined by researchers after a comprehensive assessment of radiological data, biopsy results, and clinical status.

[0427] Intolerable toxicities associated with G9.2-17, including the development of irreversible adverse events (irAEs) determined by investigators to be unacceptable due to the individual patient's likely response to the therapy and the severity of the events.

[0428] If the patient continues to receive research treatment, any medical conditions that may jeopardize the patient's safety will be considered.

[0429] Using another non-formula cancer treatment

[0430] (C) Objective

[0431] Part 1 (Issue 1a)

[0432] Primary objectives: safety, tolerability, optimal biological dose (OBD) or maximum administered dose (MAD), and recommended phase 2 dose (RP2D).

[0433] Secondary objectives: pharmacokinetic (PK), pharmacodynamic (PD) parameters, immunogenicity

[0434] Exploratory goals: In addition to the following exploratory endpoints, the exploratory endpoints for Part 1 are: objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), and patient survival at 3 months (Part 1), 6 months, and 12 months (Part 1 and Part 2).

[0435] Part 2 (Phase 1b) of CRC and CCA

[0436] Primary objective: Objective response rate (ORR)

[0437] Secondary objectives: progression-free survival (PFS), disease control rate (DCR), duration and depth of response according to RECIST 1.1, patient survival at 6 and 12 months, time to response, safety, and tolerability.

[0438] Part 2 (Phase 1b) of PDAC

[0439] Primary objective: Progression-free survival (PFS) at 6 months.

[0440] Secondary objectives: Objective response rate (ORR), disease control rate (DCR) at 6 and 12 months, patient survival at 6 and 12 months, time to response, duration and depth of response according to RECIST 1.1 criteria, safety and tolerability.

[0441] Exploratory endpoints for all research sections:

[0442] The iRECIST criteria include immunophenotypic analysis from blood and tumors, cytokine profiles (serum), soluble galactagogue-9 levels in blood (serum or plasma), galactagogue-9 expression levels and immunohistochemical expression patterns (tumor, stroma, immune cells) of galactagogue-9, tumor mutational burden (TMB), immunohistochemical PDL-1 expression, mismatch repair status, disease-related tumor markers, ctDNA, and the correlation between these parameters and response. Time to response (TTR) is also considered. Quality of life and symptom control are also assessed.

[0443] (D) Research Procedures

[0444] (i) Assessment schedule

[0445] The assessment schedule is divided into four-week cycles following one pre-dosing screening cycle (which can occur up to four weeks before treatment begins). Table 8 lists the pre-dosing screening assessments and tests, and shows which ones will be performed during the treatment cycles. Optional access is permitted during each cycle if there is a medical indication, during which any research assessments may be conducted.

[0446] (ii) Screening and evaluation procedures

[0447] The following procedures must be performed within 4 weeks of starting treatment (as outlined in Table 8. Assessment Schedule):

[0448] Written informed consent form

[0449] Verify the selection and exclusion criteria

[0450] Previous COVID-19 infection history and most recent RT PCR and / or SARS CoV2 IgG / IgM test results (if performed).

[0451] Record the patient's intention to receive the COVID-19 vaccine (if available).

[0452] Record seasonal influenza vaccination status

[0453] Preferred tumor imaging assessment CT with or without contrast agents; if necessary, MRI with or without contrast agents; if necessary, PET-CT (diagnostic CT) based on investigator's judgment.

[0454] Tumor biopsy (before first administration and repeat biopsy) – as scheduled by scanning

[0455] For pre-screening of galactagogue-9 expression via immunohistochemistry, archived tumor tissue (if available) can be used, provided it was obtained within a 5-year timeframe and details of treatment administered after tissue acquisition are known and documented. This is not a prerequisite for inclusion, and researchers will provide archived specimens whenever possible.

[0456] Tumor markers relevant to the tumor type, such as CA15-3, CA-125, CEA, CA19-9, and alpha-fetoprotein, will be evaluated before each cycle of dosing (this may be reduced to every 3 cycles after 6 months of treatment, following the same schedule as the re-staging scan), depending on the situation.

[0457] Patient demographic data

[0458] Personal medical history, including previous treatments / surgeries, records of any in-situ implants or past implants, previous and / or current medical devices, concomitant medications (name, indication, dosage, route of administration, start and end dates and reasons for dosage adjustments (if any), pre-existing symptoms and adverse events), and risky hereditary diseases based on family history and a complete family history best known to the patient.

[0459] Any and all previously obtained test results (next-generation and / or whole-exome sequencing results, circulating tumor DNA test, germline sequencing results, DPD test results, G6PD test results, Oncotype Dx and / or Endopredict test results, common molecular subtype (CMS) classification, DXA scan (if any)). These are not prerequisites for inclusion.

[0460] Records of any dental / root canal or eye surgery performed in the past 12 months

[0461] History of mandibular or maxillary osteonecrosis

[0462] Any history of port-a-cath infection requiring prior intravenous antibiotics and / or antifungal agents, or port-a-cath replacement.

[0463] If the investigator's assessment indicates that the patient is dehydrated, oral and / or intravenous rehydration is permitted and recommended prior to any cycle of administration. The investigator may decide to perform a BUN test on the day of administration to guide decision-making.

[0464] Physical examination and visual signs record

[0465] ECHO, Ejection Fraction (EF)

[0466] 12-lead ECG

[0467] Record of the location and condition / size of any keloid scar.

[0468] Neurological examination in patients with stable, pre-treated brain metastases

[0469] Records of any dietary requirements or preferences (e.g., implementation of specific dietary plans: intermittent fasting, ketogenic diet, etc.)

[0470] Supplement use (current and past 12 months), type of use, duration of use, dosage, and frequency

[0471] ECOG and / or Karnofsky status

[0472] Vital signs (body temperature, heart rate, blood pressure, respiratory rate, and body weight) after 5 minutes of lying supine.

[0473] For women of childbearing age, a pregnancy test is required. If there is a history of bilateral salpingo-oophorectomy and / or hysterectomy, this is not necessary, but these procedures should be documented.

[0474] Past and / or current allergy history (allergens, severity)

[0475] Complete blood count (CBC), differential count, platelets, hemoglobin

[0476] Blood chemistry (glucose, total protein, albumin, electrolytes [sodium, potassium, chloride, total CO2], calcium, phosphorus, magnesium, uric acid, bilirubin (total bilirubin, direct bilirubin), SGPT (ALT) or SGOT (AST), alkaline phosphatase, bilirubin, lactate dehydrogenase (LDH), creatinine, blood urea nitrogen, CPK, TSH, fT4, lipase, amylase, PTH, FSH, LH, CRP, and / or troponin)

[0477] Coagulation (PT, PTT, APTT)

[0478] Urine analysis

[0479] PD Blood-Biomarker Analysis

[0480] PD Tumors: Biomarker Analysis of Pre-treatment Biopsy

[0481] (iii) Ongoing projects

[0482] Ensure PD blood biomarker analysis is performed at each blood draw; perform tumor biomarker analysis in pre- and during / post-study biopsies. Screening procedures must include and document neurological examinations. Any grade >2 irAE will be referred to the relevant specialist and documented accordingly. Management of irAEs will be conducted in accordance with the management of immunotherapy-related toxicities (NCCN Guideline Version 1.2020). Study-related procedures and assessments performed during study treatment are detailed below and in Table 8 (Assessment Schedule).

[0483] For COVID-19 infections diagnosed during treatment, investigators and sponsors will follow FDA guidance and local policies, and investigators should contact the medical monitor to discuss the best course of action.

[0484] Cycle 1 Procedure

[0485] (a) Day 1 of Cycle 1

[0486] After all previous screening and baseline procedures are completed, the following procedures will be performed on Day 1.

[0487] 12-lead ECG

[0488] Physical examination

[0489] ECOG

[0490] Vital signs (body temperature, heart rate, blood pressure, respiratory rate, respiratory rate, including weight and / or body salinity) after 5 minutes of supine position.

[0491] Concomitant medications (name, indication, dosage, route of administration, start and end dates, any and all dosage adjustments, their timing and reason)

[0492] Adverse events

[0493] Complete blood count (CBC), differential count, platelets, hemoglobin

[0494] All C1D1 results within 7 days: Blood chemistry (glucose, Hgb, A1c (if there is a history of DM1 or DM2), total protein, albumin, electrolytes [sodium, potassium, chloride, total CO2], calcium, phosphorus, magnesium, uric acid, bilirubin (total bilirubin, direct bilirubin), SGPT (ALT) or SGOT (AST), alkaline phosphatase, bilirubin, lactate dehydrogenase (LDH), creatinine, blood urea nitrogen, CPK) – Fasting glucose is only performed before administration if clinically indicated. TSH, fT4, lipase, amylase, PTH, FSH, LH, CRP, troponin

[0495] Relevant tumor markers, such as CA15-3, CA-125, CEA, CA19-9, and alpha-fetoprotein, will be evaluated before each cycle of dosing (this can be reduced to every 3 cycles after 6 months of treatment, following the same schedule as re-staging scans), depending on the situation.

[0496] Coagulation (PT, PTT, APTT)

[0497] Urine analysis (including protein and culture / antibacterial spectrum)

[0498] PD Blood Biomarker Analysis (Soluble Gagrenin 9, Tissue IHC of Gagrenin-9 from Pretreatment Biopsy and Immunophenotyping Analysis)

[0499] PK blood samples at the annotated time points in Table 8

[0500] (b) Day 2 of Cycle 1

[0501] The following procedure will be performed on the second day of the first cycle.

[0502] Concomitant medications (name, indication, dosage, route of administration, start and end dates)

[0503] Adverse events

[0504] PD Blood-Biomarker Analysis

[0505] PK blood samples

[0506] (c) Day 4 of Cycle 1

[0507] The following procedure will be performed on the 4th day of the first cycle.

[0508] Concomitant medications (name, indication, dosage, route of administration, start and end dates)

[0509] Adverse events

[0510] PD Blood-Biomarker Analysis

[0511] PK blood samples

[0512] (d) Cycle 1, Day 7 Procedure

[0513] The following procedure will be performed on the 7th day of the first cycle.

[0514] Vital signs (body temperature, heart rate, blood pressure, respiratory rate, and body weight) after 5 minutes of lying supine.

[0515] Concomitant medication

[0516] Adverse events

[0517] Complete blood count (CBC), differential count, platelets, hemoglobin

[0518] Blood chemistry (glucose, total protein, albumin, electrolytes [sodium, potassium, chloride, total CO2], calcium, phosphorus, magnesium, uric acid, bilirubin (total bilirubin, direct bilirubin), SGPT (ALT) or SGOT (AST), alkaline phosphatase, bilirubin, lactate dehydrogenase (LDH), creatinine, blood urea nitrogen, CPK)

[0519] PD Tumor - Biomarker Analysis

[0520] PK blood samples

[0521] (e) Cycle 1, Day 15

[0522] Perform the following procedure on day 15 of cycle 1.

[0523] Complete blood count (CBC), differential count, platelets, hemoglobin

[0524] Blood chemistry (glucose, total protein, albumin, electrolytes [sodium, potassium, chloride, total CO2], calcium, phosphorus, magnesium, uric acid, bilirubin (total bilirubin, direct bilirubin), SGPT (ALT) or SGOT (AST), alkaline phosphatase, bilirubin, lactate dehydrogenase (LDH), creatinine, blood urea nitrogen, CPK) ECOG

[0525] Vital signs (body temperature, heart rate, blood pressure, respiratory rate, and body weight) after 5 minutes of lying supine.

[0526] Adverse events

[0527] PD Blood-Biomarker Analysis

[0528] PK blood samples

[0529] Second cycle procedure

[0530] (a) Day 1 of Cycle 2

[0531] Perform the following procedure on day 1 of cycle 2.

[0532] Complete blood count (CBC), differential count, platelets, hemoglobin

[0533] Blood chemistry (glucose, total protein, albumin, electrolytes [sodium, potassium, chloride, total CO2], calcium, phosphorus, magnesium, uric acid, bilirubin (total bilirubin, direct bilirubin), SGPT (ALT) or SGOT (AST), alkaline phosphatase, bilirubin, lactate dehydrogenase (LDH), creatinine, blood urea nitrogen, CPK), TSH, fT4

[0534] Relevant tumor markers, such as CA15-3, CA-125, CEA, CA19-9, and alpha-fetoprotein, will be evaluated before each cycle of dosing (this can be reduced to every 3 cycles after 6 months of treatment, following the same schedule as the re-staging scan), depending on the situation.

[0535] Physical examination

[0536] Adverse events

[0537] ECOG

[0538] Vital signs (body temperature, heart rate, blood pressure, respiratory rate, and body weight) after 5 minutes of lying supine.

[0539] PK blood samples

[0540] PD Blood-Biomarker Analysis

[0541] Concomitant medications (name, indication, dosage, route of administration, start and end dates, any and all dosage adjustments, their timing and reason)

[0542] (b) Cycle 2, Day 7 Procedure

[0543] The following procedure will be performed on the 7th day of the 2nd cycle.

[0544] 12-lead ECG

[0545] Vital signs (body temperature, heart rate, blood pressure, respiratory rate, and body weight) after 5 minutes of lying supine.

[0546] Concomitant medications (name, indication, dosage, route of administration, start and end dates)

[0547] Adverse events

[0548] Complete blood count (CBC), differential count, platelets, hemoglobin

[0549] Blood chemistry (glucose, total protein, albumin, electrolytes [sodium, potassium, chloride, total CO2], calcium, phosphorus, magnesium, uric acid, bilirubin (total bilirubin, direct bilirubin), SGPT (ALT) or SGOT (AST), alkaline phosphatase, bilirubin, lactate dehydrogenase (LDH), creatinine, blood urea nitrogen, CPK)

[0550] PD Blood-Biomarker Analysis

[0551] PK blood samples

[0552] Pregnancy test, if the woman is of childbearing age and her ovaries and uterus are in their original position.

[0553] (c) Cycle 2, Day 15

[0554] Perform the following procedure on day 15 of cycle 2.

[0555] Re-staging scans (CT, MRI, PET-CT, or X-ray with contrast agents) – can be performed 6 to 8 weeks after the start of study drug administration, scheduled as an additional separate visit.

[0556] Tumor biopsy -3 / +12 days, if feasible and scheduled as a stand-up visit / can be performed concurrently with scanning, as imaging guidance may be required to facilitate tissue sample acquisition (biopsy of target lesions should not be performed).

[0557] PD Tumor-Biomarker Analysis

[0558] ECOG

[0559] Vital signs (body temperature, heart rate, blood pressure, respiratory rate, and body weight) after 5 minutes of lying supine.

[0560] Adverse events

[0561] PK blood samples

[0562] PD Blood-Biomarker Analysis

[0563] 3rd cycle procedure

[0564] (a) Day 1 of Cycle 3

[0565] Perform the following procedure on day 1 of cycle 3.

[0566] Complete blood count (CBC), differential count, platelets, hemoglobin

[0567] Blood chemistry (glucose, total protein, albumin, electrolytes [sodium, potassium, chloride, total CO2], calcium, phosphorus, magnesium, uric acid, bilirubin (total bilirubin, direct bilirubin), SGPT (ALT) or SGOT (AST), alkaline phosphatase, bilirubin, lactate dehydrogenase (LDH), creatinine, blood urea nitrogen, CPK), TSH, fT4

[0568] Relevant tumor markers, such as CA15-3, CA-125, CEA, CA19-9, and alpha-fetoprotein, will be evaluated before each cycle of administration (this can be reduced to every 3 cycles after 6 months of treatment, following the same schedule as the re-staging scan), depending on the situation.

[0569] Physical examination

[0570] ECOG

[0571] Vital signs (body temperature, heart rate, blood pressure, respiratory rate, and body weight) after 5 minutes of lying supine.

[0572] Concomitant medications (name, indication, dosage, route of administration, start and end dates)

[0573] Adverse events

[0574] PD Blood-Biomarker Analysis

[0575] PK blood samples

[0576] Pregnancy test, if the woman is of childbearing age

[0577] (b) Day 7 of Cycle 3

[0578] Perform the following procedure on day 7 of cycle 3.

[0579] ECHO

[0580] Vital signs (body temperature, heart rate, blood pressure, respiratory rate, and body weight) after 5 minutes of lying supine.

[0581] Concomitant medications (name, indication, dosage, route of administration, start and end dates)

[0582] Adverse events

[0583] Complete blood count (CBC), differential count, platelets, hemoglobin

[0584] Blood chemistry (glucose, total protein, albumin, electrolytes [sodium, potassium, chloride, total CO2], calcium, phosphorus, magnesium, uric acid, bilirubin (total bilirubin, direct bilirubin), SGPT (ALT) or SGOT (AST), alkaline phosphatase, bilirubin, lactate dehydrogenase (LDH), creatinine, blood urea nitrogen, CPK)

[0585] PD Blood-Biomarker Analysis

[0586] PK blood samples

[0587] (c) Cycle 3, Day 15

[0588] Perform the following procedure on day 15 of cycle 3.

[0589] ECOG

[0590] Vital signs (body temperature, heart rate, blood pressure, respiratory rate, and body weight) after 5 minutes of lying supine.

[0591] Adverse events

[0592] PK blood samples

[0593] PD Blood-Biomarker Analysis

[0594] Procedures from the 4th cycle onwards

[0595] (a) Day 1 of Cycle 4

[0596] The following procedure is performed on day 1 of cycle 4 and in subsequent cycles.

[0597] Complete blood count (CBC), differential count, platelets, hemoglobin

[0598] Blood chemistry (glucose, total protein, albumin, electrolytes [sodium, potassium, chloride, total CO2], calcium, phosphorus, magnesium, uric acid, bilirubin (total bilirubin, direct bilirubin), SGPT (ALT) or SGOT (AST), alkaline phosphatase, bilirubin, lactate dehydrogenase (LDH), creatinine, blood urea nitrogen, CPK), TSH, fT4, lipase, amylase, PTH, troponin, FSH, LH, CRP)

[0599] Re-staged scans (CT, MRI, PET-CT, or X-ray with contrast agents) – can be completed 6 to 8 weeks after the start of investigational drug administration.

[0600] Relevant tumor markers, such as CA15-3, CA-125, CEA, CA19-9, and alpha-fetoprotein, will be evaluated before each cycle of dosing (this can be reduced to every 3 cycles after 6 months of treatment, following the same schedule as the re-staging scan), depending on the situation.

[0601] Physical examination

[0602] ECOG

[0603] Adverse events

[0604] PK blood samples

[0605] PD Blood-Biomarker Analysis

[0606] Pregnancy test, if the woman is of childbearing age

[0607] (b) The procedure for day 7 of cycle 4 (same as C3 D7)

[0608] The following procedure will be performed on day 7 of cycle 4.

[0609] (c) The procedure for day 15 of cycle 4 (same as C3D15)

[0610] The following procedure will be performed on day 15 of cycle 4.

[0611] (iv) Study termination or early termination procedures

[0612] The following procedure is performed on day 59 or 30 days after the last dose, including patients who discontinue treatment early.

[0613] Re-staged scanning (preferably CT with or without contrast agent, MRI with or without contrast agent, or PET-CT if the investigator requires) – if the study ends >6 to 8 weeks after the last cycle and the interval is shorter, the investigator decides to repeat the study.

[0614] Relevant tumor markers, such as Ca15-3, CA-125, CEA, CA19-9, and alpha-fetoprotein, will be evaluated before each cycle of dosing (this can be reduced to every 3 cycles after 6 months of treatment, following the same schedule as the re-staging scan), depending on the situation.

[0615] 12-lead ECG

[0616] Physical examination

[0617] ECOG

[0618] Vital signs (body temperature, heart rate, blood pressure, respiratory rate, and body weight) after 5 minutes of lying supine.

[0619] Concomitant medications (name, indication, dosage, route of administration, start and end dates)

[0620] Adverse events

[0621] A pregnancy test, if the mother is female and her ovaries and uterus are in situ.

[0622] Complete blood count (CBC), differential count, platelets, hemoglobin

[0623] Blood chemistry (glucose, total protein, albumin, electrolytes [sodium, potassium, chloride, total CO2], calcium, phosphorus, magnesium, uric acid, bilirubin (total bilirubin, direct bilirubin), SGPT (ALT) or SGOT (AST), alkaline phosphatase, bilirubin, lactate dehydrogenase (LDH), creatinine, blood urea nitrogen, CPK), TSH, fT4, PTH, estradiol, prolactin, testosterone, FSH, LH)

[0624] Coagulation (PT, PTT)

[0625] Urine analysis

[0626] PD Blood-Biomarker Analysis

[0627] PK blood samples

[0628] (v) Long-term follow-up

[0629] Once patients complete their treatment, overall survival will be monitored every 3 months for up to 2 years. For patients who withdraw due to clinical progression, radiological evaluation will continue as possible.

[0630] Survival data, as well as information on any new anticancer treatments initiated after disease progression, are collected approximately every 3 months. Follow-up can be conducted via telephone interviews or case reviews and reported on a case report form. During follow-up, deaths (regardless of causation) and serious adverse events deemed relevant to the study treatment are collected and reported within 24 hours of discovery or event notification.

[0631] (vi) Research Evaluation

[0632] (a) Physical examination

[0633] Medical and physical examinations must be performed by qualified physicians, nurse practitioners, or physician assistants and should include a thorough examination of the entire body during screening, treatment, and at the end of the study. Physical examinations include a breast examination (if clinically indicated) and vital signs—temperature, heart rate (HR), blood pressure (BP), and respiratory rate (RR)—measured after the patient has rested in a supine position for 5 minutes. Patient weight is also measured and recorded.

[0634] (b) Medical history

[0635] Medical history includes cancer history, radiation therapy history, surgical history, and current and past medication history.

[0636] Personal medical history, including previous treatments / surgeries, records of any in-situ implants or past implants, previous and / or current medical devices, concomitant medications (name, indication, dosage, route of administration, start and end dates, dosage modifications (if any) and reason), pre-existing symptoms and adverse events, and risky hereditary diseases based on family history and a complete family history best known to the patient.

[0637] Any and all previously obtained test results (next-generation and / or whole-exome sequencing results, circulating tumor cell-free DNA test results, germline sequencing results, DPD test results, G6PD test results, Oncotype Dx and / or Endopredict test results)

[0638] Any dental treatment records performed in the past 12 months

[0639] Record the location, condition, and size of any keloid scars.

[0640] For patients who have previously undergone resection of pancreatic adenocarcinoma, record whether the primary tumor was located in the pancreatic head, body, or tail.

[0641] Bowel habits / typical frequency and consistency

[0642] (c) Clinical laboratory assessment

[0643] According to the assessment schedule, patient blood samples will be collected for routine clinical laboratory testing. Clinical laboratory parameters will be analyzed at the local laboratory on-site. The laboratory assessments to be completed include hematology and serology, defined as follows:

[0644] Serum chemistry: including glucose, total protein, albumin, electrolytes [sodium, potassium, chloride, total CO2], calcium, phosphorus, magnesium, uric acid, bilirubin (total bilirubin, direct bilirubin), SGPT (ALT) or SGOT (AST), alkaline phosphatase, bilirubin, lactate dehydrogenase (LDH), creatinine, HgbA1c, blood urea nitrogen, CPK, TSH, fT4, lipase, amylase, PTH, testosterone, estradiol, prolactin, FSH, LH, and CRP. Fasting glucose should be administered on C1D1, C2D1, C3D1, C4D1, and other days (only if clinically indicated).

[0645] Hematology: including complete blood cell count, differential count, platelet count, and hemoglobin.

[0646] Coagulation includes prothrombin time (PT), partial prothrombin kinase time (PTT), and activated partial prothrombin kinase time (APTT).

[0647] Biomarker analysis (PD blood): including galactagogue-9 levels in patient serum / plasma, peripheral blood immunophenotyping analysis, and cytokine measurement.

[0648] Pharmacokinetic (PK) blood sampling: If the investigator determines that administration of the study drug should be interrupted, additional PK and safety assessments will be collected before administration (within 2 hours of administration) and 4 hours + / - 30 minutes after administration. Centers unable to accommodate patients for more than 2 hours after administration due to COVID-19 restrictions will only provide samples 2 hours after administration.

[0649] If administration is interrupted for any reason and then resumed, additional PK assessments may be performed during the interruption period at the investigator's discretion. If the dose of the study drug is reduced after administration is resumed, additional PK assessments will be collected before resumption of administration and 2 hours + / - 15 minutes after administration. Additional PK and other hematologic assessments may be performed if there is a clinical indication at the investigator's discretion.

[0650] Where possible, blood may be obtained for additional PK or PD assessments up to 4 weeks after the last administration of the study drug to patients discontinuing the study, approximately every 7 to 14 days. Blood for PK assessments may be collected before administration, 2 hours + / - 15 minutes after administration, and 4 hours (+ / - 15 minutes) after administration of the study drug.

[0651] (d) Urine analysis

[0652] Urine samples were collected from patients for routine urinalysis. The urinalysis included color, appearance and specific gravity test strips, protein, leukocyte esterase, glucose, ketones, urobilinogen, nitrite, WBC, RBC, and pH, as well as urine cultures at the time of screening.

[0653] (e) Electrocardiogram (ECG)

[0654] Evaluate the following parameters from a 12-lead electrocardiogram: heart rate, PR interval, QRS duration, QT interval, and QTcF interval.

[0655] (f) Tumor imaging assessment

[0656] Contrast-enhanced CT is the preferred method (if CT is not feasible or appropriate, MRI, PET-CT, and / or other imaging modalities may be used to replace or supplement CT scans, taking into account the location of the disease). Evaluation should include at least the neck / chest / abdomen / pelvis and should include other anatomical regions as indicated by the patient's tumor type and medical history. Imaging scans must be de-identified and archived in their original DICOM format as part of the patient's study file. While the type of scan obtained is determined by the investigator to be appropriate for the disease, the same approach should be used throughout the study. Evaluations should be performed every 6 to 8 weeks + / - 1 week, or at the end of treatment if no evaluation has been performed in the past 4 to 6 weeks.

[0657] (g) Tumor biopsy

[0658] Collect biopsies before and during / after treatment. Pre-treatment samples should be collected prior to the first dose. During treatment, collection can be performed on any treatment day after cycle 1 where a biopsy is feasible. The next biopsy is preferably performed before the first study scan. If the procedure cannot be performed within the timeframe specified in the protocol, alternative procedures may be permitted, but must be discussed with the study director / medical monitor. Several clinical factors are generally considered to contribute to difficulties in obtaining sufficient specimens. The decision not to complete a biopsy during treatment should be discussed with the medical monitor.

[0659] (h) Tumor markers

[0660] Exploratory biomarkers, such as CA15-3, CA-125, CEA, CA19-9, and alpha-fetoprotein, should be evaluated before each cycle of administration (this can be reduced to every 3 cycles after 6 months of treatment, following the same schedule as re-staging scans), depending on the situation.

[0661] (i) Adverse events

[0662] Record any adverse events (AEs) that begin or worsen after administration of the study drug. AEs should be monitored until they subside to baseline, stabilize, or are deemed irreversible. All serious AEs (SAEs) must be collected from the date of the patient's written consent until 30 days after discontinuation of administration or cessation of study participation (if the last scheduled visit occurs at a later time).

[0663] Table 8. Evaluation Timeline

[0664]

[0665]

[0666] A Study drug administration: Treatment was administered on day 1 and day xxx of cycle 1, and patients were assessed for 4 hours as outpatients.

[0667] B Re-staging scans (CT, MRI, PET-CT, or X-ray): CT with contrast agent is preferred (MRI if CT is not feasible or appropriate at a given disease location). Evaluation should include at least the neck / chest / abdomen / pelvis and should include other anatomical regions indicated based on the patient's tumor type and medical history. Imaging scans must be de-identified and archived in their original DICOM format as part of the patient's study file. While the type of scan obtained is determined by the investigator to be appropriate for the disease, the same approach should be used throughout the study. Evaluations should be performed every 6 to 8 weeks + / - 1 week and at the end of treatment (if no evaluation has been performed in the past 4 to 6 weeks).

[0668] C Tumor biopsy: Biopsies are collected before and during / after treatment. Pre-treatment biopsies are collected prior to the first dose. During treatment, biopsies may be collected on any treatment day after cycle 1, if feasible. The next biopsy is preferably performed before the first study scan. If the procedure cannot be performed within the timeframe specified in the protocol, alternative procedures may be permitted, but must be discussed with the study director / medical monitor. Several clinical factors are generally considered to contribute to difficulties in obtaining sufficient specimens. The decision not to perform a biopsy during treatment should be discussed with the medical monitor.

[0669] D Relevant tumor markers: Exploratory markers, such as CA15-3, CA-125, CEA, CA19-9, alpha-fetoprotein, etc., should be evaluated before each cycle of administration (after 6 months of treatment, the evaluation can be reduced to every 3 cycles, following the same schedule as the re-staging scan), depending on the situation.

[0670] E Demographic data: including date of birth, sex, height, race, and ethnicity.

[0671] F Medical history: including history of cancer, history of radiation therapy, history of surgery, and current and past medication history.

[0672] G MUGA / ECHO: Repeat tests will only be collected if there is a clinical indication during the study period.

[0673] H Physical examination: If there are clinical indications, including breast examination

[0674] I Concomitant medications: Collect name, indication, dosage, route of administration, start and end dates.

[0675] J Adverse events: Record the onset or worsening of the study drug after administration. AEs should be monitored until they subside to baseline, stabilize, or are deemed irreversible. All serious AEs (SAEs) must be collected from the date of the patient's written consent until 30 days after discontinuation of administration or the patient's study participation (if the last scheduled visit occurs at a later time).

[0676] K Pregnancy test: Must have an HCG sensitivity ≤ IU / L or an equivalent unit of HCG, and must be performed within 24 hours of the first treatment cycle.

[0677] L Hematology: Complete blood count, differential count, platelets, hemoglobin

[0678] MBiochemistry: Glucose, total protein, albumin, electrolytes [sodium, potassium, chloride, total CO2], calcium, phosphorus, magnesium, uric acid, bilirubin (total bilirubin, direct bilirubin), SGPT (ALT) or SGOT (AST), alkaline phosphatase, bilirubin, lactate dehydrogenase (LDH), creatinine, blood urea nitrogen, CPK

[0679] N Coagulation, glucose, and urine analysis: collect PT, PTT, glucose, and UA. Collections in cycle 3 and beyond will only be performed when there are clinical indications (e.g., signs of bleeding, especially gastrointestinal bleeding). Fasting glucose should only be administered on C1D1, C3D1, and other clinically indicated days.

[0680] O PD blood – Biomarker analysis: gene expression, metabolites, oxygen consumption rate (OCR), other biomarker analysis, and PDX formation. Additional cycles are performed according to the same schedule as the re-staging scan.

[0681] P PK Blood Samples: If the investigator decides that the dose of the study drug should be interrupted, additional PK and safety assessments should be collected before administration (within 2 hours of administration) and 4 hours + / - 30 minutes after administration of the study drug upon resumption of administration; additional PK assessments may be performed at the investigator's discretion during the interruption period. If the dose of the study drug is reduced, additional PK assessments should be collected before administration (within 2 hours of administration) and after the start of the dose reduction. Additional PK and other blood assessments may be performed at the investigator's discretion if there is a clinical indication. Where possible, PK blood samples may be obtained for additional PK or PD assessments up to 4 weeks after the last administration of the study drug to patients discontinuing the study. In addition to the time points shown in the assessment schedule, the investigator may obtain blood for additional PK assessments at the investigator's discretion.

[0682] @ Optional access is permitted within each cycle (if medically indicated), during which any research evaluation may be conducted.

[0683] §LYT-200 should be applied every two weeks.

[0684] Administration: The dose is administered on day 1 and day 15 of cycle 1; thereafter, it will be administered according to this schedule.

[0685] 1 Data were collected before administration (within 2 hours of administration) and 4 hours (+ / - 30 minutes) after administration of the study drug.

[0686] 2Data were collected before administration (within 2 hours of administration) and at 2, 4, 6, 8, and 12 hours (+ / - 15 minutes) after administration of the study drug.

[0687] 3 Collect before administration (within 2 hours of administration)

[0688] (E) Security Assessment

[0689] All observed or proactively reported adverse events, regardless of treatment group or causal relationship with the investigational drug, were recorded on the adverse events page of the Case Report Form (CRF). Adverse events were coded using the MedDRA coding system, and all AEs were graded according to the National Cancer Institute Common Terminology Standard for Adverse Events version 5.0 (NCI-CTCAE) [NCI, 2017].

[0690] Adverse events

[0691] Adverse events are defined in the International Conference on Harmonisation (ICH) Good Clinical Practice Guidelines as "any adverse medical event that occurs in a patient or clinical trial subject who is administering a drug product and is not necessarily causally related to the treatment."

[0692] In this study, the definition of adverse events was expanded to include the occurrence of any such event (e.g., signs, symptoms, or diagnosis) or exacerbation of a pre-existing medical condition from the time the subject signed the informed consent form to the time the study drug was first used. Exacerbation indicates an increase in the severity, frequency, or duration of a pre-existing medical condition (e.g., diabetes, migraine, gout, hypertension, etc.) or is associated with a significantly worse outcome.

[0693] For all adverse events, investigators must seek and obtain sufficient information to determine the outcome of the adverse event and assess whether it meets the criteria for classification as a serious adverse event, thus requiring immediate notification to the sponsor or its designated representative. For all adverse events, investigators should obtain sufficient information to determine the causal relationship of the adverse event. Investigators need to assess the causal relationship. For adverse events causally related to the investigation product, investigators need to follow up until the event subsides or stabilizes at a level acceptable to the investigator and the sponsor's clinical monitor or its designated representative.

[0694] Serious adverse events

[0695] A serious adverse event (SAE) is defined as the following adverse event:

[0696] Caused death;

[0697] Life-threatening (putting the subject at direct risk of death);

[0698] Hospitalization or extension of the current hospital stay is required;

[0699] This results in persistent or severe disability / incapacity; or

[0700] Congenital abnormalities / birth defects

[0701] An event may be considered a SAE if, based on appropriate medical judgment, it is unlikely to result in death, is life-threatening, or requires hospitalization, but may endanger the patient and may require medical or surgical intervention to prevent one of the outcomes listed in this definition. Examples of such medical events include allergic bronchospasm, blood cachexia, or seizures requiring intensive treatment in the emergency room or at home, but not requiring hospitalization. Hospitalization meeting the definition of “severe” refers to any hospitalized patient admission that includes at least an overnight stay in a healthcare facility.

[0702] Inpatient admissions do not include: rehabilitation facilities, hospice facilities, specialized nursing facilities, nursing homes, routine emergency room admissions, same-day surgeries (as outpatient / day / outpatient surgeries) or community admissions (e.g., where the subject has no place to sleep).

[0703] Safety will be assessed by qualified physicians, physician assistants, or nurses throughout the study. Measurements used to assess safety include medical history, physical examination, vital signs, clinical laboratory tests, urinalysis, 12-lead ECG, and adverse event (AE) monitoring.

[0704] Repeatable laboratory measurements that clinically deviate significantly from previous measurements (as determined by the investigator). If necessary, additional or more frequent tests should be performed than specified in the protocol to provide sufficient AE recording and AE interpretation.

[0705] For all adverse events, researchers should obtain sufficient information to determine the causal relationship between the adverse event and the study treatment (e.g., the study drug or other disease). The relationship between the adverse event and the study treatment should be assessed according to the following definitions:

[0706] Unrelated Any timing sequence that does not conform to the reasonable timing of investigational drug administration and An event that may arise from the patient’s clinical condition or other treatment modalities given to the patient.

[0707] unlikelyAny timing sequence that does not conform to the reasonable timing of investigational drug administration or An event that may arise from the patient’s clinical condition or other treatment modalities given to the patient.

[0708] possible : It conforms to the reasonable time sequence of drug administration in the study. or It conforms to the known response pattern to the suspected drug. and Any response that cannot be reasonably explained by known characteristics of the patient’s clinical condition or by other modalities of treatment given to the patient.

[0709] Related It conforms to the reasonable time sequence for the administration of the research drug. and It conforms to a known response pattern to the suspected drug. and Relapse upon further challenges and / or Any response that improves by stopping the medication or reducing the dose.

[0710] (a) Dose reduction procedures for adverse event management

[0711] If dose reduction is used for AE management, two dose reductions are permitted. Each dose is a 30% reduction from the baseline dose. Dose reductions are sought when clinical benefit is anticipated and is likely to continue to be obtained.

[0712] (b) Criteria for discontinuing research treatment

[0713] Patients should generally continue study treatment until validated radiographic progression. If a patient has radiographic progression but no clear clinical progression and has not started alternative therapy, the patient may continue study treatment at the investigator's discretion. However, if a patient has clear clinical progression but no radiographic progression, study treatment should be discontinued, and the patient should be advised of available treatment options.

[0714] (c) Continuous security review

[0715] G9.2-17 should be discontinued if a serious or life-threatening immune-related adverse reaction (IMAR) occurs or if systemic steroid therapy is indicated, except in certain exceptions (e.g., certain endocrine disorders in clinically stable patients).

[0716] Provide a detailed monitoring plan to limit the severity and duration of IMARs that occur during joint drug development.

[0717] On days 1, 8, and 15 of each 28-day cycle, at 125 mg / m² 2Abraxane is administered intravenously over 30 to 40 minutes. Gemcitabine is administered immediately after Abraxane on days 1, 8, and 15 of each 28-day cycle. One or more of the following may be administered based on the development of potential adverse events in the patient:

[0718] Patients with mild liver function impairment do not require adjustment.

[0719] If AST > 10x ULN or bilirubin > 5x ULN, discontinue Abraxane.

[0720] Reduce the starting dose in patients with moderate to severe hepatic impairment.

[0721] Due to severe blood, neurological, skin, or gastrointestinal toxicity, dose reduction or discontinuation may be necessary.

[0722] Contraindications:

[0723] Neutrophil count < 1,500 cells / mm² 3

[0724] Severe hypersensitivity to abraxane. Patients known to be hypersensitive to gemcitabine.

[0725] Tables 9 through 12 below provide exemplary guidance on recommended and reduced doses of Abraxane and gemcitabine. See also Abraxane monograph: Abraxis BioScience, LLC. Highlights of Prescribing Information [Internet]. Summit (NJ): Celgene Corporation; 2019 Dec [cited May 7, 2020].

[0726] Table 9. Recommended starting dose of Abraxane in patients with liver injury

[0727]

[0728] a Patients with bilirubin levels higher than the upper limit of normal were excluded from clinical trials for pancreatic or lung cancer.

[0729] Table 10. Decreased dose levels of Abraxane and gemcitabine in patients with pancreatic adenocarcinoma

[0730]

[0731] Table 11. Effects of neutropenia and / or neutropenia at the start of a cycle or during a cycle in patients with pancreatic adenocarcinoma

[0732] Dosage recommendations and adjustments for Abraxane and gemcitabine in patients with thrombocytopenia i

[0733]

[0734] ANC = Absolute Neutrophil Count

[0735] Table 12. Dosage modifications of Abraxane and gemcitabine for other adverse drug reactions in patients with pancreatic adenocarcinoma

[0736]

[0737] Given the possibility of extravasation, close monitoring of the infusion site is recommended during administration. Limiting Abraxane infusion to 30 minutes as directed can reduce the likelihood of infusion-related reactions.

[0738] (d) Identify potential security issues

[0739] Dose-limiting toxicity (DLT) period: one (1) cycle.

[0740] A cycle consists of C1D1 (day 1 of cycle 1) and C1D15 (day 15 of cycle 1).

[0741] Monitoring Plan

[0742] In the dose expansion phase of Part 1, the dose will be expanded to the next cohort after a review of the first cycle of each cohort. Safety and available PK data will be used by the SMC to evaluate DLT in all patients in each cohort. As a safety precaution, during the dose expansion, new patients will only be enrolled and treated if the first patient in each cohort has received G9.2-17 and at least 7 to 14 days have passed since treatment. A selected DLT safety analysis will be performed for each patient after the completion of the first cycle. Toxicity will be monitored by the SMC during the expansion phase, which will hold a meeting to review the overall toxicity rate before each dose expansion. The frequency of SMC meetings will increase with the toxicity rate. The SMC reserves the right to recommend termination or changes to the study design of this clinical study at any time, including but not limited to testing intermediate dose levels or initiating an intermittent dosing regimen.

[0743] (e) Dose-limiting toxicity standards

[0744] Dose-limiting toxicities (DLTs) are defined as clinically important non-hematologic adverse events or abnormal laboratory values ​​that are assessed as unrelated to metastatic disease progression, interstitial disease, or concomitant medication, are related to the study drug, occur in cycle 1 of the study, and meet any of the following criteria:

[0745] All Grade 4 non-hematologic toxicities for any duration

[0746] All are grade 3 non-hematologic toxicities. Exceptions are:

[0747] Grade 3 nausea, vomiting, and diarrhea do not require hospitalization or TPN support and can be controlled to ≤2 within 48 hours with supportive care.

[0748] Electrolyte abnormality of grade O3 should be corrected to grade ≤2 within 24 hours.

[0749] o Other level 3 asymptomatic laboratory abnormalities

[0750] The DLT cycle consists of one (1) cycle, that is, four (4) weeks. One cycle consists of applying G9.2-17 on day 1 and day 15 (C1D1 and C1D15; day 1 of cycle 1 and day 15 of cycle 1, respectively).

[0751] (f) Dosage delay and reduction

[0752] Any adverse events (AEs) of grade 3 or higher that may, are highly probable, or are certain to be associated with one or more investigational drugs will be discussed with the medical monitor before continuing medication, except in the following cases where discussion with the medical monitor is not required:

[0753] Local injection site reactions lasting less than 72 hours, including pain, redness, swelling, induration, or itching.

[0754] Systemic injection reactions lasting less than 72 hours include fever, myalgia, headache, or fatigue.

[0755] If the investigator deems it appropriate (after discussion with the medical monitor), dosing may need to be delayed for grade ≥3 adverse events until the toxicity subsides (to grade 1 or lower).

[0756] In Part 2 of the protocol, if more than 3 patients develop DLT, the dose of G9.2-17 is reduced to one dose lower than the recommended Phase 2 dose (RP2D).

[0757] (F) RECIST criteria for tumor assessment

[0758] In baseline tumor assessment, tumor lesions / lymph nodes are classified as measurable or non-measurable. Measurable tumor lesions are recorded based on the longest diameter on the measurement plane (except for pathological lymph nodes, which are measured on the shortest axis). When more than one measurable lesion is present at baseline, a maximum of five lesions representing all affected organs (with a maximum of two lesions per organ) should be identified as target lesions. Target lesions should be selected based on their size (the lesion with the longest diameter). The sum of the diameters of all target lesions is calculated and reported as the baseline total diameter.

[0759] All other lesions (or disease sites), including pathological lymph nodes, should be identified as non-target lesions and should also be recorded at baseline. No measurement is required; these lesions should be tracked as "present," "absent," or "clearly progressing."

[0760] Disease response (complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD)) was assessed as outlined in Appendix 4.

[0761] Disease response measures allow for the calculation of overall disease control rate (DCR) including CR, PR, and SD, objective response rate (ORR) including CR and PR, progression-free survival (PFS), and time to progression (TTP).

[0762] (G) Patient completes or withdraws

[0763] The patient completed

[0764] Part 1 - Dosage Exploration :

[0765] Patients receive one of five dose levels of the study drug every two weeks until they withdraw from the study due to disease progression, unacceptable toxicity, or dose-limiting toxicity (DLT).

[0766] Two patients were administered the drug, with a maximum usable sample size of 24. Dosage scaling was initiated only upon approval from the SMC. At each dose scaling, new patients were enrolled and treated only if two patients in the previous cohort had received G9.2-17 and at least 7 days had passed since their treatment.

[0767] Part 1 was completed when 6 consecutive patients received the same dose and the dose was determined to be OBD.

[0768] Part 2 – Tumor Type-Specific Treatment :

[0769] An expanded cohort of patients with metastatic PDAC requires combination therapy with G9.2-17 and gemcitabine / abraxane. Study completion depends on patient response at 3 months and survival of responding patients at 12 months.

[0770] Part 2 - Extension :

[0771] If a promising efficacy signal attributable to the tumor type is identified in one of the five experimental groups, an expanded cohort will be initiated to confirm the finding. This will be completed as described in Part 2.

[0772] Discontinued research treatment

[0773] Patients may be discontinued before completing the study treatment for any of the following reasons:

[0774] Dose-limiting toxicity – defined as a clinically significant non-hematologic adverse event or abnormal laboratory value that is assessed as unrelated to metastatic disease progression, interstitial disease, or concomitant medication, and is associated with the study drug and occurs within the first cycle of the study, meeting any of the following criteria:

[0775] o All grade 4 non-hematologic toxicities for any duration

[0776] o All grade 3 non-hematologic toxicities. Exceptions are as follows:

[0777] Grade 3 nausea, vomiting, and diarrhea do not require hospitalization or TPN support and can be controlled to ≤ Grade 2 within 48 hours with supportive care.

[0778] Grade 3 electrolyte abnormality, corrected to ≤ Grade 2 within 24 hours.

[0779] Progressive disease according to RESIST criteria or significant clinical progression at an earlier time point (if the investigator judges it to be in the best interests of the patient).

[0780] Intermittent diseases that hinder further treatment

[0781] Example 3. Non-GLP single-dose, range-based exploratory intravenous toxicity study in male Sprague Dawley rats at 1-week and 3-week observation periods after administration.

[0782] This study evaluated the anatomical endpoint of G9.2-17 IgG4 following a single intravenous bolus injection in Sprague Dawley rats, followed by 1-week (terminal) and 3-week (recovery) necropsies on days 8 and 22. All animals survived to the scheduled necropsy time. In this study, no macroscopic findings, organ weight changes, or microscopic findings related to the test sample were observed in animals at either the terminal or recovery necropsy.

[0783] The objective of this non-GLP exploratory, single-dose, range-exploratory, intravenous toxicity study was to identify and characterize the acute toxicity of G9.2-17 IgG4 by administering an intravenous bolus injection over 2 minutes to Sprague Dawley rats, followed by observation periods of 1 week (terminal) and 3 weeks (recovery).

[0784] This non-GLP single-dose toxicity study was conducted in 24 male Sprague Dawley rats to determine the toxicokinetics and potential toxicities of different doses of G9.2-17 IgG4 administered in a single dose. On day 1, animals were administered the medium or 10 mg / kg, 30 mg / kg, or 70 mg / kg G9.2-17 IgG4 via slow intravenous bolus injection over at least 2 minutes, followed by administration for 1 week (termination, day 8) or 3 weeks (recovery, day 22). Study endpoints included death, clinical observation, body weight and food consumption, clinicopathology (hematology, coagulation, clinical chemistry, and urinalysis), toxicokinetic parameters, ADA assessment, and anatomical pathology (gross autopsy, organ weight, and histopathology). Table 13 below provides a summary of the experimental design.

[0785] Table 13. Experimental Design

[0786]

[0787] On day 8 or day 22, submit all surviving animals for necropsy. Perform a full necropsy and collect organ weights. Weigh all organs at terminal and recovery times. Trim, routinely process, embed in paraffin, and stain with hematoxylin and eosin for tissues required for microscopic evaluation.

[0788] No unplanned deaths occurred during this study. All animals survived to terminal diagnosis or were returned for necropsy. Noticeable histological changes were considered incidental or related to certain aspects of the experimental procedure, rather than to the administration of the test substance. The morbidity, severity, or histological characteristics of those incidental tissue changes were not associated with the test substance. No results related to G9.2-17 IgG4 were found in clinical observation, body weight, food consumption, clinicopathology, or anatomical pathology. In conclusion, Sprague-Dawley rats tolerated single intravenous administrations of 10, 30, and 70 mg / kg G9.2-17 IgG4 without adverse outcomes. Therefore, the NOEL under the conditions of this study was 70 mg / kg.

[0789] Example 4. Non-GLP single-dose, range-based intravenous infusion toxicity study of G9.2-17 IgG4 in cynomolgus monkeys during a 3-week observation period after administration.

[0790] This non-GLP single-dose toxicity study was conducted in eight cynomolgus monkeys to identify and characterize the acute toxicity of G9.2-17 IgG4 administered as a single dose at different doses. The vehicle or 30 mg / kg, 100 mg / kg, or 200 mg / kg G9.2-17 IgG4 was administered to animals (1 male [M] / 1 female [F] / group) via intravenous (IV) infusion over 30 minutes, followed by a 3-week observation period. Study endpoints included: mortality, clinical observation, body weight, and qualitative food consumption; clinicopathology (hematology, coagulation, clinical chemistry, immunophenotyping analysis, and galactagogue-9 expression in leukocyte subsets, as well as cytokine analysis); toxicokinetic parameters; serum collection for potential antidrug antibody assessment (ADA); soluble galactagogue-9 analysis; and anatomical pathology (gross necropsy, organ weight, and histopathology).

[0791] No findings associated with G9.2-17 IgG4 were noted in clinical observation, body weight, food consumption, clinicopathology (hematology, clinical chemistry, coagulation or cytokine analysis), immunophenotypic analysis, galactagogue-9 expression on leukocyte subsets, soluble galactagogue-9, or anatomical pathology.

[0792] In summary, cynomolgus monkeys tolerated single intravenous infusions of 30, 100, and 200 mg / kg G9.2-17 IgG4 without adverse outcomes. Therefore, under the conditions of this study, the no-observed-adverse-effect level (NOAEL) was 200 mg / kg, which was the highest dose level assessed. The study design is shown in Table 14.

[0793] Table 14. Experimental Design

[0794]

[0795] a Group 4 was administered one week after the administration of Groups 1 to 3.

[0796]

[0797] a Group 4 was administered one week after the administration of Groups 1 to 3.

[0798] During the study, the mediator and test sample were administered via intravenous infusion over 30 minutes through a catheter placed percutaneously in the saphenous vein. Dosage levels were 30, 100, and 200 mg / kg, administered at a dose volume of 20 mL / kg. The control group received the mediator in the same manner as the treatment group.

[0799] During administration, the animal should be sling-restrained. The medium or test sample should be administered based on the animal's most recent body weight using an infusion pump and a sterile, disposable syringe. The administration syringe should be filled with the appropriate volume of medium or test sample (20 mL / kg, plus an additional 2 mL). Upon completion of administration, the animal should be removed from the infusion system. The weight of each administration syringe should be recorded before the start and end of each infusion to determine dose-response accounting.

[0800] Detailed clinical observation

[0801] Animals were removed from their cages and subjected to a detailed clinical examination at 1 hour and 4.5 hours after the start of the infusion (SOI) on Day 1, and then daily thereafter. The weight of all animals was measured and recorded at transfer, before randomization, on Day -1, and weekly during the study period.

[0802] All animals underwent clinicopathological evaluation (hematology, coagulation, and clinical chemistry) before testing and on day 1 (before administration), day 3, day 8, and day 21. Additional samples used to determine hematological parameters and for peripheral blood lymphocyte and cytokine analysis were collected 30 minutes after SOI (immediately after infusion) and at 4.5, 8.5, 24.5, and 72.5 hours (relative to day 1). Bone marrow smears were collected and preserved.

[0803] Blood samples (approximately 0.5 mL) were collected from all animals via the femoral vein to determine the serum concentration of the test sample (see Table 15) (for deviations, see Appendix 1). Animals were not fasted prior to blood collection, except for a time interval consistent with that used for clinical pathology collection.

[0804] Table 15 Bioanalytical Sample Collection Schedule

[0805]

[0806] For processing, blood samples were collected in additive-free, barrier-free microtubes and centrifuged at controlled room temperature within 1 hour of collection. The resulting serum was aliquoted into two approximately equal aliquots in pre-labeled cryovials. All aliquots were frozen at -60°C to -90°C within 2 hours of collection.

[0807] Postmortem evaluations were conducted on all animals euthanized at the scheduled autopsy.

[0808] A necropsy was performed following procedures approved by a veterinary pathologist. The animal's external abnormalities, including palpable masses, were carefully examined. The skin was exposed through a midline ventral incision to identify any subcutaneous masses and correlate them with findings from the pre-mortem examination. The abdominal, thoracic, and cranial cavities were examined for abnormalities. Organs were removed, examined, and placed in fixatives as needed. All designated tissues, except for the eyes (including the optic nerve) and testes, were fixed in neutral buffered formalin (NBF). The eyes (including the optic nerve) and testes were placed in a modified Davidson's fixative and then transferred to 70% ethanol for up to three days before final placement in NBF. Formalin was administered into the lungs via the trachea. All intact tissues and organs were harvested from all animals.

[0809] Record the weight of all animals and the weight of the organs specified in the protocol at the scheduled necropsy, and calculate the appropriate organ-to-weight ratio (relative to body weight and brain weight). Weigh organs in pairs. Collect the total weight of the thyroid and parathyroid glands.

[0810] result

[0811] All animals survived to their scheduled necropsy on day 22. No test-product-related clinical or veterinary observations were observed in treated animals. No test-product-related effects on body weight were observed in treated animals during treatment or recovery. No G9.2-17 IgG4-related effects on hematological endpoints were observed in either sex at any time interval and at any dose level.

[0812] There were no effects of G9.2-17 IgG4 on clotting time (i.e., activated partial thromboplastin kinase time [APTT] and prothrombin time) or fibrinogen concentration in either sex at any dose level and at any time interval. All fluctuations in individual coagulation values ​​were considered incidental, consistent with biological and procedural variations, and / or negligible in magnitude, and unrelated to G9.2-17 IgG4 administration.

[0813] There were no G9.2-17 IgG4-related effects on clinical chemistry endpoints for either sex at any dose level and at any time interval. All fluctuations in individual clinical chemistry values ​​were considered incidental, consistent with biological and procedural variations, and / or negligible in magnitude and unrelated to G9.2-17 IgG4 administration.

[0814] No G9.2-17 IgG4-related effects on cytokine endpoints were observed in either sex at any time interval and at any dose level. All fluctuations in individual cytokine values ​​were considered incidental, consistent with biological and procedural variations, and / or negligible in magnitude, and unrelated to G9.2-17 IgG4 administration.

[0815] A review of the gross autopsy findings revealed no findings deemed relevant to the test sample. No organ weight changes were deemed relevant to the test sample. No changes were found that were relevant to the test sample.

[0816] In summary, cynomolgus monkeys tolerated single intravenous infusions of 30, 100, and 200 mg / kg G9.2-17 IgG4 without adverse outcomes. Therefore, the no-adverse-effect level (NOAEL) under the conditions of this study was 200 mg / kg, which was the highest dose level assessed.

[0817] Animals were removed from their cages and each animal underwent a detailed clinical examination 1 hour and 4.5 hours after the start of the infusion (SOI) on Day 1, and then once daily during the study period.

[0818] Example 5. Study on intravenous infusion of G9.2-17 in cynomolgus monkeys

[0819] The goal of this study was to further characterize the toxicity and toxicokinetics of different doses of the test product G9.2-17 (an hIgG4 monoclonal antibody bound to galactagogue-9) after weekly 30-minute intravenous (IV) infusions for 5 weeks in cynomolgus monkeys, and to assess the reversibility, progression, or delayed appearance of any observed changes after a 3-week recovery period.

[0820] Experimental Design

[0821] Table 16 summarizes the research design.

[0822] Table 16. Experimental Design

[0823]

[0824] a Based on the latest actual weight measurements.

[0825] The animals (cynomolgus monkeys) used in this study were assigned to study groups according to a standard randomization procedure based on body weight, aimed at achieving similar group average weights. Males and females were randomized separately. The body weights of the animals assigned to the study were [amount missing]% of the average body weight for each sex. Within 20%.

[0826] Animals were administered the formulation lacking G9.2-17 (“the medium”) or containing G9.2-17 (“the test article”) once weekly for 5 weeks (days 1, 8, 15, 22, and 29) via 30-minute IV infusion during the study period. Dosage levels were 0, 100, and 300 mg / kg / dose, administered in a dose volume of 10 mL / kg. Control animals received the medium in the same manner as the treatment group. Doses were administered via a percutaneously placed catheter through the saphenous vein, using a new sterile disposable syringe for each administration. Dosage balance measurements were measured and recorded on the toxicokinetic sample collection days (days 1, 15, and 29) before and at the end of administration to ensure administration of ±10% of the target dose. Individual doses were based on most recent body weight. The last administration site was marked for collection at terminal and post-mortem examinations. All doses were administered within 8 hours of test article preparation.

[0827] The following example illustrates the in-life procedures, observations, and measurements performed on animals.

[0828] All animals underwent electrocardiographic (ECG) examinations. Care was taken to avoid causing excessive excitement in the animals before recording the ECG to minimize extreme fluctuations or artifacts in these measurements. A standard ECG (10 leads) was recorded at 50 mm / s. Using appropriate leads, the RR, PR, and QT intervals, as well as the QRS duration, were measured to determine the heart rate. The corrected QT (QTc) interval was calculated using a procedure based on the method described by Bazett (1920). All traces were evaluated and reported by a consulting veterinary cardiologist.

[0829] To help improve continuity and reliability, the Functional Observation Bundle (FOB) assessment was conducted by two independent assessors for all cases and consisted of a detailed home cage and open area neurobehavioral assessment (Gauvin and Baird, 2008). Each technician independently scored each monkey observed in each home cage and outside the cage (without sharing results), and then assessed whether the individual scores were consistent with their peers' scores after the test was completed. FOB assessments were performed before each animal was administered (on day -9 or day 8) to determine baseline differences, and 2 to 4 hours after the start of infusion on day 1 and day 15, and before terminal and post-mortem examinations. Observations included, but were not limited to, assessments of activity level, posture, lacrimation, salivation, tremors, convulsions, fasciculations, stereotyped behaviors, facial muscle movements, eyelid closure, pupillary response, response to stimuli (visual, auditory, and food), body temperature, Chaddock and Babinski reflexes, proprioception, paralysis, ataxia, distance perception impairment, and slope assessment, movement, and gait.

[0830] Blood pressure was measured and recorded for each animal, consisting of systolic pressure, diastolic pressure, and mean arterial pressure (MAP). Blood pressure measurements were reported using three readings within a 20 mmHg MAP range.

[0831] Respiratory rate was measured and recorded for each animal at 3 collection intervals using visual assessment according to the testing facility's SOP. The average of the 3 collections was reported as the average value.

[0832] All animals underwent clinicopathological evaluation (e.g., immunophenotypic analysis and cytokine assessment) at predetermined time intervals. Bone marrow smears were collected and preserved. Blood samples (approximately 0.5 mL) were collected from all animals via the femoral vein for determination of serum concentrations of the test samples. Animals were fasted prior to blood collection, except for the time intervals consistent with the fasting used for clinicopathological collection. At the end of the study (day 36 or day 50), animals were euthanized and tissues were collected for histological processing and microscopic evaluation.

[0833] Soluble galactagogue-9 was evaluated as follows. Blood samples (approximately 1 mL) were collected from all animals via the femoral vein before administration and 24 hours after the start of infusion on days 1, 8, 15, and 29, and prior to final and / or resumed necropsy for the determination of serum soluble galactagogue-9. Animals were not fasted prior to blood collection, except at intervals consistent with those used for clinicopathological collection.

[0834] Soluble galactagogue 9 samples were processed as follows: Blood samples were collected in additive-free, barrier-free tubes, allowed to coagulate at ambient temperature, and centrifuged at ambient temperature. The resulting serum was aliquoted into two equal aliquots in pre-labeled cryovials (100 µL in aliquot 1, the remainder in aliquot 2). All aliquots were rapidly frozen on dry ice within 2 hours of collection and stored at -60°C to 90°C.

[0835] All results shown in the report tables are calculated using the original data rounding procedure with non-rounded values ​​and may not be accurately reproduced from the single data provided.

[0836] result

[0837] mortality rate

[0838] All animals survived until the scheduled final autopsy on day 36 and the recovery autopsy on day 50.

[0839] Detailed clinical and veterinary observation

[0840] No clinical or veterinary observations related to the test product were observed in treated animals during treatment or recovery.

[0841] Functional observation combination

[0842] No FOB observations related to the test article were found in treated animals during treatment or recovery.

[0843] Weight and weight gain

[0844] No effects on body weight and weight gain related to the test substance were observed in treated animals during treatment or recovery.

[0845] ophthalmological examination

[0846] During treatment or recovery, no effects on ophthalmic examinations related to the test substance were observed in treated animals.

[0847] blood pressure value

[0848] No effects on blood pressure values ​​related to the test substance were observed in treated animals during treatment or recovery.

[0849] respiratory rate value

[0850] No effect on respiratory rate values ​​related to the test substance was found in treated animals during treatment or recovery.

[0851] electrocardiography

[0852] During treatment or recovery, no effect on electrocardiogram assessment related to the test article was found in treated animals.

[0853] hematology

[0854] No G9.2-17-related effects on hematological parameters were observed at any dose level, at any time point, and in any sex.

[0855] Blood clotting

[0856] No G9.2-17-related effects on coagulation parameters were observed at any dose level, at any time point, and in any sex.

[0857] Clinical Chemistry

[0858] No G9.2-17-related effects on clinical chemistry parameters were observed at any dose level, at any time point, and in any sex.

[0859] Urine analysis

[0860] During the mid-13-week period, no changes in urinalysis parameters related to G9.2-17 were observed in either sex at any dose level.

[0861] Cytokines

[0862] No clear G9.2-17-related effects on cytokines were observed at any dose level or time point.

[0863] Peripheral blood leukocyte analysis (PBLA)

[0864] No G9.2-17-related effect on PBLA endpoints was observed at any time point, at any dose level, and in any sex.

[0865] Bioanalysis, galactagogue-9 and toxicokinetic assessment

[0866] G9.2-17 was quantifiable in all cynomolgus monkey samples from all animals treated with G9.2-17 after dose administration. No measurable amounts of G9.2-17 were detected in control cynomolgus monkey samples. Soluble galactagogue-9 was quantifiable in all cynomolgus monkey samples from all animals. Serum concentrations of G9.2-17 were below the limit of quantitation (LLOQ < 0.04 μg / mL) in most G9.2-17 treated animals prior to day 1 administration and in all serum samples from control animals on days 1 and 29.

[0867] Gross pathology and organ weight

[0868] No clear macroscopic observations related to the test substance were found in the primary study or recovery animals. No changes in organ weight related to the test substance were also observed in the primary study or recovery animals.

[0869] Histopathology

[0870] No clear microscopic observations were obtained related to the test sample.

[0871] In summary, cynomolgus monkeys tolerated weekly intravenous infusions of 100 and 300 mg / kg of G9.2-17 for 5 weeks without adverse outcomes.

[0872] Example 6. Intravenous infusion study of G9.2-17 in Sprague Dawley rats

[0873] The objective of this study was to evaluate the potential toxicity of different doses of G9.2-17 (an IgG4 human monoclonal antibody against galactagogue-9) administered weekly to Sprague Dawley rats via intravenous injection for 4 weeks, followed by a 3-week recovery period. Furthermore, the toxicokinetics of G9.2-17 were determined.

[0874] Experimental Design

[0875] Table 17 summarizes the research design.

[0876] Table 17: Research Design

[0877]

[0878] a Individual dose-volume calculation based on most recent body weight

[0879] b SSD animals: 3 animals / sex / group, TK collection performed only after a single dose on day 1.

[0880] One hundred and eighty-six animals (Sprague Dawley rats) were randomly assigned to the treatment group according to body weight. The control / mediator (formulation buffer for the test product) and test product G9.2-17 were administered via a single IV injection via tail vein at dose levels of 0, 100, and 300 mg / kg on days 1, 8, 15, 22, and 29. The test product was administered on day 1 to animals assigned to the SSD subgroup at dose levels of 100 and 300 mg / kg.

[0881] Clinical observation was conducted once daily before cleaning the enclosure, starting from the second day of acclimatization. Mortality checks were performed twice daily to assess overall animal health. Food consumption was estimated by weighing the food supply and remaining amount in the containers weekly. Average grams (g) per animal per day was calculated from weekly food consumption. Body weight was measured before randomization, on day -1, then weekly throughout the study period, and on the day of each necropsy. Functional observation portfolio (FOB) observations of SSB animals were recorded approximately 24 hours after dose administration on days 1, 35, and 49. Urine was collected overnight using metabolic cages. Samples were obtained on days 36 and 50.

[0882] Animals are fasted overnight prior to each series of collections, including serum chemical samples. In these cases, the relevant clinicopathological assessment is performed on the fasted animal. Blood is collected at termination from the jugular vein of a restrained, conscious animal or from the vena cava of an anesthetized animal.

[0883] Parameters assessed during the study's survival monitoring included a combination of clinical observations, food consumption, body weight, and functional observations. Blood samples were collected at selected time points for clinicopathological (hematological, coagulation, and serological) analysis. Urine samples were collected for urinalysis. Blood samples were also collected at selected time points for toxicokinetics (TK), immunogenicity (e.g., anti-drug antibodies or ADA), and cytokine analysis. Animals underwent necropsy on days 36 and 50. At each necropsy, overall findings and organ weights were recorded, and tissues were collected for microscopic examination.

[0884] result

[0885] Survival check

[0886] mortality rate No abnormal clinical observations or weight changes were found in the animal during the study.

[0887] Clinical observation No clinical observations related to G9.2-17 were found during the study.

[0888] Food consumption / weight No changes in food consumption, weight, or weight gain associated with G9.2-17 were found during the study period.

[0889] Clinical Pathology No changes in clinicopathological parameters related to G9.2-17 were found.

[0890] Cytokine analysis No changes in serum concentrations of IL-2, IL-4, IFN-γ, IL-5, IL-6, IL-10 and / or TNF-α, MCP-1 and MIP-1b associated with G9.2-17 were observed.

[0891] General Pathology No gross observations were found related to G9.2-17. Furthermore, no changes in absolute or relative organ weight were found related to G9.2-17.

[0892] Histopathology No histological findings related to G9.2-17 were found.

[0893] In summary, intravenous administration of G9.2-17 once weekly for a total of 5 doses to Sprague Dawley rats was generally well tolerated. No changes in G9.2-17 were observed in clinical findings, food consumption, body weight, FOB parameters, clinicopathology, cytokines, overall observations, or organ weight.

[0894] Equivalent content

[0895] From the above description, those skilled in the art can readily determine the essential features of the present invention, and various changes and modifications can be made to adapt it to various uses and conditions without departing from the spirit and scope of the invention. Therefore, other embodiments are also within the scope of the claims.

[0896] Although several inventive embodiments have been described and illustrated herein, various other means and / or structures for performing the functions described herein and / or obtaining the results and / or one or more advantages will readily conceive of by those skilled in the art, and each such variation and / or modification is considered to be within the scope of the inventive embodiments described herein. More generally, it will be readily understood by those skilled in the art that all parameters, dimensions, materials, and configurations described herein are exemplary, and actual parameters, dimensions, materials, and / or configurations depend on the specific application using the teachings of the invention. Many equivalents of the specific inventive embodiments described herein can be recognized or determined by those skilled in the art using only conventional experiments. Therefore, it should be understood that the foregoing embodiments are presented by way of example only, and that the inventive embodiments may be practiced in ways other than those specifically described and claimed within the scope of the appended claims and their equivalents. The inventive embodiments of this disclosure pertain to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods, provided that such features, systems, articles, materials, kits, and / or methods do not contradict each other, is included within the scope of this disclosure.

[0897] All definitions defined and used herein should be understood to take precedence over dictionary definitions, definitions incorporated by reference in other documents, and / or the general meaning of the defined terms.

[0898] All references, patents, and patent applications disclosed in this document are incorporated into each cited subject by way of citation, and in some cases, the entire document may be covered.

[0899] The indefinite articles “a” and “an” used in the specification and claims, unless explicitly stated otherwise, shall be understood as “at least one (a)”.

[0900] As used herein in the specification and claims, the phrase “and / or” should be understood to mean “any one or two” of the elements so combined, that is, elements that exist together in some cases and separately in others. Multiple elements listed with “and / or” should be interpreted in the same way, i.e., elements so combined “one or more”. In addition to the elements specifically identified by the “and / or” clause, other elements may optionally be present, whether or not they are related to those specifically identified elements. Thus, as a non-limiting example, when used in conjunction with open-ended language such as “comprising / including,” a reference to “A and / or B” may refer only to A (optionally including elements other than B) in one embodiment; only to B (optionally including elements other than A) in another embodiment; both A and B (optionally including other elements) in yet another embodiment; and so on.

[0901] As used herein in the specification and claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” should be interpreted as inclusive, meaning it includes at least one, but also includes multiple elements or lists of elements, and (optionally) other items not listed. Only when the opposite terms are explicitly stated, such as “only one” or “exactly one”, or when used in the claims as “consisting of…”, does it mean exactly one number or one element in a list of elements. Generally, the term “or” as used herein should only be interpreted to indicate an exclusive alternative (i.e., “one or the other but not both”), if preceded by an exclusive term such as “any,” “one of,” “only one,” or “exactly one.” The phrase “consisting of…” as used in the claims should have the general meaning used in the field of patent law.

[0902] As used herein in the specification and claims, the phrase "at least one" when referring to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list, but not necessarily including at least one of each element specifically listed in the list, and does not exclude any combination of elements in the list. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements referred to by the phrase "at least one," whether or not they are related to those specifically identified elements. Therefore, as a non-limiting example, “at least one of A and B” (or equivalently, “at least one of A or B”, or equivalently, “at least one of A and / or B”) may refer to at least one, optionally including more than one A, with no B (and optionally including elements other than B) in one embodiment; in another embodiment, it may refer to at least one, optionally including more than one B, with no A (and optionally including elements other than A); in yet another embodiment, it may refer to at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); and so on.

[0903] It should also be understood that, unless there is an explicit instruction to the contrary, in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order of the steps or actions of the described method.

Claims

1. Use of a combination of an antibody conjugating human galactagogue-9 and one or more chemotherapeutic agents in a medicament for preparing a method of treating solid tumors, wherein the method comprises administering to a subject in need (a) an effective amount of the antibody conjugating human galactagogue-9 and (b) an effective amount of the one or more chemotherapeutic agents, wherein: The antibody that binds to human galactagogue-9 comprises: (a) A light chain containing a light chain variable region, the light chain variable region comprising a light chain complementarity determination region 1 as shown in SEQ ID NO: 1, a light chain complementarity determination region 2 as shown in SEQ ID NO: 2, and a light chain complementarity determination region 3 as shown in SEQ ID NO: 3, and (b) A heavy chain containing a heavy chain variable region, the heavy chain variable region including a heavy chain complement determination region 1 as shown in SEQ ID NO: 4, a heavy chain complement determination region 2 as shown in SEQ ID NO: 5, and a heavy chain complement determination region 3 as shown in SEQ ID NO: 6; The one or more chemotherapeutic agents mentioned above include gemcitabine, paclitaxel, or combinations thereof, and The solid tumors mentioned are pancreatic cancer, colorectal cancer, hepatocellular carcinoma, or bile duct cancer.

2. The use as described in claim 1, wherein the pancreatic cancer, colorectal cancer, hepatocellular carcinoma, or bile duct cancer is metastatic.

3. The use as described in claim 1, wherein the solid tumor is pancreatic ductal adenocarcinoma.

4. The use as described in any one of claims 1-3, wherein the paclitaxel is a protein-bound paclitaxel.

5. The use as described in claim 4, wherein the protein-bound paclitaxel is nanoparticle albumin-bound paclitaxel.

6. The use according to any one of claims 1-3, wherein the antibody binding human galactagogue-9 comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

8.

7. The use as described in any one of claims 1-3, wherein the antibody binding human galactagogue-9 is an IgG4 molecule.

8. The use as claimed in claim 7, wherein the antibody binding human galactagogue-9 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.

9. A combination of an antibody against human galactagogue-9 and one or more chemotherapeutic agents, wherein: The antibody binding to human galactagogue-9 comprises: (a) a light chain containing a light chain variable region, the light chain variable region comprising a light chain complementarity-determining region 1 as shown in SEQ ID NO: 1, a light chain complementarity-determining region 2 as shown in SEQ ID NO: 2, and a light chain complementarity-determining region 3 as shown in SEQ ID NO: 3, and (b) a heavy chain containing a heavy chain variable region, the heavy chain variable region comprising a heavy chain complementarity-determining region 1 as shown in SEQ ID NO: 4, a heavy chain complementarity-determining region 2 as shown in SEQ ID NO: 5, and a heavy chain complementarity-determining region 3 as shown in SEQ ID NO: 6; The one or more chemotherapeutic agents mentioned above include gemcitabine, paclitaxel, or combinations thereof.

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