HK40112915A - Anti-galectin-9 antibodies and therapeutic uses thereof - Google Patents

Description

Anti-galactoglobulin-9 antibodies and their therapeutic uses

Cross-references to related applications

This application claims the benefit of U.S. Provisional Application No. 63/251,227, filed October 1, 2021, and U.S. Provisional Application No. 63/277,384, filed November 9, 2021, pursuant to 35 U.S.C. § 119(e), each of which is incorporated herein by reference in its entirety.

sequence list

This application contains a sequence list that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy created on September 30, 2022, is named 112174-0234-NP003WO01_SEQ.xml and has a size of 25,891 bytes.

Background Technology

The immune system has enormous potential to recognize and destroy cancer cells, but the complex network controlling tumor immune escape is a barrier to broad and effective immune regulation (Martinez-Bosch N et al., Immune Evasion in Pancreatic Cancer: From Mechanisms to Therapy. Cancer (Basel). 2018; 10(1)). Approved immuno-oncology (IO) agents deliver increased survival improvements to many cancer types (e.g., melanoma, lung cancer, kidney cancer, bladder cancer, some colon cancers, etc.) and are rapidly being integrated as standard of care in addition to, and in combination with, surgery, chemotherapy, and radiotherapy. However, significant gaps remain in the treatment and survival of many other aggressive malignancies. For example, the 5-year survival rates for metastatic pancreatic ductal adenocarcinoma (PDAC or PDA), cholangiocarcinoma (CCA), and colorectal cancer (CRC) remain <9%, <5%, and <15%, respectively. These gastrointestinal tumors are highly invasive, and many patients are already in the late stages of the disease when they seek medical attention. Furthermore, the effectiveness of approved immunotherapies is not ideal (Rizvi et al., Cholangiocarcinoma-evolving concepts and therapeutic strategies; Nat Rev Clin Oncol. 2018; 15(2):95-111; Kalyan et al., Updates on immunotherapy for colorectal cancer; J Gastrointest Oncol. 2018; 9(1):160-169).

The success of first-generation checkpoint inhibitors (anti-PD-1, anti-PD-L1, and anti-CTLA4) has led to a surge in efficacy and differentiation in new IO clinical trials (Holl et al., Examining Peripheral and Tumor Cellular Immunome in Patients with Cancer; Front Immunol. 2019; 10:1767). However, alongside these successes, there have also been many unfortunate development failures, thus necessitating the development of more novel and effective treatments.

Galactolectin-9 is a tandem repeat lectin composed of two carbohydrate recognition domains (CRDs) and was first identified and described in patients with Hodgkin's lymphoma (HL) in 1997 (Tureci et al., J. Biol. Chem. 1997, 272, 6416-6422). Three isoforms exist, and it can be located intracellularly or extracellularly. Elevated levels of galactolectin-9 have been observed in a variety of 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 galactolectin-9 expression show later disease progression and larger tumor size (Kawashima et al.; BJU Int. 2014; 113:320-332). In melanoma, galactolectin-9 is expressed in 57% of tumors and is significantly increased in the plasma of patients with advanced melanoma compared with healthy controls (Enninga et al., Melanoma Res. 2016 Oct; 26(5):429-441). Numerous studies have demonstrated the usefulness of galactolectin-9 as a prognostic biomarker and it has recently been shown to be a potential target for novel drugs (Enninga et al., 2016; Kawashima et al., BJU Int 2014; 113:320-332; Kageshita et al., Int J Cancer. 2002 Jun; 99(6):809-16, and references therein).

Galactolectin-9 has been described to play important roles in many cellular processes, such as adhesion, cancer cell aggregation, apoptosis, and chemotaxis. Recent studies have shown that galactolectin-9 plays a role in supporting tumor-mediated immune regulation, for example, by negatively regulating Th1 responses, Th2 polarization, and macrophage polarization toward the M2 phenotype. This work also includes studies that have shown galactolectin-9 to participate in direct T cell inactivation through its interaction with T cell immunoglobulin and mucin protein 3 (TIM-3) receptors (Dardalhon et al., J Immunol., 2010, 185, 1383-1392; Sanchez-Fueyo et al., Nat Immunol., 2003, 4, 1093-1101).

Galactochondrin-9 has also been found to play a role in polarizing T cell differentiation toward a tumor suppressor phenotype and in promoting the programming of tolerogenic macrophages and adaptive immunosuppression (Daley et al., Nat Med., 2017, 23, 556-567). In a mouse model of pancreatic ductal adenocarcinoma (PDAC), it has been shown that blocking the checkpoint interaction between galactochondrin-9, found on innate immune cells in the tumor microenvironment (TME), and its receptor Dectin-1 can increase the antitumor immune response in the TME and slow tumor progression (Daley et al., Nat Med., 2017, 23, 556-567). It was also found that galactolectin-9 binds to CD206 (a surface marker of M2 macrophages), leading to a decrease in the secretion of CVL22 (MDC) (a macrophage-derived chemokine associated with longer survival and lower risk of recurrence in lung cancer) (Enninga et al., J Pathol. 2018 Aug; 245(4):468-477).

Summary of the Invention

This disclosure is based, at least in part, on the development of treatment options for solid tumors (e.g., metastatic solid tumors), such as head and neck cancer or urothelial carcinoma, alone or in combination with checkpoint inhibitors (such as anti-PD-1 antibodies (e.g., tislelizumab)).

Therefore, in some aspects, this disclosure provides a method for treating solid tumors, the method comprising administering to a subject in need (a) an effective amount of an antibody binding to human galactolectin-9 (anti-galactolectin-9 antibody) and (b) an effective amount of an anti-PD-1 antibody (such as tislelizumab). In some embodiments, the anti-galactolectin-9 antibody may comprise: (i) a light chain variable region ( _VL ) comprising 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; and (ii) a heavy chain variable region comprising 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. In some cases, anti-galactoglobulin-9 antibodies (e.g., G9.2-17(IgG4) as disclosed herein) may be administered to subjects at doses of about 0.2 mg/kg to about 18 mg/kg. In some instances, anti-galactoglobulin-9 antibodies may be administered to subjects once weekly.

In some embodiments, the solid tumor is head and neck cancer, urothelial carcinoma, gastric or esophageal cancer, or non-small cell lung cancer. In some embodiments, the solid tumor is a metastatic tumor (e.g., locally advanced or metastatic solid tumor). In some embodiments, the solid tumor is refractory and/or recurrent. In some embodiments, the subject to be treated by any of the methods disclosed herein is a human patient with a solid tumor.

In some embodiments, anti-galactoglobulin-9 antibody (e.g., G9.2-17(IgG4) as disclosed herein) may be administered to the subject at a dose of about 4 mg/kg to about 18 mg/kg. For example, anti-galactoglobulin-9 antibody may be administered to the subject at doses of about 4 mg/kg, about 6.3 mg/kg, about 10 mg/kg, about 12 mg/kg, about 14 mg/kg, about 16 mg/kg, or about 18 mg/kg. In one example, the dose of anti-galactoglobulin-9 antibody is about 6.3 mg/kg. In another example, the dose of anti-galactoglobulin-9 antibody is about 10 mg/kg. In yet another example, the dose of anti-galactoglobulin-9 antibody is about 16 mg/kg.

In some specific instances, anti-galactoglobulin-9 antibodies (e.g., G9.2-17(IgG4) as disclosed herein) may be administered to subjects once weekly at a dose of approximately 6.3 mg/kg. In some specific instances, anti-galactoglobulin-9 antibodies (e.g., G9.2-17(IgG4) as disclosed herein) may be administered to subjects once weekly at a dose of approximately 10 mg/kg. In other specific instances, anti-galactoglobulin-9 antibodies (e.g., G9.2-17(IgG4) as disclosed herein) may be administered to subjects once weekly at a dose of approximately 16 mg/kg. Alternatively or additionally, anti-galactoglobulin-9 antibodies may be administered to subjects by intravenous infusion.

In some implementations, tislelizumab is administered to subjects at a dose of about 200 mg every 3 weeks, about 300 mg every 4 weeks, or about 400 mg every 6 weeks. In one instance, tislelizumab was administered to subjects at a dose of about 300 mg every 4 weeks. Alternatively or additionally, tislelizumab is administered to subjects by intravenous infusion.

In one example, the method disclosed herein includes weekly administration of an anti-galactoglobulin-9 antibody (e.g., G9.2-17(IgG4)) at a dose of approximately 6.3 mg/kg and weekly administration of tislelizumab at a dose of approximately 300 mg every 4 weeks. Both antibodies can be administered via intravenous infusion.

In one example, the method disclosed herein includes administering an anti-galactoglobulin-9 antibody (e.g., G9.2-17(IgG4)) once weekly at a dose of about 10 mg/kg and tislelizumab once every 4 weeks at a dose of about 300 mg. Both antibodies can be administered via intravenous infusion.

In one example, the method disclosed herein includes administering an anti-galactoglobulin-9 antibody (e.g., G9.2-17(IgG4)) once weekly at a dose of about 16 mg/kg and tislelizumab once every 4 weeks at a dose of about 300 mg. Both antibodies can be administered via intravenous infusion.

In some instances, tislelizumab was administered to the subject on the same day as the subject received anti-galactolectin 9 antibody. Alternatively, tislelizumab and anti-galactolectin 9 antibody were administered on consecutive days. In some instances, tislelizumab was administered prior to anti-galactolectin 9 antibody administration.

In any of the methods disclosed herein, the anti-galactoglobulin-9 antibody may comprise a V _L chain containing the amino acid sequence of SEQ ID NO:8 and a V _H chain containing the amino acid sequence of SEQ ID NO:7. In some cases, the anti-galactoglobulin-9 antibody is an IgG1 or IgG4 molecule. For example, the anti-galactoglobulin-9 antibody is an IgG4 molecule having a modified Fc region of human IgG4. In some instances, the modified Fc region of human IgG4 comprises the amino acid sequence of SEQ ID NO:14. In one instance, the anti-galactoglobulin-9 antibody comprises 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.

In any of the methods disclosed herein, the subject has received one or more prior anticancer therapies. In some instances, one or more prior anticancer therapies include chemotherapy, immunotherapy, radiation therapy, therapies involving biological agents, or combinations thereof. In some cases, the subject's disease has progressed or they have developed resistance to one or more prior therapies.

In some implementations, the subject is a human patient with elevated galectin-9 levels relative to control values. For example, the human patient has elevated serum or plasma galectin-9 levels relative to control values. In some cases, the human patient has cancer cells expressing galectin-9, immune cells expressing galectin-9, or both.

Any method disclosed herein may also include monitoring for the occurrence of side effects in subjects. Alternatively or additionally, the method may also include reducing the dose of anti-galactoglobulin-9 antibody, the dose of tislelizumab, or both, in the event of a side effect.

The following are also within the scope of this disclosure: pharmaceutical compositions for treating solid tumors (e.g., those described herein and including metastatic solid tumors), and the use of any one of anti-galactoglobulin-9 antibodies and anti-PD-1 antibodies (such as tislelizumab) for manufacturing a medicament for treating solid tumors, wherein in some embodiments, the uses disclosed herein relate to one or more treatment conditions (e.g., dosage, dosing schedule, route of administration, etc.) as also disclosed herein.

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 the appended claims.

Attached Figure Description

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 in conjunction with the detailed description of the specific embodiments presented herein.

Figure 1 depicts the results of a study showing mice treated with G9.2-17mIgG2a alone or in combination with αPD-1 mAb. Mice with orthotopically implanted KPC tumors (n = 10/group) were treated weekly with commercial αPD-1 (200 μg) mAb or G9.2-17mIg2a (200 μg), or a combination of G9.2-17 and αPD-1, or a matched allotype, for three weeks. Tumors were removed and weighed. Each point represents one mouse; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; by unpaired Student t-test.

Figures 2A and 2B depict the role of G9.2-17 in the B16F10 subcutaneous homology model. Tumors were subcutaneously implanted and treated with either G9.2-17 IgG1 mouse mAb, anti-PD-1 antibody, or a combination of G9.2-17 IgG1 mouse mAb and anti-PD-1 antibody. Figure 9A depicts the effect on tumor volume. Figure 9B depicts the infiltration of CD8 T cells within the tumor. The results indicate that, in the combination group, the presence of effector T cells within the tumor was enhanced.

Figures 3A and 3B include images showing patient-derived ex vivo tumor cultures (organoids) from cholangiocarcinoma treated with G9.2-17. Patient-derived ex vivo tumor cultures (organoids) were treated with G9.2-17 or an isotype control for three days. The expression of CD44 (Figure 3A) and TNFα (Figure 3B) in CD3+ T cells derived from PDOT was assessed.

Detailed Implementation

This document provides methods for treating solid tumors (e.g., head and neck cancer, urothelial carcinoma, and other solid tumors as disclosed herein) using a combination of an anti-galactoglobulin-9 antibody (e.g., G9.2-17) and a checkpoint inhibitor (e.g., an anti-PD-1 antibody (e.g., tislelizumab)). In some embodiments, the cancer is metastatic. In some embodiments, the methods disclosed herein provide specific dosages and/or dosing schedules. In some cases, the methods disclosed herein target specific patient populations, such as patients who have undergone prior treatment and have shown disease progression with prior treatment, or patients who are resistant to prior treatment (de novo or acquired).

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

Galectin-9 has been found to interact with Dectin-1 (an innate immune receptor highly expressed on macrophages in PDAC and on cancer cells) (Daley et al., Nat Med. 2017; 23(5):556-6). Regardless of its origin, disruption of the interaction between galectin-9 and Dectin-1 has been shown to lead to the reprogramming of CD4 ⁺ and CD8 ⁺ cells into indispensable mediators of antitumor immunity. Therefore, galectin-9 serves as a valuable therapeutic target for blocking Dectin-1-mediated signaling. Consequently, in some embodiments, the anti-galectin-9 antibody described herein disrupts the interaction between galectin-9 and Dectin-1.

It was also found that galactolectin-9 can interact 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 galactolectin-9 has been found to have pleiotropic effects on immune cells, inducing apoptosis in Th1 cells (Zhu et al., Nat Immunol., 2005, 6:1245-1252) and stimulating the secretion of tumor necrosis factor-α (TNF-α), leading to the maturation of monocytes into dendritic cells and resulting in innate immune-induced inflammation (Kuchroo et al., Nat Rev Immunol., 2008, 8:577-580). It has been found that galectin-9/TIM-3 signaling co-activates NF-κB and β-catenin signaling (two pathways promoting LSC self-renewal) (Kikushige et al., Cell Stem Cell, 2015, 17(3):341-352). Anti-galectin-9 antibodies that interfere with galectin-9/TIM-3 binding can have therapeutic effects, particularly for leukemia and other hematologic malignancies. Therefore, in some embodiments, the anti-galectin-9 antibodies described herein disrupt the interaction between galectin-9 and TIM-3.

Furthermore, galactolectin-9 was found to interact with CD206 (a mannose receptor highly expressed on M2-polarized macrophages, thereby promoting tumor survival) (Enninga et al., J Pathol. 2018 Aug; 245(4):468-477). Tumor-associated macrophages expressing CD206 are mediators of tumor immunosuppression, angiogenesis, metastasis, and recurrence (see, for example, Scodeller et al., Sci Rep. 2017 Nov 7; 7(1):14655, and references therein). Specifically, M1 (also known as classically activated macrophages) are triggered by Th1-associated cytokines and bacterial products to 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-tumorigenic effects of M2 include promoting angiogenesis, promoting invasion and metastasis, and protecting tumor cells from chemotherapy-induced apoptosis (Hu et al., Tumour Biol. 2015 Dec; 36(12):9119-9126, and references therein). Tumor-associated macrophages are considered to have an M2-like phenotype and pro-tumorigenic effects. Galactochondrin-9 has been shown to mediate the differentiation of bone marrow cells into the M2 phenotype (Enninga et al., Melanoma Res. 2016 Oct; 26(5):429-41). Galactohemagglutinin-9 binding to CD206 could potentially reprogram TAM to the M2 phenotype, similar to what was previously shown with Dectin-1. Not wishing to be bound by theory, blocking the interaction between galactocelemin-9 and CD206 could provide a mechanism by which anti-galactocelemin-9 antibodies (e.g., G9.2-17 antibodies) could be therapeutically beneficial. Therefore, in some embodiments, the anti-galactocelemin-9 antibodies described herein disrupt the interaction between galactocelemin-9 and CD206.

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

Anti-galactoglobulin-9 antibodies can act as therapeutic agents for treating diseases associated with galactoglobulin-9 (e.g., those 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, antibodies can interfere with the interaction between galactoglobulin-9 and its binding mate (e.g., Dectin-1, TIM-3, or CD206), thereby blocking signaling triggered by galactoglobulin-9/ligand interactions. Alternatively, or additionally, anti-galactoglobulin-9 antibodies can also exert their therapeutic effect by inducing blocking and/or cytotoxicity (e.g., ADCC, CDC, or ADCP against pathological cells expressing galactoglobulin-9). Pathological cells are those that directly or indirectly contribute to the occurrence and/or development of disease.

The anti-galactolectin-9 antibody disclosed herein can inhibit galactolectin-9-mediated signaling (e.g., galactolectin-9/Dectin-1 or galactolectin-9/Tim-3 mediated signaling pathways) or eliminate pathological cells expressing galactolectin-9 via, for example, ADCC. Therefore, the anti-galactolectin-9 antibody described herein can be used to inhibit any galactolectin-9 signaling and/or eliminate galactolectin-9-positive pathological cells, thereby benefiting the treatment of galactolectin-9-related diseases.

Anti-galactolectin-9 antibodies (such as G9.2-17) have been found to effectively induce apoptosis in cells expressing galactolectin-9. Furthermore, the antitumor effects of anti-galactolectin-9 antibodies (such as G9.2-17) alone or in combination with checkpoint inhibitors (e.g., anti-PD-1 antibodies) have been demonstrated in mouse models. As reported in this paper, the efficacy of G9.2-17 was tested in mouse models of PDAC and melanoma, as well as in a patient-derived organoid tumor model (PDOT). The orthotopic PDAC KPC mouse models used (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) encapsulate many characteristics of the human disease, including non-responsiveness to approved checkpoint inhibitors (Bisht and Feldmann G; Animal models for modeling pancreatic cancer and novel drug discovery; Expert Opin Drug Discov. 2019; 14(2):127-142; Weidenhofer et al., Animal models of pancreatic cancer and their application in clinical research; Gastrointestinal Cancer: Targets and Therapy 2016; 6). The B16F10 melanoma mouse model has long been the standard for testing immunotherapy (Curran et al., PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors; Proc Natl Acad Sci U S A. 2010; 107(9):4275-4280).

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

In vivo studies using G9.2-17 mouse IgG1 (G9.2-17mIgG1 contains the exact same binding epitopes and has the same effector functions as G9.2-17 human IgG4) have shown significant reductions in tumor growth as a single agent in in situ KPC models where approved checkpoint inhibitors are ineffective. In the B16F10 model, G9.2-17 significantly outperformed anti-PD-1 efficacy. In both models, the use of G9.2-17mIgG1 has been demonstrated to modulate the intratumoral immune microenvironment by upregulating effector T cell activity and inhibiting immunosuppressive signaling, as well as to enhance intratumoral CD8 T cell infiltration.

These results demonstrate that the antitumor approach disclosed in this paper (involving an anti-galactoglobulin-9 antibody, optionally combined with a checkpoint inhibitor) will achieve excellent therapeutic efficacy against target solid tumors.

Therefore, this article describes the therapeutic use of anti-galactoglobulin-9 antibodies for the treatment of certain cancers as disclosed herein.

Antibodies that bind to galactolectin-9

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

An antibody (which may be used interchangeably in the plural form) is an immunoglobulin molecule capable of specifically binding to a target (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” (e.g., anti-galactoglobulin-9 antibody) encompasses not only complete (e.g., full-length) polyclonal or monoclonal antibodies, but also its antigen-binding fragments (such as Fab, Fab', F(ab')2, Fv), single chains (scFv), mutants thereof, fusion proteins containing antibody portions, humanized antibodies, chimeric antibodies, bivalent antibodies, 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 (e.g., anti-galactoglobulin-9 antibody) include any class of antibodies, such as IgD, IgE, IgG, IgA, or IgM (or their subclasses), and antibodies do not need to belong to any particular class. Immunoglobulins can be designated into different classes depending on the amino acid sequence of the antibody's heavy chain constant domain. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these 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 called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known.

Typical antibody molecules contain heavy chain variable regions ( _VH ) and light chain variable regions ( _VL ), which are typically involved in antigen binding. _{The VH} and _VL regions can be further subdivided into hypervariable regions, also known as “complementarity-determining regions” (“CDRs”), interspersed with more conserved regions called “framework regions” (“FRs”). Each _VH and _VL typically 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 framework regions 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 contact definition, all of which are well-known in the art. See, for example, Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., Department of Health and Human Services, NIH Publication No. 91-3242; Chothia et al. (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; Allazikani et al. (1997) J. Molec. Biol. 273:927-948; Edelman et al., Proc Natl Acad Sci U S A. May 1969; 63(1):78-85; and Almagro, J. Mol. Recognit. 17:132-143 (2004); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Lefranc M P et al., Dev Comp Immunol, January 2003; 27(1):55-77; and Honegger A and Pluckthun A, J Mol Biol, 8 June 2001; 309(3):657-70. See also hgmp.mrc.ac.uk and bioinf.org.uk/abs).

In some embodiments, the anti-galactoglobulin-9 antibody described herein is a full-length antibody containing two heavy chains and two light chains, each chain 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 encompassed within the term "antigen-binding fragment" in full-length antibodies include: (i) Fab fragments, a monovalent fragment consisting of _VL , _VH , _CL , and _CH1 domains; (ii) F(ab') ₂ fragments, a bivalent fragment comprising two Fab fragments linked by disulfide bonds through hinge regions; (iii) Fd fragments consisting of _VH and _CH1 domains; (iv) Fv fragments consisting of the _VL and _VH domains of a single arm of the antibody; (v) dAb fragments (Ward et al., (1989) Nature 341:544-546) consisting of a _VH domain; and (vi) functionally separate complementarity-determining regions (CDRs). Furthermore, although the two domains _VL and _VH of an Fv fragment are encoded by different genes, they can be linked together using recombinant methods via synthetic linkers, enabling them to be made into a single protein chain where the _VL and _VH regions pair to form 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.

Any antibody described herein (e.g., anti-galactoglobulin-9 antibody) may be monoclonal or polyclonal. "Monoclonal antibody" refers to a homogeneous population of antibodies, and "polyclonal antibody" refers to a heterogeneous population of antibodies. Neither term limits the source of the antibody or its method of manufacture.

Reference antibody G9.2-17 refers to an antibody capable of binding to human galactoglobulin-9 and comprising the heavy chain variable region of SEQ ID NO:7 and the light chain variable region of SEQ ID NO:8, both provided below. In some embodiments, the anti-galactoglobulin-9 antibody used in the methods disclosed herein is the G9.2-17 antibody. 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 the reference antibody G9.2-17. Two antibodies having the same V _H and/or V _L CDRs 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/).

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

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

In some instances, 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. Anti-galactoglobulin-9 antibodies (including 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(IgG4)” refers to a G9.2-17 antibody, which is an IgG4 molecule (e.g., having a heavy chain comprising SEQ ID NO:19 and a light chain comprising SEQ ID NO:15). Similarly, the term “G9.2-17(Fab)” refers to a G9.2-17 antibody, which is a Fab molecule.

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 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 increment thereof) sequence identity with the heavy chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO:4, 5, and 6.

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

In some embodiments, the anti-galactoglobulin-9 antibody disclosed herein comprises a light chain _CDR that has, alone or together with, 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 corresponding V _L CDR of the reference antibody G9.2-17. Alternatively or additionally, in some embodiments, the anti-galactoglobulin-9 antibody comprises a heavy chain CDR that has, alone or together with, 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 corresponding V H CDR of the reference antibody G9.2-17.

The "percentage of identity" between the two amino acid sequences was determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, with modifications as described in 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. A BLAST protein search can be performed using the XBLAST program (score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecule of this invention. When vacancies exist between the two sequences, vacancy-adding BLAST can be used, as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using BLAST and BLAST with gaps, you can use the default parameters of their respective procedures (e.g., XBLAST and NBLAST).

In other embodiments, the anti-galactoglobulin-9 antibody described herein comprises _VH , which includes HC CDR1, HC CDR2, and HC CDR3, and which, relative to the HC CDR1, HC CDR2, and HC CDR3 of reference antibody G9.2-17, contain a total of up to 8 amino acid residue variations (8, 7, 6, 5, 4, 3, 2, or 1 variation, including addition, deletion, and/or substitution). Alternatively or additionally, in some embodiments, the anti-galactoglobulin-9 antibody described herein comprises _VH , which includes LC CDR1, LCCDR2, and LC CDR3, and which, relative to the LC CDR1, LC CDR2, and LC CDR3 of reference antibody G9.2-17, contain a total of up to 8 amino acid residue variations (8, 7, 6, 5, 4, 3, 2, or 1 variation, including addition, deletion, and/or substitution).

In one instance, 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 amino acid substitution is made. Variants can be prepared according to methods known to those skilled in the art for altering polypeptide sequences, such as those found in references editing such methods, for example, *Molecular Cloning: A Laboratory Manual*, eds. J. Sambrook et al., 2nd ed., *Cold Spring Harbor Laboratory Press*, *Cold Spring Harbor*, *New York*, 1989, or *Current Protocols in Molecular Biology*, eds. F.M. Ausubel et al., *John Wiley & Sons*, *Inc.*, *New York*. Conservative substitutions of amino acids include substitutions between amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

In some embodiments, the anti-galactohemagglutinin-9 antibodies disclosed herein, having the heavy chain CDR disclosed herein, contain a frame region derived from a germline _VH fragment subclass. Such germline _VH regions 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, and IGHV3-30). IGHV3-33, IGHV3-43, IGHV3-48, IGHV3-49, IGHV3-53, IGHV3-64, IGHV3-66, IGHV3-72 and IGHV3-73, IGHV3-74), the IGHV4 subfamily (e.g., IGHV4-4, IGHV4-28, 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).

Alternatively or additionally, in some embodiments, the anti-galactolectin-9 antibody having the light chain CDR disclosed herein contains a frame region derived from the germline Vκ 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, the anti-galactolectin-9 antibody comprises a light chain variable region containing a frame derived from the germline Vλ fragment. Examples include the IGλ1 framework (e.g., IGλV1-36, IGλV1-40, IGλV1-44, IGλV1-47, IGλV1-51), the IGλ2 framework (e.g., IGλV2-8, IGλV2-11, IGλV2-14, IGλV2-18, IGλV2-23), and the IGλ3 framework (e.g., IGλV3-1, IGλV3-9, IGλV3-10, IGλV3-12, IGλV3-16, IGλV3-19, IGλV3-21, IGλV3). -25, IGλV3-27), IGλ4 frame (e.g., IGλV4-3, IGλV4-60, IGλV4-69), IGλ5 frame (e.g., IGλV5-39, IGλV5-45), IGλ6 frame (e.g., IGλV6-57), IGλ7 frame (e.g., IGλV7-43, IGλV7-46), IGλ8 frame (e.g., IGλV8-61), IGλ9 frame (e.g., IGλV9-49), or IGλ10 frame (e.g., IGλV10-54).

In some embodiments, the anti-galactohemagglutinin-9 antibody used in the methods disclosed herein may be an antibody having the same heavy chain variable region ( _VH ) and/or the same light chain variable _region ( _VL ) as the reference antibody _G9.2-17 , the amino acid sequences of which are provided below:

V _H :

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPG KGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSL RAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSS(SEQ ID NO:7)

V _L :

DIQMTQSPSSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGK APKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQ QSSTDPITFGQGTKVEIKR(SEQ ID NO:8)

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).

In some cases, the anti-galactolectin-9 antibodies disclosed herein are functional variants of the reference antibody G9.2-17. Functional variants may be structurally similar to the reference antibody (e.g., containing a limited number of amino acid residue variations in one or more of the heavy and/or light chain CDRs of G9.2-17 as disclosed herein, or having sequence identity relative to the heavy and/or light chain CDRs of G9.2-17, or the VH and/or VL of G9.2-17), and have binding affinity substantially similar to human galactolectin-9 (e.g., having the same _KD value).

In some embodiments, the anti-galactolectin-9 antibody as described herein can bind to galactolectin-9 and inhibit its activity by at least 20% (e.g., 31%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or greater, including any increments thereof). The apparent inhibition constant (Ki ^app or Ki _,app ) providing inhibitory efficacy is related to the inhibitor concentration required to reduce enzyme activity and is independent of enzyme concentration. The inhibitory activity of the anti-galactolectin-9 antibody described herein can be determined by conventional methods known in the art.

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

Where A equals v_{_o} /E, the initial rate of the enzymatic reaction (v _{_o} ) in the absence of an inhibitor (I) divided by the total enzyme concentration (E). In some embodiments, the anti-galactoglobin-9 antibody described herein has a Ki_^app value of 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 pM or less for the target antigen or epitope. In some embodiments, the anti-galactoglobin-9 antibody has a lower Ki_^app for the first target (e.g., CRD2 of galactoglobin-9) relative to the second target (e.g., CRD1 of galactoglobin-9). The difference in Ki ^app (e.g., 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 10⁵ times. In some instances, the anti-galactoglobin-9 antibody inhibits the first antigen (e.g., the first protein or its mimicry in the first conformation) more strongly than the second antigen (e.g., the same first protein or its mimicry in the second conformation). In some embodiments, any of the anti-galactoglobin-9 antibodies undergoes further affinity maturation to reduce the Ki ^app of the antibody against the target antigen or its epitope.

In some embodiments, the anti-galactolectin-9 antibody inhibits Dectin-1 signaling in tumor-infiltrating immune cells such as macrophages. In some embodiments, the anti-galactolectin-9 antibody inhibits Dectin-1 signaling triggered by galactolectin-9 by at least 30% (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 routine assays. Alternatively or additionally, the anti-galactolectin-9 antibody inhibits T-cell immunoglobulin mucin-3 (TIM-3) signaling induced by galactolectin-9. In some embodiments, the anti-galactoglobulin-9 antibody inhibits T-cell immunoglobulin mucin-3 (TIM-3) signaling, for example, 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 greater, including any increments thereof). This inhibitory activity can be determined by conventional methods such as routine assays.

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

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

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 representing the main pathway.

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

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 greater, including any increments thereof).

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

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

In some embodiments, the anti-galactoglobulin-9 antibody, as described herein, has a suitable binding affinity to the target antigen (e.g., galactoglobulin-9) or its epitope. As used herein, “binding affinity” refers to the apparent association constant, or _KA . _KA is the reciprocal of the dissociation constant ( _KD ). The anti-galactoglobulin-9 antibody described herein may have a binding affinity ( _KD ) of at least ^10⁻⁵ , ^10⁻⁶ , ^10⁻⁷ , ^10⁻⁸ , ^10⁻⁹ , ^10⁻¹⁰ M or lower to the target antigen or epitope. An increase in binding affinity corresponds to a decrease in _KD . 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 evaluating binding affinity were in HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v/v) surfactant P20).

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 generally related to the total target protein concentration ([target]) by the following equation:

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

However, precise determination of _KA is not always necessary, as sometimes obtaining a quantitative measurement of affinity (e.g., determined using methods such as ELISA or FACS analysis, proportional to _KA , and thus usable for comparisons, such as determining whether a higher affinity is, for example, 2-fold higher), a qualitative measurement of affinity, or an inferred result of affinity (e.g., activity in a functional assay, such as in vitro or in vivo) is sufficient. In some cases, in vitro binding assays indicate in vivo activity. In others, in vitro binding assays do not necessarily indicate in vivo activity. In some cases, tight binding is beneficial, but in others, tight binding in vivo is not ideal, and antibodies with lower binding affinity are preferred.

In some embodiments, the heavy chain of any of the anti-galactolectin-9 antibodies 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 one specific example, the heavy chain constant region is derived from human IgG (γ heavy chain) of any of the IgG subfamilies described herein.

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: 4, 5, 6). 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 numbers) that 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 that inhibits Fab arm exchange (S228P; EU number). 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, which is cleaved during secretion and is therefore not in the mature antibody molecule. The heavy chain of mature proteins (post-secretionary) begins with "EVQ", and the light chain begins with "DIM". An exemplary amino acid sequence of the heavy chain constant region is provided below:

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

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

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

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL G APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

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

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK*

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

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

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

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP P CPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK*

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

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP P CPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

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

Light chain constant region (SEQ ID NO:11)

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

An example full-length anti-galactohemagglutinin-9 antibody is provided as follows:

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

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

G9.2-17hIgG1 LALA Heavy Chain (SEQ ID NO:17)

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL G APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

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

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK*

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

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

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

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP P CPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK*

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

EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP P CPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

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

DIQMTQSPSSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSTDPITFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

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 a heavy chain IgG4 constant region containing SEQ ID NO:10. In one embodiment, the constant region of the anti-galactoglobulin-9 antibody comprises a heavy chain IgG1 constant region consisting of SEQ ID NO:10.

In some embodiments, 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 constant region of the anti-galactoglobulin-9 antibody comprises the heavy chain IgG4 constant region containing SEQ ID NO:20. In one embodiment, the constant region of the anti-galactoglobulin-9 antibody comprises the heavy chain IgG4 constant region consisting of SEQ ID NO:20.

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 increment 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.

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 increment 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.

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 a 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.

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.

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 assessed using the methods disclosed in U.S. Patent No. 5,500,362. In other embodiments, the constant region is modified as described in Eur. J. Immunol. (1999) 29:2613-2624, PCT application No. PCT/GB99/01441, and/or UK Patent Application No. 9809951.8. In some embodiments, the IgG4 constant region is a mutant with reduced heavy chain exchange. In some embodiments, the constant region is derived from the human IgG4 Fab arm exchange mutant S228P.

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 increment 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.

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 increment 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.

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

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

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 a heavy chain sequence containing SEQ ID NO:16. In one embodiment, the anti-galactoglobulin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO:16.

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 a heavy chain sequence containing SEQ ID NO:17. In one embodiment, the anti-galactoglobulin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO:17.

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 a heavy chain sequence containing SEQ ID NO:18. In one embodiment, the anti-galactoglobulin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO:18.

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 a heavy chain sequence containing SEQ ID NO:22. In one embodiment, the anti-galactoglobulin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO:22.

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 containing 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 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 a heavy chain sequence containing SEQ ID NO:23. In one embodiment, the anti-galactoglobulin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO:23.

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 increments thereof) 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.

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.

In some embodiments, any anti-galactoglobulin-9 antibody disclosed herein (e.g., G9.2-17(IgG4)) may have a missing C-terminal lysine residue of the heavy chain.

Preparation of anti-galactolectin-9 antibody

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

Nucleic acids encoding the heavy and light chains of the anti-galactolectin-9 antibody as described herein can be cloned into an expression vector, with each nucleotide sequence operatively linked to a suitable promoter. In one example, each of the nucleotide sequences 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.

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

Generally, a nucleic acid sequence encoding one or all strands of an antibody can be cloned into a suitable expression vector operatively linked 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 that can pair with each other and be linked together with a ligase. Alternatively, synthetic nucleic acid adapters can be ligated to the ends of the 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.

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

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

Adjustable promoters including repressors with operons can be used. In one embodiment, a lac repressor from *Escherichia coli* can act as a transcription regulator to regulate transcription of mammalian cell promoters with lac operons (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)). A tetracycline repressor (tetR) is combined with a transcription activator (VP16) to produce the tetR-mammalian cell transcription activator fusion protein tTa (tetR-VP16), wherein the minimal promoter with tetO is derived from the major immediate early promoter of human cytomegalovirus (hCMV) to produce the tetR-tet operon gene system, thereby controlling gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. When the tetracycline operon is correctly positioned downstream of the TATA element of the CMVIE promoter, the tetracycline repressor (tetR), alone rather than a tetR-mammalian cell transcription factor fusion derivative, can act as a potent trans-regulator to modulate gene expression in mammalian cells (Yao et al., Human Gene Therapy, 10(16):1392-1399(2003)). A particular advantage of this tetracycline-inducible switch is that it does not require the use of tetracycline repressor-mammalian cell trans-activators or repressor fusion proteins (which may be cellularly toxic in some cases) to achieve its modulatory effect (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551(1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526(1995)).

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

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

One or more vectors (e.g., expression vectors) containing a nucleic acid encoding any of the antibodies can be introduced into suitable host cells to produce antibodies. Host cells can be cultured under suitable conditions 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) via conventional methods (e.g., affinity purification). If desired, the polypeptide chain of the antibody can be incubated under suitable conditions for a suitable period of time to allow for antibody production.

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

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

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

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

Anti-galactolectin-9 antibodies prepared in this manner can be characterized using methods known in the art, thereby detecting and/or measuring a reduction, improvement, or neutralization of galactolectin-9 biological activity. For example, in some embodiments, ELISA-type assays are suitable for qualitative or quantitative measurements of galactolectin-9 inhibition of Dectin-1 or TIM-3 signaling.

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

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 typing.

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, for example, from the M2 to the M1 phenotype (in vitro or in vivo) (e.g., increased MHCII, decreased CD206, increased TNFα and iNOS). Alternatively, ADCC levels can be assessed, for example, in in vitro assays as described herein.

Treatment

This disclosure provides methods for treating solid tumors, including but not limited to head and neck cancer, urothelial carcinoma, gastric and esophageal cancer, or non-small cell lung cancer, using any anti-galactoglobulin antibody (e.g., G9.2-17, such as G9.2-17IgG4) alone or in combination with a checkpoint inhibitor (e.g., an anti-PD-1 antibody, such as tislelizumab). Other target solid tumors that can be treated by the methods disclosed herein may include pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), cholangiocarcinoma, renal cell carcinoma, and breast cancer.

Depending on the type or site 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, anti-galactoglobulin-9 antibody and/or anti-PD-1 antibody may be administered to the subject via intravenous infusion.

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

As used herein, the term “treatment” means the application or administration of a composition comprising one or more active agents to a subject suffering from a target disease or condition, symptoms of the disease/condition, or a predisposition to the disease/condition, with the aim of curing, healing, alleviating, mitigating, altering, remedying, reducing, improving, or influencing the condition, symptoms of the disease or condition, or a predisposition to the disease or condition.

Relieving a target disease/disorder includes delaying the development or progression of the disease, 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 condition means postponing, hindering, slowing, delaying, stabilizing, and/or slowing the progression of the disease. This delay can be of varying lengths, depending on the history of the disease and/or the individual being treated. Methods of “delaying” or mitigating the development of a disease or delaying its onset are those that reduce the likelihood of the occurrence of one or more symptoms of the disease within a given timeframe and/or alleviate the degree of symptoms within a given timeframe, compared to not using such methods. Such comparisons are typically based on clinical studies using a sufficient number of participants to yield statistically significant results.

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 initial onset and/or relapse.

(i) Treatment with anti-galactoglobulin 9 antibody

Any of the anti-galactoglucoagulation-9 antibodies described herein can be used in any of the methods described herein. In some embodiments, the anti-galactoglucoagulation-9 antibody is G9.2-17, such as G9.2-17 (IgG4). Such antibodies can be used to treat diseases associated with galactoglucoagulation-9. In some aspects, the present invention provides methods for treating cancer. In some embodiments, this disclosure provides methods for reducing, improving, or eliminating one or more symptoms associated with cancer.

In some embodiments, the anti-galactoglobulin 9 antibody is an antibody having the same heavy chain CDR sequence and/or the same light chain CDR sequence as the reference antibody G9.2-17. In some embodiments, the anti-galactoglobulin 9 antibody is an antibody having the same VH and VL sequences as the reference antibody G9.2-17. In some embodiments, such an antibody is an IgG1 molecule (e.g., having a wild-type IgG1 constant region or a mutant thereof as disclosed herein). Alternatively, the antibody is an IgG4 molecule (e.g., having a wild-type IgG4 constant region or a mutant thereof as described herein). In some embodiments, the antibody comprises 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, and/or comprises 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. In some embodiments, the antibody comprises a heavy chain variable region containing SEQ ID NO:7. In some embodiments, the antibody comprises a light chain variable region containing SEQ ID NO:8. In some embodiments, the antibody comprises a heavy chain variable region containing SEQ ID NO:7 and a light chain variable region containing SEQ ID NO:8. In some embodiments, the antibody comprises a heavy chain containing SEQ ID NO:19. In some embodiments, the antibody comprises a light chain containing SEQ ID NO:15. In a specific instance, the anti-galactoglobulin-9 antibody (G9.2-17(IgG4)) used in this paper has the heavy chain of SEQ ID NO:19 and the light chain of SEQ ID NO:15.

An effective amount of the anti-galactoglobulin-9 antibody described herein (e.g., G9.2-17(IgG4)) can be administered systemically or locally to a subject requiring treatment (e.g., a human) via a suitable route. In some embodiments, the anti-galactoglobulin-9 antibody is administered intravenously, for example as a bolus or by continuous infusion over a period of time, via intramuscular, intraperitoneal, intraspinal, subcutaneous, intraarticular, intra-articular, intrasynovial, intrathecal, intratumoral, suburethral, oral, inhalation, or local routes. In one embodiment, the anti-galactoglobulin-9 antibody is administered to the subject via intravenous infusion. In one embodiment, the anti-galactoglobulin-9 antibody is administered to the subject intraperitoneally.

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 achieved by reducing the activity and/or amount/expression of galactolectin-9 in the tumor microenvironment, reducing Dectin-1 signaling, reducing TIM-3 signaling, reducing CD206 signaling, or increasing the antitumor immune response. Non-limiting examples of increasing the antitumor response include increasing the activation level of effector T cells or converting TAMs from the M2 phenotype to the M1 phenotype. In some cases, the antitumor response includes increasing the ADCC response. Determining whether a certain 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 depending on the specific disease being treated, the severity of the disease, individual patient parameters (including age, physical condition, body size, sex, and weight), the duration of treatment, the nature of concurrent therapy (if any), the specific route of administration, and similar factors within the knowledge and expertise of a healthcare professional. These factors are well known to those skilled in the art and can be resolved with only routine experimental procedures. It is generally preferred to use the maximum dose of the individual components or their combination, that is, the highest safe dose based on reasonable medical judgment.

Empirical considerations (such as half-life) often help determine dosage. For example, antibodies compatible with the human immune system (such as humanized antibodies or fully 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. Dosage frequency can be determined and adjusted during treatment and is usually, but not necessarily, based on the treatment and/or inhibition 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.

In one instance, the antibody dosage, as described herein, was determined empirically in individuals who had received one or more antibody administrations. The antagonist was administered to the individual in escalating doses. To assess the efficacy of the antagonist, indicators of the disease/symptom could be followed.

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

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

In some embodiments, the antibody is G9.2-17 IgG4. In some embodiments, the anti-galactoglobulin-9 antibody is administered to the subject at a dose of about 0.2 mg/kg to about 32 mg/kg, such as 0.2 mg/kg, 0.63 mg/kg, 2 mg/kg, 4 mg/kg, 6 mg/kg, 6.3 mg/kg, 8 mg/kg, 10 mg/kg, 12 mg/kg, and 16 mg/kg or higher. In some embodiments, the anti-galactoglobulin-9 antibody is administered to the subject at a dose of about 1 mg/kg to about 32 mg/kg, such as at doses selected from 2 mg/kg, 4 mg/kg, 8 mg/kg, 12 mg/kg, and 16 mg/kg or higher. In some embodiments, the anti-galactoglobulin-9 antibody is administered to the subject at a dose of about 0.2 mg/kg to about 32 mg/kg, for example, at dose levels selected from 0.2 mg/kg, 0.63 mg/kg, 2 mg/kg, 4 mg/kg, 6 mg/kg, 6.3 mg/kg, 10 mg/kg, and 16 mg/kg or higher. In some embodiments, the antibody is administered, for example, via intravenous infusion every two weeks.

In some embodiments, the anti-galactolectin 9 antibody disclosed herein (e.g., G9.2-17 IgG4) is administered via intravenous infusion over 30 minutes to 6 hours. In some instances, intravenous infusion of the anti-galactolectin-9 antibody can be performed over 30 minutes to 2 hours. In other instances, the anti-galactolectin 9 antibody can be administered over a longer infusion period (e.g., about 2-6 hours, such as about 2-4 hours or about 4-6 hours). In specific instances, exemplary anti-galactolectin-9 antibodies can be administered intravenously over periods of about 3 hours, about 4 hours, about 5 hours, or about 6 hours.

In some embodiments, the anti-galactoglobulin-9 antibody (e.g., G9.2-17(IgG4) as disclosed herein) for use in any of the methods disclosed herein may be administered to the subject at doses of about 0.2 mg/kg to about 32 mg/kg, for example, at doses selected from 0.2 mg/kg, 0.63 mg/kg, 2 mg/kg, 4 mg/kg, 6 mg/kg, 6.3 mg/kg, 8 mg/kg, 10 mg/kg, 12 mg/kg, and 16 mg/kg or higher. In some embodiments, the anti-galactoglobulin-9 antibody may be administered to the subject at doses of about 1 mg/kg to about 32 mg/kg, for example, at doses selected from 2 mg/kg, 4 mg/kg, 8 mg/kg, 12 mg/kg, and 16 mg/kg or higher. In some implementations, the anti-galactoglobulin-9 antibody is administered to the subject at a dose of about 0.2 mg/kg to about 32 mg/kg, for example, at a dose selectable from 0.2 mg/kg, 0.63 mg/kg, 2 mg/kg, 4 mg/kg, 6 mg/kg, 6.3 mg/kg, 10 mg/kg or 16 mg/kg or higher.

In some embodiments, an anti-galactoglobulin-9 antibody (e.g., G9.2-17(IgG4) as disclosed herein) used in any of the methods disclosed herein can be administered to the patient requiring treatment, for example, via intravenous infusion once weekly. Alternatively, the anti-galactoglobulin-9 antibody can be administered to the patient every two weeks (e.g., via intravenous infusion). In some embodiments, the anti-galactoglobulin-9 antibody is administered once weekly for one cycle, once weekly for two cycles, once weekly for three cycles, once weekly for four cycles, or once weekly for more than four cycles. In other embodiments, the anti-galactoglobulin-9 antibody is administered once every two weeks for one cycle, once every two weeks for two cycles, once every two weeks for three cycles, once every two weeks for four cycles, or once every two weeks for more than four cycles.

In some implementations, the treatment duration is 12-24 months or longer. In some implementations, the cycle extends to a duration of 3 to 6 months, 6 to 12 months, or 12 to 24 months or longer. In some implementations, the cycle length is adjusted, for example, temporarily or permanently, to a longer duration, such as 3 weeks or 4 weeks.

Given that the pro-tumorigenic effects of galactolectin-9 are mediated through interactions with immune cells (e.g., via TIM-3, CD44, and 41BB with lymphoid cells, and via dectin-1 and CD206 with macrophages), and given that galactolectin-9 is expressed in a wide range of tumors, targeting galactolectin-9 (e.g., using galactolectin-9 binding antibodies to inhibit its interaction with its receptors) offers a therapeutic approach applicable to a variety of different tumor types.

(ii) Combination therapy with anti-PD-1 antibodies

Any method disclosed herein may also include administering an effective amount of an anti-PD-1 antibody (e.g., tislelizumab) to a patient. Examples of PD-1 inhibitors include anti-PD-1 antibodies such as pembrolizumab, nivolumab, tislelizumab, dotalipimab, and cimipril. Such checkpoint inhibitors may be administered simultaneously or sequentially (in any order) with an anti-galactolectin 9 antibody according to this disclosure. In some embodiments, the checkpoint molecule is PD-L1. Examples of PD-L1 inhibitors include anti-PD-L1 antibodies such as durvalumab, avelumab, and ateslelizumab.

In some embodiments, the antibody binding to PD-1 is tislelizumab. In some embodiments, the method described herein includes administering tislelizumab intravenously to the subject at a dose of about 200 mg every 3 weeks. In some embodiments, the method described herein includes administering tislelizumab intravenously to the subject at a dose of about 400 mg every 6 weeks. In some embodiments, the method described herein includes administering tislelizumab to the subject at a dose of about 300 mg every 4 weeks. In some embodiments, tislelizumab is administered intravenously at a dose of about 300 mg every 4 weeks in a 28-day cycle. Alternatively or additionally, tislelizumab may be administered via intravenous infusion, for example, over about 30 minutes. In some embodiments, the antibody binding to PD-1 is dotalimab. In some embodiments, the method described herein includes administering dotalimab intravenously to the subject at a dose of about 500 mg every 3 weeks or at a dose of about 1000 mg every 6 weeks.

In some cases, checkpoint inhibitors (such as any anti-PD-1 antibody disclosed herein (e.g., tislelizumab) and any anti-galactoglobulin-9 antibody disclosed herein, such as (G9.2-17(IgG4))) may be administered on the same day. In some instances, the checkpoint inhibitor may be administered to the subject prior to administration of the anti-galactoglobulin-9 antibody. In other cases, administration of the checkpoint inhibitor (e.g., anti-PD-1 antibody) and the anti-galactoglobulin-9 antibody may be performed on two consecutive days. The checkpoint inhibitor (e.g., anti-PD-1 antibody) may be administered to the subject on the first day of administration, and the anti-galactoglobulin-9 antibody may be administered to the subject on the second day.

In other cases, checkpoint inhibitors (such as any anti-PD-1 antibody disclosed herein) may be administered approximately 1–7 days prior to administration of anti-galactoglobin 9 antibodies (such as G9.2-17) disclosed herein (e.g., day 1, day 2, day 3, day 4, day 5, day 6, or day 7).

In some instances, anti-galactoglobulin-9 antibody may be administered to the subject prior to the administration of a checkpoint inhibitor (e.g., anti-PD-1 antibody). In other cases, administration of both anti-galactoglobulin-9 antibody and checkpoint inhibitor (e.g., anti-PD-1 antibody) is performed on two consecutive days. Anti-galactoglobulin-9 antibody may be administered to the subject on the first day of administration, and the checkpoint inhibitor (e.g., anti-PD-1 antibody) may be administered to the subject on the second day.

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

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

In some instances, the methods described herein involve administering, to subjects requiring treatment (e.g., human patients with head and neck cancer, urothelial carcinoma, or other solid tumors as disclosed herein) once weekly with a dose of anti-galactoglobin-9 antibody (such as G9.2-17(IgG4)) and, for example, a dose of anti-PD-1 antibody (such as tislelizumab) once every 4 weeks. In one instance, a patient was given a dose of G9.2-17(IgG4) once weekly and a dose of tislelizumab once every 4 weeks. In another instance, a patient was given a dose of G9.2-17(IgG4) once weekly and a dose of tislelizumab once every 4 weeks. In another instance, the patient was given 10 mg/kg of G9.2-17 (IgG4) once weekly and 300 mg of tislelizumab every 4 weeks. Alternatively, the patient was given 16 mg/kg of G9.2-17 (IgG4) once weekly and 300 mg of tislelizumab every 4 weeks.

(iii) Patients for treatment

Subjects with any of the aforementioned cancers 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, subjects to be treated by the methods described herein are human cancer patients who have experienced or undergone any combination or sequence of systemic and/or locally delivered anticancer therapy regimens (e.g., chemotherapy, radiation therapy, tumor therapeutic electric fields (TTFields), immunotherapy, biotherapy, small molecule inhibitors, antihormonal therapy, cell-based therapies, and/or surgery) of the treatment modalities outlined. In some embodiments, subjects have received prior immunomodulatory agents or any other antitumor agents or treatment modalities listed above. Non-limiting examples of such immunomodulatory agents include, but are not limited to, antiPD-1, antiPD-L1, antiCTLA-4, antiTIGIT, antiPVRIG, antiLAG-3, antiCD47, antiCD40, antiCSFR1, antiCD73, antiSIRP, antiA2AR, antiOX40, antiCD137, etc. In some embodiments, subjects have shown disease progression upon treatment. In other embodiments, the subject is resistant to treatment (de novo or acquired). In some embodiments, such a subject is shown to have advanced malignancy (e.g., inoperable or metastatic). Alternatively or additionally, in some embodiments, the subject does not have available standard treatment options or does not meet the criteria for standard treatment options, which are therapies commonly used in clinical settings to treat the corresponding solid tumor.

Tumor-treating fields (TTFields) are cancer treatment methods that use alternating electric fields of medium frequency (~100-500 kHz) and low intensity (1-3 V/cm) to disrupt cell division. In any of the embodiments described herein, anti-galactoglobulin-9 antibodies may be administered before, simultaneously with, or after a tumor-treating electric field (TTFields) protocol, alone or in combination with checkpoint inhibitors such as anti-PD-1 antibodies.

In some cases, subjects may be human patients with refractory diseases (such as refractory head and neck cancer or refractory urothelial carcinoma). As used herein, “refractory” means a tumor that does not respond to treatment or becomes resistant. In some cases, subjects may be human patients with recurrent diseases (such as recurrent head and neck cancer or recurrent urothelial carcinoma). As used herein, “recurrent” or “relapse” means a tumor that reappears or progresses after a period of treatment improvement (e.g., partial or complete response).

In some implementations, human patients to be treated by the methods disclosed herein meet one or more of the inclusion and exclusion criteria disclosed in Example 3 below. For example, human patients may be 18 years of age or older; have histologically confirmed unresectable metastatic or inoperable cancer (e.g., no standard treatment options available), have a life expectancy of >3 months, have a recently archived tumor sample available for biomarker analysis (e.g., archived species with galactolectin-9 tumor tissue expression levels assessed by IHC); have measurable disease according to RECIST v1.1, have an Eastern Cooperative Oncology Group (ECOG) performance status of 0–1 or a Karnofsky score >70; have no available standard of care options, have high MSI-H (high microsatellite instability) and MSS (microsatellite stable); have received at least one line of systemic therapy in an advanced/metastatic setting; have adequate hematological and end-organ function (defined in Example 1 below; e.g., neutrophil count ≥1 x ^10⁹ /L, platelet count ≥100 x ^10⁹ /L (for HCC ≥50 x 10⁹/ ^L in Part 1)). /L); hemoglobin ≥9.0 g/dL, creatinine ≤1.5x ULN, AST (SGOT) ≤3x ULN (≤5x ULN in the presence of HCC or liver metastasis), ALT (SGPT) ≤3x ULN (≤5x ULN in the presence of HCC or liver metastasis), bilirubin ≤1.5x ULN (bilirubin may be ≤3.0x ULN in patients with known Gilbert's disease), albumin ≥3.0 g/dL, INR and PTT ≤1.5x ULN; and/or amylase and lipase ≤1.5x ULN); treatment for brain metastases has been completed (if any) (see Example 1 below); no active infection and no evidence of serious infection in the past month; at least four (4) weeks or five half-lives (whichever is shorter) since the last dose of anticancer therapy prior to the first administration of anti-Gal-9 antibody.

Alternatively or additionally, subjects suitable for the treatments disclosed herein may not have one or more of the following: a diagnosis of metastatic cancer of unknown primary origin; any active, uncontrolled bleeding and any patient with a bleeding constitution (e.g., active peptic ulcer disease); receiving any other investigational agent within 4 weeks or 5 half-lives of the administration of anti-galactoglobulin-9 antibody; receiving radiotherapy within 4 weeks of the first dose of anti-galactoglobulin-9 antibody, except for a limited range of palliative radiotherapy, such as for the treatment of bone pain or focal pain tumor masses; having fungal tumor masses; Active clinically severe infection >2 NCI-CTCAE version 5.0; symptomatic or active brain metastases; ≥CTCAE grade 3 toxicity (see details and exceptions in Example 1); history of a second malignancy (see exceptions in Example 1); evidence of severe or uncontrolled systemic disease or congestive heart failure; severe non-healing wounds, active ulcers, or untreated fractures; uncontrolled pleural effusion, pericardial effusion, or ascites requiring repeated drainage procedures; spinal cord compression without a clear history of surgery and/or radiation therapy. Active or previously treated leptomeningeal disease; significant vascular disease; active autoimmune disease (see exceptions in Example 1); requiring systemic immunosuppressive therapy; tumor-related pain (> grade 3) unresponsive to extensive analgesic interventions (oral and/or patch); uncontrolled hypercalcemia despite bisphosphonate use; any history of immune-related grade 4 adverse events attributable to prior checkpoint inhibitor therapy (CIT); receiving organ transplantation; and/or undergoing dialysis; and/or a Child-Pugh score ≥ 7. In some cases, human patients may not have metastatic hepatocellular carcinoma that has progressed while receiving at least one prior line of systemic therapy; have refused or are intolerant of sorafenib; or have considered standard therapy ineffective, intolerable, or inappropriate, or no effective standard therapy is available.

Alternatively or additionally, human patients receiving any of the treatments disclosed herein may not have: (i) metastatic cancer of unknown primary origin; (ii) clinically significant, active, uncontrolled bleeding, any bleeding predisposition (e.g., active peptic ulcer disease); (iii) radiation therapy within 4 weeks of the first dose of treatment; (iv) fungal tumor masses; (v) ≥CTCAE grade 3 toxicity due to prior cancer treatment (excluding alopecia and vitiligo); (v) a history of a second malignancy; (vi) severe or uncontrolled systemic disease, congestive heart failure >NYHA grade 2 or evidence of myocardial infarction (MI) within 6 months; (vii) severe unhealed wound, active ulcer, or untreated fracture; (viii) uncontrolled pleural effusion requiring repeated drainage procedures. (ix) History of severe allergic, anaphylactic, or other hypersensitivity reactions to chimeric or humanized antibodies or fusion proteins; (x) Significant vascular disease within 6 months of treatment (e.g., aortic aneurysm requiring surgical repair or recent arterial thrombosis), history of pulmonary embolism, stroke, or transient ischemic attack within 3 months of treatment, and/or history of abdominal fistula or gastrointestinal perforation within 6 months of treatment; (xi) Active autoimmune disease (excluding 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) Uncontrolled hypercalcemia despite bisphosphonate use; (xiv) Receiving organ transplantation.

In some cases, the subject is a human patient with elevated galectin-9 levels, such as those relative to control levels. The galectin-9 level can be the plasma or serum level of galectin-9 in a human patient. In other instances, the galectin-9 level is the level of galectin-9 in cancer cells within a tumor. In other instances, the galectin-9 level is the level of galectin-9 in immune cells within a tumor. In other instances, the galectin-9 level can be the level of galectin-9 on the cell surface, such as on cancer cells. In one instance, the galectin-9 level can be, for example, the level of galectin-9 expressed on the surface of cancer cells, or the level of galectin-9 expressed in immune cells, measured in a patient-derived organoid tumor spheroid (PDOT), which can be prepared by methods disclosed, for example, in the following examples. The control level may refer to the level of galactolectin-9 in matched samples from subjects of the same species (e.g., humans) without solid tumors. In some instances, the control level represents the level of galactolectin-9 in healthy subjects. In some implementations, the control level may be the baseline level before treatment.

To identify such subjects, suitable biological samples can be obtained from subjects suspected of having solid tumors, and these samples can be analyzed using conventional methods (e.g., ELISA or FACS) to determine the levels of galectin-9 contained therein (e.g., free, cell surface expressed, or total). In some embodiments, organoid cultures are prepared, for example, as described herein, and used to assess galectin-9 levels in subjects. Single cells obtained from certain fractions as part of the organoid preparation process are also suitable for assessing galectin-9 levels in subjects. In some cases, assays for measuring levels of free form or expressed on the cell surface of galectin-9 involve the use of an antibody that specifically binds to galectin-9 (e.g., specifically human galectin-9). Any anti-galectin-9 antibody known in the art can be tested for suitability in any of the above assays and then used in such assays in a conventional manner. In some embodiments, antibodies described herein (e.g., G9.2-17 antibody) can be used in such assays. In some embodiments, the antibody is described in U.S. Patent Nos. 10,344,091 and WO2019/084553, the relevant disclosures of which are incorporated herein by reference for the purposes and subject matter of this document. In some instances, the anti-galactolectin-9 antibody is a Fab molecule. Assays for determining galactolectin-9 levels as disclosed herein are also within the scope of this disclosure.

(iv) Response to treatment

The efficacy of the treatments disclosed herein can be assessed through routine practice. In some embodiments, any of the methods disclosed herein may increase antitumor activity (e.g., reduction of cell proliferation, tumor growth, tumor volume and/or tumor burden or load, or reduction of the number of metastatic lesions over time) by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to levels in pre-treatment or control subjects. In some embodiments, the reduction is measured by comparing cell proliferation, tumor growth and/or tumor volume in subjects before and after administration of the pharmaceutical composition. In some embodiments, the methods disclosed herein may improve one or more symptoms of cancer by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some embodiments, cancer cells and/or biomarkers in a 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, burden, or load in a subject to an undetectable size or to about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the tumor volume, size, burden, or load in the subject before treatment. In other embodiments, methods are provided for reducing the cell proliferation rate or tumor growth rate in a subject to an undetectable rate or to about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the rate before treatment. 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 about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the rate before treatment.

The response to treatment (e.g., treatment of solid tumors as described herein) may be evaluated according to RECIST or RECIST 1.1 criteria and/or irRC, irRECIST, iRECIST, imRECISTPDAC, as described in Example 1 below and Eisenhower et al., New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1); European Journal of Cancer 45 (2009) 228-247; or Borcoman et al., Annals of Oncology 30:385-396, 2019; Nishino et al., Clin Cancer Res 2013; 19(14):3936-3943, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, methods are provided for improving and/or controlling overall response/tumor burden/tumor size (e.g., at approximately 2, 3, 6, or 12 months or later) compared to baseline levels obtained prior to the initiation of a G9.2-17 IgG4 treatment regimen, said methods comprising administering an anti-galactoglobulin-9 antibody as described herein. In some embodiments, said methods are used to improve and/or control overall response/tumor burden/tumor size at approximately 2 months. In some embodiments, when an anti-galactoglobulin-9 antibody (e.g., G9.2-17(IgG4)) is administered in combination with a checkpoint inhibitor (e.g., an anti-PD-1 antibody, such as tislelizumab), overall response/tumor burden/tumor size (e.g., at approximately 2, 3, 6, or 12 months or later), for example, compared to baseline levels obtained prior to the initiation of treatment, may be improved or controlled. In some implementations, methods are provided for producing a complete response, a partial response, or stable disease (e.g., as measured at approximately 2, 3, 6, or 12 months, or at a later time, or at any other clinically indicated time point), said methods comprising administering the anti-galactohemagglutinin-9 antibody described herein. Such a response may be transient or permanent over a period of time.

In some embodiments, the methods disclosed herein may improve the likelihood of a complete response, a partial response, or stable disease (e.g., measured at approximately 2, 3, 6, or 12 months, or at a later time, or at any other clinically indicated time point), for example, compared to baseline levels obtained prior to initiation of a G9.2-17 IgG4 treatment regimen. This response may be transient or permanent over a period of time. In some embodiments, treatment may produce, for example, a reduction or attenuation of progressive disease compared to baseline levels obtained prior to initiation of a G9.2-17 IgG4 treatment regimen (e.g., measured at approximately 2, 3, 6, or 12 months, or at a later time, or at any other clinically indicated time point). This attenuation may be transient or permanent. In any of these embodiments, the anti-galactohemagglutinin-9 antibody may be administered in combination with a checkpoint inhibitor (e.g., an anti-PD-1 antibody).

In some embodiments, the methods disclosed herein may attenuate disease progression or reduce progressive disease (e.g., as measured at approximately 3 months, 6 months, or 12 months, or at a later time or any other clinically indicated time point). The methods involve administering a therapeutically effective amount of an anti-galactolectin-9 antibody, as disclosed herein, to a subject. In any of these embodiments, the anti-galactolectin-9 antibody may be administered in combination with a checkpoint inhibitor (e.g., an anti-PD-1 antibody).

In any of the methods described herein, partial response, stable disease, complete response, partial response, stable disease, progressive disease, and disease progression (e.g., measured at approximately 2, 3, 6, or 12 months, or at a later time or at any other clinically indicated time point) may be assessed according to the irC criteria, RECIST criteria, RECIST 1.1, irRECIST or iRECIST, or imRECIST criteria, or other criteria known in the art (see, for example, Borcoman et al., Annalsof Oncology 30:385-396, 2019; iRC: Hoos et al., J. Immunother. 30(1):1-15).

A partial response is a reduction in tumor size or degree of cancer in vivo (i.e., tumor burden) compared to baseline levels before treatment. For example, according to RECIST response criteria, a partial response is defined as a reduction of at least 30% in the sum of the diameters of the target lesions, with reference to the baseline total diameter. Progressive disease is a disease that is growing, spreading, or worsening. For example, according to RECIST response criteria, progressive disease includes a condition in which an increase of at least 20% in the sum of the diameters of the target lesions is observed, and the sum must also show an absolute increase of at least 5 mm. Furthermore, the appearance of one or more new lesions is also considered progressive. A tumor that neither decreases nor increases in degree or severity compared to baseline levels before treatment is considered stable disease. For example, according to RECIST response criteria, stable disease occurs when there is neither sufficient contraction to meet the criteria for a partial response nor sufficient increase to meet the criteria for progressive disease, with reference to the minimum total diameter at the time of study.

In some embodiments, this disclosure provides methods for permanently or for a minimal period of time reducing or maintaining tumor size in a subject (including human subjects) relative to a baseline tumor size prior to treatment initiation (e.g., measured at approximately 2, 3, 6, or 12 months, or at a later time, or at any other clinically indicated time point), said methods comprising administering a therapeutically effective amount of anti-galactohemagglutinin-9 antibody alone or in combination with a checkpoint inhibitor (e.g., anti-PD-1 antibody) to the subject. Tumor size, such as tumor diameter, can be measured according to methods known in the art, including measurements from CT and MRI images according to specific measurement protocols, such as those described above in Eisenhower et al., in combination with various software tools. Thus, in some embodiments, tumor size is measured in periodically scheduled restaging scans (e.g., CT with/without contrast, MRI with/without contrast, PET-CT (diagnostic CT) and/or X-ray, ultrasound, and/or other relevant imaging modalities). In some embodiments, tumor size reduction or maintenance refers to the size of a target lesion. In some embodiments, tumor size reduction or maintenance refers to the size of a non-target lesion. According to RECIST 1.1 criteria, when more than one measurable lesion is present at baseline, all lesions representing a maximum of five lesions in total (with a maximum of two lesions per organ) representing all relevant organs should be identified as target lesions. All other lesions (or disease sites), including pathological lymph nodes, should be identified as non-target lesions.

In some embodiments, this disclosure provides methods for increasing the likelihood of reducing or maintaining tumor burden (e.g., as measured at approximately 2, 3, 6, or 12 months, or at a later time, or at any other clinically indicated time point), said methods comprising administering a therapeutically effective amount of a single anti-galactohemagglutinin-9 antibody, as disclosed herein, or in combination with a checkpoint inhibitor (e.g., an anti-PD-1 antibody, such as tislelizumab), to a subject. In some embodiments, treatment may result in a greater likelihood of reducing or maintaining tumor burden (e.g., as measured at approximately 2, 3, 6, or 12 months, or at a later time, or at any other clinically indicated time point). As used herein, tumor burden refers to the number, size, or volume of cancers in a subject, taking into account all disease sites. Tumor burden can be measured using methods known in the art, including but not limited to FDG positron emission tomography (FDG-PET), magnetic resonance imaging (MRI), and optical imaging, including bioluminescence imaging (BLI) and fluorescence imaging (FLI).

In some embodiments, the methods described herein increase the time to disease progression or progression-free survival (e.g., measured at approximately 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated time point after treatment initiation). Progression-free survival can be permanent or progress-free survival over a certain period of time. In some embodiments, the methods provide a greater probability of progression-free survival (permanent progression-free survival or progression-free survival over a certain period of time, such as 3, 6, or 12 months, or measured at approximately 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated time point after treatment initiation). Progression-free survival (PFS) is defined as the time from randomization (e.g., from treatment initiation) in a clinical trial to disease progression or death from any cause. In some embodiments, the methods achieve longer survival or a greater probability of survival, for example, over a certain period of time, such as 6 or 12 months.

Response to treatment (e.g., treatment of solid tumors as described herein) can be assessed according to iRECIST criteria, as described in Seymour et al., iRECIST: guidelines for response criteria for use in trials; The Lancet, Vol. 18, March 2017 (the contents of which are incorporated herein by reference in their entirety). iRECIST was developed specifically for the use of modified RECIST 1.1 criteria in cancer immunotherapy trials to ensure consistent design and data collection, and to serve as a guideline for standard methods of measuring solid tumors, as well as for defining objective changes in tumor size used in trials using immunotherapy. iRECIST is based on RECIST 1.1. Responses assigned using iRECIST have the prefix "i" (i.e., immune)—for example, "immune" complete response (iCR) or partial response (iPR), and unconfirmed progressive disease (iUPD) or confirmed progressive disease (iCPD) or stable disease (iSD)—to distinguish them from responses assigned using RECIST 1.1, all of which are defined in RECIST 1.1 by Seymour et al. In some embodiments, the criteria may be compared to baseline levels prior to treatment initiation. In any of these embodiments, anti-galactoglobulin-9 antibodies may be administered alone or in combination with checkpoint inhibitors, such as anti-PD-1 antibodies, as disclosed herein.

Therefore, in some embodiments, this disclosure provides methods for improving the overall response (iOR) or achieving an “immune” complete response (iCR), partial response (iPR), or stable disease (iSD) compared to the baseline level of the disease prior to treatment initiation (e.g., as measured at approximately 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated time point). The reduction in the “immune” response (e.g., iCR, iPR, or iSD) may be temporary or permanent over a period of time. In some embodiments, treatment may increase the likelihood of a complete response (iCR), partial response (iPR), or stable disease (iSD) (e.g., as measured at approximately 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated time point). For example, in some embodiments, this disclosure provides a method for attenuating disease progression or mitigating progressive disease (e.g., mitigating unproven progressive disease (iUPD) or proven progressive disease (iCPD)) (e.g., as measured at approximately 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated time point), said method comprising administering to a subject a therapeutically effective amount of an anti-galactolectin-9 antibody as disclosed herein. Any of these iRECIST criteria mentioned above can be compared to baseline levels prior to treatment initiation. In any of these methods, the anti-galactolectin-9 antibody can be administered alone or in combination with a checkpoint inhibitor (e.g., an anti-PD-1 antibody).

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

Other criteria can be used to measure treatment response. For example, tumor burden can be measured according to the irRC criteria (Hoos et al., 2007). In the irRC, tumor burden is measured by combining “indicator” lesions with new lesions, where new lesions are considered changes in tumor burden. In the irRC, immune-related complete response (irCR) is the disappearance of all lesions, whether measured or not, with no new lesions; immune-related partial response (irPR) is a 50% decrease in tumor burden relative to baseline as defined by the irRC; and immune-related progressive disease (irPD) is a 25% increase in tumor burden relative to the lowest recorded level. Everything else is considered immune-related stable disease (irSD).

Immune-related RECIST (irRECIST) is based on a one-dimensional measure of RECIST and further redefines specific immune-related criteria within irRECIST. Recently, a new criterion was evaluated based on atezolizumab data in NSCLC (immunomodified RECIST (imRECIST)), requiring confirmation of disease progression at least 4 weeks after initial assessment (Hodi et al., JCO 2018; 36(9):850-858). For comparisons of RECIST 1.1, irRC, irRECIST, iRECIST, and imRECIST, see, for example, Figure 4 in Borcoman et al., Annals of Oncology 30:385-396, 2019; and Nishino et al., ClinCancer Res 2013; 19(14):3936-3943, the contents of which are incorporated herein by reference in their entirety. Any of these criteria is suitable for determining the response rate of any of the methods described herein.

(v) Monitoring adverse events and adjusting treatment conditions

In addition, the occurrence of side effects (e.g., serious side effects) can be monitored in subjects treated alone with any of the anti-galactoglobulin-9 antibodies disclosed herein (e.g., G9.2-17) or in combination with a checkpoint inhibitor disclosed herein (e.g., anti-PD-1, such as tislelizumab). Exemplary side effects monitored are provided in Example 3 below. If a side effect is observed, the treatment condition for that subject can be changed. For example, the dose of the anti-galactoglobulin-9 antibody can be reduced and/or the dosing interval can be extended. The appropriateness and extent of the reduction can be assessed by a qualified clinician. In one embodiment, a reduction of 30% or 50% of the previous dose level is implemented. In one specific example, a reduction of at least 30% is implemented as assessed by a clinician (reduction to dose level 1, the level at the time of the first dose reduction). If necessary, a further dose reduction of 30% of dose level 1 is implemented (dose level 2, the level at the time of the second dose reduction). In another example, a further dose reduction of 50% of dose level 1 is implemented (dose level 2). In some embodiments, one or more doses are administered to reduce the previous dose level by about 10% to about 80%. In some embodiments, one or more doses are administered to reduce the previous dose level by about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, or about 70% to about 80%. In some embodiments, one or more doses are administered to reduce the previous dose level by 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, or 70% to 80%. In some embodiments, one or more doses are administered to reduce the previous dose level by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%. In some implementations, one or more doses are administered at a reduction of 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the previous dose level. Alternatively or additionally, the dose of the checkpoint inhibitor may be reduced and/or the dosing interval of the checkpoint inhibitor may be extended. In some cases (e.g., in the event of life-threatening side effects), treatment may be discontinued.

In some cases, if side effects are observed in patients, the dose of anti-galactoglobulin-9 antibody (such as G9.2-17 (IgG4)) and/or anti-PD-1 antibody (such as tislelizumab) may be reduced. In some cases, the dose may be reduced by 50%. When necessary, the dose may be reduced by a further 50%. See, for example, Example 3 below.

(vi) Biomarkers used to assess response to treatment

The response to treatment can also be characterized by one or more of the following: blood and tumor immunophenotype, cytokine profile (serum), soluble galactolectin-9 levels in blood (serum or plasma), galactolectin-9 tumor tissue expression levels and patterns (by immunohistochemistry) (tumor, stroma, immune cells), tumor mutational burden (TMB), PD-L1 expression (e.g., by immunohistochemistry), mismatch repair status, or disease-associated tumor markers (e.g., measured at approximately 3 months, 6 months, or 12 months, or at a later time or any other clinically indicated time point). Examples of such tumor markers include, but are not limited to, CA15-3, CA-125, CEA, CA19-9, and alpha-fetoprotein. These parameters can be compared to baseline levels prior to treatment initiation. In any of these implementation schemes, anti-galactolectin-9 antibodies may be administered alone or in combination with checkpoint inhibitors (e.g., anti-PD-1 antibodies).

In any of the methods disclosed herein, subjects may be examined for one or more of the following characteristics 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) galactolectin 9 serum/plasma levels; (d) peripheral blood mononuclear cell immunophenotyping; (e) multiple immunophenotyping of tumor tissue biopsy/resected specimens; (f) galactolectin 9 expression levels and patterns in tumor tissue biopsy/resected specimens; and (g) any other immunohistochemical tests, such as PD-L1 immunohistochemistry, tumor mutational burden (TMB), tumor microsatellite instability status, and groups such as: - HalioDx, ImmunoSeq-Adaptive Biotechnologies, NanoString gene expression system, 18-gene signature, PanCancer IO 360 ^TIS , developed on NanoString Technologies, is another example. Other suitable biomarkers specific to target tumors (such as PDAC) can also be used. In a non-limiting example, PD-L1 (SP263) (Roche, Ventana) can be used to detect PD-L1 in cancer tissues using immunohistochemistry.

In some implementations, this document describes methods for altering the levels of immune cells and immune cell markers (e.g., immune activation) in the blood or tumor, including the administration of a single anti-Gal-9 antibody or a combination thereof with a checkpoint inhibitor (e.g., an anti-PD-1 antibody). Such changes can be measured in patient blood and tissue samples using methods known in the art, such as multiplex flow cytometry and multiplex immunohistochemistry. 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 2 lists non-limiting examples of markers that can be used in these evaluation methods. Flow cytometry (FC) is a rapid and informative technique of choice for analyzing cell phenotypes and functions, and has gained prominence in immunophenotypic surveillance. It allows for the characterization of many cell subpopulations, including rare subpopulations, in complex mixtures such as blood and represents a rapid method for acquiring large amounts of data. FC offers advantages such as speed, high sensitivity, and high specificity. Standardized antibody sets and procedures can be used to analyze and classify immune cell subtypes. Multiplex IHC is a powerful research tool that provides objective, quantitative data describing the tumor immune background in terms of the number and location of immune subsets, and allows for the evaluation of multiple biomarkers on a single tissue slice. Computer algorithms can be used to quantify the levels of IHC-based biomarkers across entire slices of a patient biopsy, thus combining chromogenic IHC methods and staining with digital pathology approaches.

Therefore, in some embodiments, methods for modulating the immune response, such as modulating those immune activation markers as shown in Table 2, are described herein, including administration of anti-gal9 antibodies alone or in combination with checkpoint inhibitor therapy. In some embodiments, modulation includes one or more of the following: (1) an increase in more CD8 cells in plasma or tumor tissue, (2) a decrease in T regulatory cells (Tregs) in plasma or tumor tissue, (3) an increase in M1 macrophages in plasma or tumor tissue, and (4) a decrease in MDSCs in plasma or tumor tissue, and (5) a decrease in M2 macrophages in plasma or tumor tissue (e.g., as measured at approximately 2, 3, 6, or 12 months, or at a later time or at any other clinically indicated time point). In some embodiments, the markers evaluated using the techniques described above or known in the art are selected from CD4, CD8, CD14, CD11b/c, and CD25. These parameters can be compared to baseline levels prior to the start of treatment.

Table 2. PBMC typing markers

(vii) Regulation of immune response

In some embodiments, methods for modulating pro-inflammatory and anti-inflammatory cytokines are described herein, including administration of antigal9 alone or in combination with checkpoint inhibitor therapy. In some embodiments, methods are provided for one or more of the following: (1) increasing the level of IFNγ in plasma or tumor tissue; (2) increasing the level of TNFα in plasma or tumor tissue; (3) decreasing the level of IL-10 in plasma or tumor tissue (e.g., as measured at approximately 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated time point). These parameters can be compared to baseline levels prior to the initiation of treatment.

In some embodiments, cytokine or immune cell levels may be assessed between a single tumor biopsy prior to administration and a repeat biopsy performed at a feasible time. In some embodiments, cytokine or immune cell levels may be assessed between two repeat biopsies. In some embodiments, methods are provided for modulating one or more of the following: soluble galactolectin-9 levels in blood (serum or plasma) or galactolectin-9 tumor tissue expression levels and expression patterns (tumor, stroma, immune cells) obtained by immunohistochemistry (e.g., as measured at approximately 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated time point). In some embodiments, said methods reduce soluble galactolectin-9 levels in blood (serum or plasma) or galactolectin-9 tumor tissue expression levels or expression patterns (tumor, stroma, immune cells) obtained by immunohistochemistry (e.g., as measured at approximately 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated time point). Galactolectin-9 levels may be compared to baseline levels prior to treatment initiation. In some embodiments, galactolectin-9 levels can be compared to untreated controls or healthy subjects. In any of these embodiments, anti-galactolectin-9 antibodies can be administered alone or in combination with a checkpoint inhibitor (e.g., an anti-PD-1 antibody). In some embodiments, methods for modulating PD-L1 expression (e.g., as assessed by immunohistochemistry) are provided, the methods comprising administering anti-galactolectin-9 antibodies alone or in combination with a checkpoint inhibitor (e.g., an anti-galactolectin-9 antibody). In some embodiments, the methods modulate one or more disease-related tumor markers (increases or decreases) (e.g., as measured at approximately 2, 3, 6, or 12 months, or at later times, or at any other clinically indicated time point). Examples of such tumor markers include, but are not limited to, CA15-3, CA-125, CEA, CA19-9, and alpha-fetoprotein. These parameters can be compared to baseline levels prior to the initiation of treatment. In any of these implementations, the anti-galactoglobulin-9 antibody may be administered alone or in combination with a checkpoint inhibitor (e.g., an anti-PD-1 antibody).

In some embodiments, this disclosure provides methods for modulating an immune response in a subject. As used herein, the term "immune response" includes T-cell-mediated and/or B-cell-mediated immune responses influenced by 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 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.

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 causes an increase (or upregulation) in one or more immune response parameters and a decrease (or downregulation) in one or more other immune response parameters, resulting in an overall increase in the immune response, such as an overall increase in the inflammatory immune response. In another instance, modulating the immune response causes an increase (or upregulation) in one or more immune response parameters and a decrease (or downregulation) in one or more other immune response parameters, resulting in an overall decrease in the immune response, such as an overall decrease in the inflammatory response. In some embodiments, the increase in the overall immune response, i.e., the increase in the overall inflammatory immune response, is determined by a reduction in tumor weight, tumor size, or tumor burden, or by any RECIST or iRECIST criteria described herein. In some embodiments, the increase in the overall immune response is determined by an increase in the levels of one or more pro-inflammatory cytokines (e.g., including two or more, three or more, etc.) or most pro-inflammatory cytokines (or a decrease or constant of one or more of the most potent anti-inflammatory or immunosuppressive cytokines and/or the most potent anti-inflammatory or immunosuppressive cytokines). In some embodiments, an increase in the overall immune response is determined by an increase in the level of one or more of the most potent pro-inflammatory cytokines (with a decrease or no change in the level of one or more anti-inflammatory and/or immunosuppressive cytokines, including one or more of the most potent cytokines). In some embodiments, an increase in the overall immune response is determined by a decrease in the level of one or more of the most potent immunosuppressive and/or anti-inflammatory cytokines (with an increase or no change in the level of one or more or most of the pro-inflammatory cytokines, including, for example, the most potent pro-inflammatory cytokines). In some embodiments, an increase in the overall immune response is determined by an increase in the level of one or more of the most potent anti-inflammatory and/or immunosuppressive cytokines (with an increase or no change in the level of one or more or most of the pro-inflammatory cytokines, including, for example, the most potent pro-inflammatory cytokines). In some embodiments, an increase in the overall immune response is determined by a combination of any of the above. Furthermore, an increase (or upregulation) in one type of immune response parameter can lead to a corresponding decrease (or downregulation) in another type of immune response parameter. For example, an increase in the production of certain pro-inflammatory cytokines can lead to a downregulation of certain anti-inflammatory and/or immunosuppressive cytokines, and vice versa.

In some embodiments, this disclosure provides methods for modulating the immune response of a subject (including human subjects) (e.g., as measured at approximately 2, 3, 6, or 12 months, or at a later time, or at any other clinically indicated time point), said methods comprising administering to the subject a therapeutically effective amount of an anti-galactoglobulin-9 antibody as disclosed herein. In some embodiments, this disclosure provides methods for modulating the levels of immune cells and immune cell markers (including, but not limited to, those described herein in Table 2) in the blood or tumor of a subject (including human subjects), for example, compared to baseline levels prior to the start of treatment (e.g., compared to baseline levels obtained prior to the start of an anti-Gal9 antibody treatment regimen), said methods comprising administering to the subject a therapeutically effective amount of an anti-galactoglobulin-9 antibody as disclosed herein. In some embodiments, the overall result of the modulation is the upregulation of pro-inflammatory immune cells and/or the downregulation of immunosuppressive immune cells. In some embodiments, this disclosure provides methods for modulating the levels of immune cells, said modulation comprising one or more of the following: (1) increasing CD8 cells in plasma or tumor tissue, (2) decreasing Tregs in plasma or tumor tissue, (3) increasing M1 macrophages in plasma or tumor tissue, (4) decreasing MDSCs in plasma or tumor tissue, and (5) decreasing M2 macrophages in plasma or tumor tissue, and said method comprising administering to a subject a therapeutically effective amount of an anti-galactohemagglutinin-9 antibody as disclosed herein. In some embodiments, biomarkers for assessing the levels of such immune cells include, but are not limited to, CD4, CD8, CD14, CD11b/c, and CD25. In some embodiments, this disclosure provides a method for modulating the levels of pro-inflammatory and immunosuppressive cytokines in the blood or tumor of a subject (including human subjects) (e.g., measured at approximately 2, 3, 6, or 12 months, or at a later time, or at any other clinically indicated time point) (e.g., compared to baseline levels prior to treatment initiation), said method comprising administering a therapeutically effective amount of an anti-galactoglobin-9 antibody, as disclosed herein, to the subject. In some embodiments, the overall result of the modulation is the upregulation of pro-inflammatory cytokines and/or the downregulation of immunosuppressive cytokines. In some embodiments, this disclosure provides a method for modulating the levels of cytokines in cells, wherein the modulation encompasses one or more of the following: (1) increasing the level of IFNγ in plasma or tumor tissue; (2) increasing the level of TNFα in plasma or tumor tissue; (3) decreasing the level of IL-10 in plasma or tumor tissue.

In some embodiments, this disclosure provides a method for altering one or more of the following: soluble galactolectin-9 levels in blood (serum or plasma) or galactolectin-9 tumor tissue expression levels and expression patterns (tumor, stroma, immune cells) obtained by immunohistochemistry (e.g., measured at 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated time point). The method includes administering a therapeutically effective amount of an anti-galactolectin-9 antibody, as disclosed herein, to a subject. In some embodiments of the method, one or more of the following remain unchanged: soluble galactolectin-9 levels in blood (serum or plasma) or galactolectin-9 tumor tissue expression levels and expression patterns (tumor, stroma, immune cells) obtained by immunohistochemistry. In some embodiments, the methods described herein reduce one or more of the following: soluble galactolectin-9 levels in blood (serum or plasma), or galactolectin-9 expression levels and patterns in tumor tissue (via immunohistochemistry) (tumor, stroma, immune cells) (e.g., measured at 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, or 12 months, or at a later time or any other clinically indicated time point). Galactolectin-9 levels can be compared to baseline levels prior to treatment initiation. In some embodiments, galactolectin-9 levels can be compared to healthy subjects. In some embodiments, treatment produces changes in PD-L1 expression (e.g., via immunohistochemistry). Dosage levels of 16 mg/kg or higher.

In some embodiments, this disclosure provides methods for altering PD-L1 expression (e.g., as assessed by immunohistochemistry) (e.g., measured at 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated time point), said methods comprising administering to a subject a therapeutically effective amount of an anti-galactolectin-9 antibody as disclosed herein. In some embodiments of the method, PD-L1 expression (e.g., as assessed by immunohistochemistry) remains unchanged. PD-L1 levels can be compared to baseline levels prior to treatment initiation. In some embodiments, the methods provided herein reduce PD-L1 expression, for example, as assessed by immunohistochemistry. PD-L1 levels can be measured using conventional methods known in the art. In one non-limiting example, PD-L1 (SP263) (Roche, Ventana) can be used to detect PD-L1 in cancer tissue using immunohistochemistry at dose levels of 16 mg/kg or higher.

In some embodiments, this disclosure provides methods for altering (increasing or decreasing) one or more disease-related tumor markers (e.g., measured at 2 weeks, 4 weeks, 1 month, 3 months, 6 months, or 12 months, or at a later time or any other clinically indicated time point), said methods comprising administering a therapeutically effective amount of an anti-galactohemagglutinin-9 antibody, as disclosed herein, to a subject. In some embodiments of said methods, the one or more disease-related tumor markers (increase or decrease) remain constant. Examples of such tumor markers include, but are not limited to, CA15-3, CA-125, CEA, CA19-9, and alpha-fetoprotein. The levels of the tumor markers can be compared to baseline levels prior to the initiation of treatment. In some embodiments, the methods provided herein reduce the occurrence of one or more disease-related tumor markers.

In some embodiments, this disclosure provides methods for altering (increasing or decreasing) one or more disease-related biomarkers (e.g., as measured at 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, or 12 months, or at a later time, or at any other clinically indicated time point), said methods comprising administering to a subject a therapeutically effective amount of an anti-galactohemagglutinin-9 antibody as disclosed herein. The levels of biomarkers in clinical tissues from a patient can be measured using conventional methods, such as multiplex immunofluorescence (mIF) techniques, as described herein in the embodiments. Exemplary groups of biomarkers may include CD3, CD4, CD8, CD45RO, FoxP3, CD11b, CD14, CD15, CD16, CD33, CD68, CD163, HLA-DR, arginase 1, granzyme B, Ki67, PD-1, PD-L1, and PanCK.

Kits for treating solid tumors

This disclosure also provides kits for treating or alleviating solid tumors (such as those disclosed herein) (e.g., head and neck cancer or urothelial carcinoma). Such kits may include one or more containers containing an anti-galactoglobulin-9 antibody (e.g., any of those described herein) (e.g., G9.2-17(IgG4)) and a checkpoint inhibitor (such as an anti-PD-1 antibody, e.g., tislelizumab, disclosed herein) used in conjunction with the anti-galactoglobulin-9 antibody, as also described herein.

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 anti-galactoglobulin-9 antibodies and anti-PD-1 antibodies to treat target diseases (as described herein), delay the onset of target diseases (as described herein), or alleviate target diseases (as described herein). In some embodiments, the kit may also include a description of selecting individuals suitable for treatment based on, for example, the application of diagnostic methods as described herein to identify whether an individual has a target disease. In still other embodiments, the instructions include a description of administering the antibodies to individuals at risk of developing the target disease.

Instructions for use involving the use of anti-galactoglobulin-9 antibodies and anti-PD-1 antibodies as disclosed herein generally include information on the intended treatment dose, dosing schedule, and route of administration. Containers may be unit doses, bulk packaging (e.g., multi-dose packs), or subunit doses. Instructions provided with kits of the present invention are typically written instructions on a label or packaging insert (e.g., a piece of paper included in the kit), but machine-readable instructions (e.g., instructions carried on a disk or optical storage disc) are also acceptable.

The kit of the present invention is packaged in suitable packaging. 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 conjunction with specific devices (such as inhalers, nasal application devices (e.g., nebulizers)) or infusion devices (such as micropumps) is also covered. In some embodiments, the kit has a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). In some embodiments, the container also has a sterile access port (e.g., the container is an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). At least one active agent in the composition is the anti-galactohemagglutinin-9 antibody described herein.

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 associated with the container. In some embodiments, the invention provides an article of manufacture containing the contents of the kit described above.

General Technology

Unless otherwise indicated, 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. Such techniques are well explained in the literature, such as *Molecular Cloning: A Laboratory Manual, 2nd Edition* (Sambrook et al., 1989), *Cold Spring Harbor Press*; *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Methods in Molecular Biology*, *Humana Press*; *Cell Biology: A Laboratory Notebook* (edited by J.E. Cellis, 1998), *Academic Press*; *Animal Cell Culture* (edited by R.I. Freshney, 1987); *Introduction to Cell and...* d Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (edited by A. Doyle, J.B. Griffiths and D.G. Newell, 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (edited by D.M. Weir and C.C. Blackwell); Gene Transfer Vectors for Mammalian Cells (J.M.Mi Muller and M.P. Calos (eds., 1987); Current Protocols in Molecular Biology (eds., F.M. Ausubel et al., 1987); PCR: The Polymerase Chain Reaction (eds., Mullis et al., 1994); Current Protocols in Immunology (eds., J.E. Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibod ies: a practical approach (edited by D. Catty., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using an tibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (edited by M. Zanetti and J.D. Capra, Harwood Academic Publishers, 1995).

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 illustrative only and not as limiting the remainder of this disclosure in any way. All publications cited herein are incorporated by reference for the purposes or subject matter of this document.

Example

Example 1: Evaluation of anti-Gal-9 antibodies, alone or in combination with checkpoint inhibitors, in a mouse model of pancreatic cancer, and tumor quality and immune characteristics in mice treated with G9.2-17mIgG1.

The effects of G9.2-17mIgG1 on tumor weight and immunomodulatory characteristics were evaluated in a mouse model of pancreatic cancer. Eight-week-old male C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were administered intrapancreatic injections of FC1242 PDAC cells derived from Pdx1Cre;KrasG12D;Trp53R172H (KPC) mice (Zambirinis CP et al., TLR9 ligation in pancreatic stellate cells promotes tumorigenesis. J Exp Med. 2015; 212:2077-94). Tumor cells were suspended in PBS containing 50% Matrigel (BD Biosciences, Franklin Lakes, NJ) and 1 x ^10⁵ tumor cells were injected into the pancreatic body via laparotomy. Mice (n = 10/group) received an intraperitoneal (IP) pretreatment dose, followed by three doses (qw) of commercial α-galactolectin-9 mAb (RG9-1, 200 μg, BioXcell, Lebanon, NH) or G9.2-17 mIgG1 (200 μg) or paired isotype G9.2-Iso or rat IgG2a (LTF-2, BioXcell, Lebanon, NH) (200 μg) (one dose per week for three weeks). Mice were sacrificed after three weeks, and tumors were harvested for flow cytometry analysis. Tissues were processed and prepared according to standard practice and analyzed by flow cytometry. See, for example, U.S. Patent No. 10,450,374.

Tumor quality and immune characteristics of mice treated with G9.2-17mIgG2a alone or in combination with αPD-1mAb

The effects of G9.2-17mIgG2a on tumor weight and immunomodulatory characteristics were evaluated, alone or in combination with immunotherapy, in a mouse model of pancreatic cancer. FC1242 PDAC cells derived from Pdx1Cre, KrasG12D, and Trp53R172H (KPC) mice were injected intrapancreatically into 8-week-old male C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME). Tumor cells were suspended in PBS containing 50% Matrigel (BD Biosciences, Franklin Lakes, NJ), and 1 x 10⁵ tumor cells were injected into the pancreatic body via laparotomy. Mice were intraperitoneally (i.p.) given a pretreatment dose, followed by three doses (q.w.) of G9.2-17mIgG2a (200 μg) alone or in combination, or neutralized αPD-1 mAb (29F.1A12, 200 μg, BioXcell, Lebanon, NH) or paired isotypes as indicated (LTF-2 and C1.18.4, BioXcell, Lebanon, NH). Mice were sacrificed on day 26, and tumors were harvested for analysis. Tissues were processed and prepared according to standard practice and analyzed by flow cytometry. See, for example, US 10,450,374. Each point represents one mouse; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; by unpaired Student t-test. These results showed that treatment with both dose levels of G9.2-17mIgG2a monotherapy reduced tumor growth, while anti-PD-1 alone had no effect on tumor size. Figure 1.

Compared to anti-galactoglobulin-9 mIgG1 200 μg, treatment with anti-galactoglobulin-9 mIgG1 200 μg plus anti-PD-1 showed a significant increase in cytotoxic T cell (CD8) levels (p<0.01), and compared to anti-PD-1 alone, the levels were significantly increased between anti-galactoglobulin-9 IgG1 200 μg and anti-PD-1 (p<0.001). These results suggest that the combination of anti-Gal9 and anti-PD-1 antibodies is expected to achieve excellent therapeutic effects.

Furthermore, the tumor immune response to treatment with G9.2-17 IgG1 mouse mAb (also known as G9.2-17mIgG), anti-PD-1 antibody, or a combination of G9.2-17 IgG1 mouse mAb and anti-PD-1 antibody was investigated in the B16F10 subcutaneous homology model described in this paper. As shown in Figures 2A and 2B, in the mouse model, the combination of G9.2-17 and anti-PD-1 showed a synergistic effect in reducing tumor volume and increasing CD8+ cells. Figures 3A and 3B show that the G9.2-17 antibody increased the expression of CD44 and TNFα in tumor-bearing T cells.

Example 2: Pharmacokinetics of G9.2-17 (IgG4) in human studies

This study is a phase 1/2, open-label, multicenter study evaluating the safety, pharmacokinetics, and antitumor activity of G9.2-17 (IgG4) alone or in combination with chemotherapy or an immune checkpoint inhibitor (e.g., a PD-1 antagonist) in patients with metastatic solid tumors. This study evaluated G9.2-17 (IgG4) administered every 2 weeks (Q2W) at dose levels of 0.2, 0.63, 2.0, 6.3, 10, or 16 mg/kg, and weekly (QW) at dose levels of 10 and 16 mg/kg. See WO2020/223702, WO2022/109302, International Patent Application No. PCT/US2022/027127, and International Patent Application No. PCT/US2022/027142, the relevant disclosures of which are incorporated herein by reference for the purposes cited.

Preliminary pharmacokinetic (PK) data from the 13 subjects in this study were available for analysis. Based on the preliminary data, the β-half-life (i.e., elimination half-life in a 2-compartment model) of G9.2-17 (IgG4) was found to be longer than that from non-compartmental analysis (NCA) sources. These results suggest that the elimination half-life of G9.2-17 (IgG4) ranges from 102 to 224 hours (i.e., 4.3 to 9.3 days) across a dose range of 0.2 mg/kg to 16 mg/kg, indicating that weekly dosing of G9.2-17 (IgG4) may be an appropriate dosing schedule for this antibody in human treatment.

Example 3: A Phase 1/2, open-label, multicenter study of the safety, pharmacokinetics, and antitumor activity of G9.2-17 (IgG4) as a single agent and in combination with tislelizumab in patients with locally advanced or metastatic solid tumors.

This is an open-label, non-randomized, multicenter phase 1/2 study in patients with recurrent and/or refractory unresectable locally advanced or metastatic solid tumors, with a dose-escalation phase (Part 1) and a cohort extension phase (Part 2). The study will be conducted at up to 20 sites in the United States. The study duration is estimated to be 12–24 months. Survival follow-up will continue for up to 2 years.

Treatment duration

The study drug will continue to be administered until disease progression, unacceptable toxicity, or withdrawal from the study. Patients who discontinue the study drug before disease progression and are not being treated with other anticancer therapies will continue the study until disease progression.

Treatment period

This study consists of the following time periods in Part 1 and Part 2 (see the following public information):

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

Treatment period : 28-day treatment cycle

Post-treatment timeframe : 30 days after the last treatment (end of treatment visit/early termination of visit).

Follow-up period for immune-mediated adverse reactions (IMAR) : All patients treated with G9.2-17 IgG4+ tislelizumab must return 90 days +/- 7 days after the last dose of study drug to assess potential IMAR.

Follow-up period : Long-term follow-up of up to 2 years (follow-up every 3 months).

Research Design

Part 1: Single Agent: Dosage Escalation Period

A dose-finding study will be conducted using a continuous reassessment (CRM) approach to establish dose-limiting toxicities (DLTs) and help evaluate possible recommended phase 2 doses (RP2D). Two to six patients in each treatment cohort 1–6 will be assigned to receive sequentially higher intravenous (IV) infusions of G9.2–17 IgG4 every two weeks (Q2W) starting at a dose of 0.2 mg/kg on days 1 and 15 of each 28-day cycle. Patients assigned to specific dose-escalation cohorts will receive the corresponding study dose for that cohort. They will receive the study drug until disease progression, unacceptable toxicity, or withdrawal from the study for other reasons.

Part 1, groups 1-8, included a maximum of approximately 44 patients. A total of 6 dose levels were evaluated:

• Dosage escalation group 1 = 0.2 mg/kg Q2W

• Dosage escalation group 2 = 0.63 mg/kg Q2W

• Dosage escalation group 3 = 2 mg/kg Q2W

• Dosage escalation group 4 = 6.3 mg/kg Q2W

• Dosage escalation group 5 = 10 mg/kg Q2W

• Dosage escalation group 6 = 16 mg/kg Q2W

If RP2D is not achieved in groups 1-6, then taking RP2D into account, two additional dose levels may be included:

• Dosage escalation group 7 = 10 mg/kg QW

• Dosage escalation group 8 = 16 mg/kg QW

In Part 1, increasing doses of G9.2-17 (IgG4) were administered sequentially via IV infusion at Q2W (groups 1-6) or QW (groups 7-8).

For groups 1–6, administer to 2 patients per dose. Initial dose escalation may be based on analysis of patient safety data focusing on the occurrence of DLT at previous dose levels, as well as other relevant safety and dose data from previous groups. Dose escalation may be performed after a minimum of 28 days (one cycle).

After completion of group 6, the once-weekly (QW) G9.2-17 IgG4 dosing schedule was evaluated, provided that RP2D had not been achieved. Patients were allowed to move to group 7 when safety data from group 6 were analyzed and no DLT was identified.

In groups 7 and 8, four patients per dose level were assigned to receive an IV infusion of G9.2-17 IgG4 QW on days 1, 8, 15, and 22 of each 28-day cycle. Starting with the first four patients in group 7, the dose was escalated to the next group if no DLT was identified.

Patients who were treated in the early groups before RP2D identification are allowed to have their dose increased to RP2D. They can continue taking RP2D until they need to discontinue treatment due to toxicity, disease progression, or other reasons.

Dose escalation is based on the generation of DLT in patients treated at previous dose levels. For each dose group, the previous DLT probability will be specified from GLP-compliant toxicity studies and preclinical models. For the total number of specified target DLT rates and dose levels, the skeleton of the power model d^exp(a) will be generated according to the method of Lee and Cheung, using previous MTD or OBD adjusted by PK/PD data at median dose levels and interval measurements δ = 0.05 (Lee and Cheung, 2011). The prior distribution of parameter “a” has a normal distribution with a mean of zero, minimizing prior variance information. RP2D is derived from the OBD/MTD dose in Part 1.

For patients experiencing toxicities (including IMAR) outside the DLT window, dose reduction is permitted only if clinical benefit is achieved and can be continued with a lower dose of G9.2-17 IgG4. The dose of G9.2-17 IgG4 will initially be reduced by 50%, and may be further reduced by 50%, as defined in the dose adjustment guidelines provided in the protocol. Further dose reduction is not permitted; detailed instructions for dose adjustment are provided in Appendix 8.

Part 1: Combination Therapy: Dosage Escalation

For Part 1 combination therapy (groups 9-14 listed below), patients were administered using a 4+2 design algorithm. Disease indications (groups 9 and 10: pancreatic ductal adenocarcinoma [PDAC]; groups 11 and 12: head and neck [H/N]; groups 13 and 14: urethral epithelial carcinoma) were administered in separate groups as follows:

• Combination group 9 = G9.2-17 IgG4 6.3mg/kg QW + gemcitabine/nab-paclitaxel (PDAC) (up to n=10)

• Combination group 10 = G9.2-17 IgG4 16.0mg/kg QW + gemcitabine/nab-paclitaxel (PDAC) (up to n=10)

• Combination group 11 = G9.2-17 IgG4 6.3mg/kg QW + tislelizumab 300mg every 4 weeks (Q4W) (H/N) (maximum n=6)

• Combination group 12 = G9.2-17 IgG4 16.0mg/kg QW + tislelizumab 300mg Q4W (H/N) (maximum n=6)

• Combination group 13 = G9.2-17 IgG4 6.3mg/kg QW + tislelizumab 300mg Q4W (urethral epithelium) (maximum n=6)

• Combination group 14 = G9.2-17 IgG4 16.0mg/kg QW + tislelizumab 300mg Q4W (urethral epithelium) (maximum n=6)

Groups 9, 11, and 13 (lower doses of 6.3 mg/kg for PDAC, H/N, and urethral epithelium, respectively) can be performed concurrently. The higher dose (16.0 mg/kg) in each of these groups is open for inclusion in DLT and evaluation of other safety parameters.

Dosage reduction

During cycle 1, the following occurs in cases of DLT events. If one out of four patients in group 9 achieves DLT in cycle 1, two more patients are added to the same group. If two or more out of four to six patients in group 9 achieve DLT, group 10 is opened at a lower dose of G9.2-17 IgG4. Groups 11-12 and 13-14 use the same procedure.

For patients experiencing toxicities (including IMAR) outside the DLT window, dose reduction is permitted only if clinical benefit is achieved and can be continued with a lower dose of G9.2-17 IgG4. The dose of G9.2-17 IgG4 may initially be reduced by 50%, and may be further reduced by 50%, as defined in the dose adjustment guidelines provided in the protocol. Further dose reduction is not permitted.

Part 2: Extension

Once safety has been established in any of the cohorts in Part 1 and preliminary efficacy and/or PD signals have been identified, one or more expansion cohorts may be initiated to further evaluate safety and efficacy in the specific tumor type. The sample size for each expansion cohort will be determined based on point estimates anticipated by (1) the criteria of care [null hypothesis] and (2) the recommended combination therapy [alternative hypothesis] for each tumor type in Part 1. Protocol amendments with details regarding the expansion cohorts, treatment regimens, and statistical methods may be submitted prior to initiation of Part 2.

In Part 2, patients received RP2D as a single agent of G9.2-17 IgG4 (as determined in Part 1), or a combination of RP2D-1 and PD-1 tislelizumab, in patients with head and neck cancer, urothelial carcinoma, or other solid tumors.

G9.2-17 IgG4. If, for any reason, administration cannot be completed on the same day, tislelizumab can be administered on day one and G9.2-17 IgG4 on day two.

Research objectives and endpoints

Part 1 - Dosage Escalation

Part 2 - Group Expansion

Single-agent and combination therapy

Treatment with single-agent or combination-agent regimens for patients with solid tumors (e.g., head and neck cancer or urothelial carcinoma) can be performed in parallel.

G9.2-17 IgG4 monotherapy

The starting dose of G9.2-17 IgG4 in single treatment may be the RP2D identified in Part 1. After testing the investigational drug in 23 patients in Phase I, the trial cohort may be terminated if ≤1 patient responds. If the trial proceeds to Phase II of Simon’s optimal design, approximately 33 additional patients will be treated in each single-drug cohort. If the total number of responding patients is ≤5, the investigational drug in that cohort will be rejected. If ≥6 patients have a confirmed ORR-3, the Part 3 extended cohort of that cohort will be activated, as described in the protocol amendment.

Dosage reduction may be permitted when clinical benefit is achieved and can continue to be obtained at a lower dose of G9.2-17 IgG4. The dose of G9.2-17 IgG4 may initially be reduced by 50%, and may be further reduced by 50%, as defined in the dosage adjustment guidelines provided in this article.

G9.2-17 IgG4 + Tislelizumab combination therapy

The dose of G9.2-17 IgG4 in combination therapy with tislelizumab can be RP2D-1, which is the dose immediately preceding the RP2D dose identified in Part 1. The optimal two-stage design can also be used to test the null hypothesis of ORR-3 ≤ 10% and the alternative hypothesis of ORR-3 ≥ 25%.

To ensure patient safety, a safety induction will be conducted, in which the first 8 patients will be administered the drug. Enrollment will continue only if ≤2 patients develop DLT (which will be below 25% of the target toxicity level (TTL)). If 3 or more patients develop DLT, this group of cancer types treated may be discontinued. If DLT occurs in any of the 8 safety inductions during the first 28 days of treatment, said patient may be permanently discontinued from treatment with the study drug.

For patients experiencing toxicity outside the DLT window, dose reduction may be permitted only if clinical benefit is achieved and continued use of a lower dose of G9.2-17 IgG4 is possible. The dose of G9.2-17 IgG4 may initially be reduced by 50%, and may be further reduced by 50%, as defined in the dose adjustment guidelines provided in the protocol. Further dose reduction is not permitted. Dose adjustment of tislelizumab may also be permitted as defined in this document and in Table 7 below.

If IMAR occurrence/relapse is not managed by reducing the dosage of either drug, then both investigational drugs can be discontinued.

Dose-limiting toxicity (DLT) criteria

In this trial, dose-limiting toxicities (DLTs) were defined as clinically significant hematologic and/or non-hematologic adverse events (AEs) or abnormal laboratory values assessed as unrelated to progression of metastatic cancer, comorbidities, or concomitant medications, and potentially related to or occurring during the first cycle (28 days) of the study. Any patient experiencing DLT during Part 1 or Part 2 of the first 28 days of treatment will be permanently discontinued from the study drug.

DLT is toxicity that meets any of the following criteria:

• Any death not clearly caused by an underlying disease or external cause

• Indications of potential drug-induced liver injury (Hy rule cases) are as follows:

○ALT or AST > 3x the upper limit of normal (ULN), confirmed by repeated testing after 24 hours, and

○ Serum total bilirubin (TBL) > 2x ULN, confirmed by repeat testing after 24 hours.

○ No other explanation can be found for elevated TBL and/or AT, such as viral hepatitis (A, B, or C), alcoholic or autoimmune hepatitis, pre-existing or acute liver disease, gallbladder obstruction or bile duct disease, Gilbert's syndrome, disease progression, or other drugs that can cause the observed effect.

• All Grade 4 non-hematological and hematological toxicities of any duration

• All Grade 3 non-hematologic and hematologic toxicities. Exceptions are as follows:

Grade 3 nausea, vomiting, and diarrhea do not require hospitalization or total parenteral nutrition support and can be managed down to grade ≤2 within 48 hours with supportive care.

Grade 3 electrolyte abnormality, which should be corrected to ≤ Grade 2 within 24 hours.

Grade 3 electrolyte abnormalities, lasting <24-72 hours, are clinically uncomplicated and resolve spontaneously or respond to routine medical interventions.

○≥ Grade 3 amylase or lipase unrelated to symptoms or clinical manifestations of pancreatitis.

Statistical methods:

Sample size

Dose escalation will be based on the presence or absence of DLT in patients treated at previous dose levels. For each dose group, the previous DLT probability will be specified from toxicity studies conforming to Good Laboratory Practice (GLP) and preclinical models. For the specified target DLT rate and total number of dose levels, the skeleton of the power model d^exp(a) will be generated according to the method of Lee and Cheung (2011), using previous MTDs adjusted for pharmacokinetic (PK)/pharmacodynamic (PD) data at median dose levels and interval measurements δ = 0.05. The prior distribution of parameter “a” has a normal distribution with a mean of zero, minimizing prior variance information. The trial will be stopped for safety reasons if the lower limit of the Agresti and Coull binomial CI at the lowest study dose level exceeds the target DLT rate.

CRM trial simulation analysis (with 1000 iterations) indicates that an average of approximately 20 patients are needed to inform the choice of RP2D, which is the maximum dose with an estimated DLT probability of less than or equal to 25% TTL.

The CRM used is based on the first 6 groups, but it does not need to determine RP2D on its own, because data from groups 7 to 14 are also used to determine RP2D.

The total sample size for Part 1 of the study is expected to be approximately 80 patients. Backfilling will provide additional patient inclusion if deemed necessary.

Part 2 of this study (the cohort expansion phase) may employ Simon’s two-stage optimal design to establish the safety and efficacy of LYT-200 in patients with the tumor types for which safety and preliminary efficacy were demonstrated in Part 1. In Part 2, the total sample size may depend on the number of expansion cohorts selected due to the safety and efficacy findings in the single-drug and combination cohorts of Part 1.

Randomized stratification:

This is an open-label study. In Part 1, patients will be assigned to treatment according to the study's CRM design. In Part 2, patients will be assigned to treatment groups, for example, based on inclusion and exclusion criteria.

Analysis of groups

Unless otherwise stated, the intention-to-treat (ITT) population is defined as patients who have received at least one dose of the investigational drug. Preliminary efficacy analyses may be performed on ITT. Patient management may be initiated for ITT.

The efficacy cohort can be defined as all patients in the ITT who have at least one measurable ORR 3 or PFS 6 assessment. This cohort can be used for sensitivity analysis.

The protocol-compliant (PP) group can be defined as any patient who has received at least one complete cycle of G9.2-17 (IgG4) without significant protocol deviation.

The safety population (SAF) can be defined as all patients who have received at least one dose of the investigational drug. Safety analyses can be performed on the SAF.

The PK/PD population can be defined as those patients who have received at least one complete cycle of G9.2-17 (IgG4).

General Statistical Plan

Database locking and preliminary analysis can be performed after the last patient experiences their primary endpoint event. Final study analysis can be conducted upon completion of the study. All analyses are likely to be descriptive.

Security Analysis

Unless otherwise stated, all security analyses can be performed on SAF and can be analyzed using descriptive statistics.

Efficacy Analysis

For ITT and PP, a descriptive summary of the disease response as assessed according to RECIST v1.1 is provided. Sensitivity analysis can be performed on efficacy populations.

Pharmacokinetics, pharmacodynamics and immunogenicity

Descriptive summaries of PK, PD, and immunogenicity in PK/PD populations.

Evaluation Timeline

Table 3 provides the evaluation schedule, except for the following administration of the study drug: G9.2-17 IgG4 treatment may be administered on days C1D1 and C1D15 of each cycle. In Part 2, tislelizumab may be administered on day 1 of each cycle of the G9.2-17 IgG4 combination regimen. The study drug may be administered on days 1, 8, and 15+/- 3 starting from C2. All patients treated with G9.2-17 IgG4 + tislelizumab must return 90 days +/- 7 days after their last dose of the study drug for evaluation of potential immune-mediated adverse events (IMAR).

research group

Patients who meet the following inclusion criteria and do not meet any exclusion criteria are eligible for the study.

Inclusion criteria

Part I

1. Written informed consent form (the patient is of sound mind, able to understand and willing to sign the informed consent form)

2. Males aged 18 years or older or non-pregnant women

3. Based on the researcher's judgment, able to adhere to the research protocol.

4. Histologically confirmed unresectable locally advanced or metastatic cancer. There were no restrictions on the prior lines of therapy received by the cancer patients included in this study.

a. For the Part 1 combined urethral epithelial carcinoma group: Histologically or cytologically confirmed unresectable locally advanced or metastatic urethral epithelial carcinoma (i.e., transitional cell carcinoma) of the renal pelvis, ureter, bladder or urethra.

b. For the Part 1 combined head and neck cancer group: histologically confirmed locally advanced or metastatic SCCHN (oral cavity, oropharynx, hypopharynx, or larynx).

5. For the combined cancer group of urothelial carcinoma and head and neck cancer, prior exposure to immunotherapy is permitted, with standard care options and/or within a clinical trial context. If patients receive an anti-PD-1 and/or anti-PD-L1-containing regimen at any point in time, they must demonstrate at least stable disease according to RECIST 1.1 or iRECIST criteria for one of these regimens (if these measurements are available). If RECIST or iRECIST measurements are not available, at least 4 months of clinical PFS must be achieved on either a prior anti-PD-1 and/or anti-PD-L1-containing regimen.

6. For the combined urethral epithelium and head and neck group in Part 1, PD-L1 expression is not required; however, fresh biopsy or archived tissue is required for PD-L1 assessment via IHC, or historical PD-L1 expression (via IHC) must be available. If PD-L1 expression data are already available, this does not overturn the protocol preference for obtaining fresh biopsy, provided it is feasible.

7. For patients with head and neck cancer of oropharyngeal origin in the Part 1 combination group: Human papillomavirus (HPV) status needs to be established at any point during the screening period or when the patient is taking the study drug, unless historically known. p16+, HPV RNA ISH, or DNA PCR are all acceptable alternatives to HPV+. This study accepts both HPV+ and HPV- patients.

8. According to the researchers' judgment, life expectancy is >3 months.

9. ECOG stamina status 0-1.

10. The patient is able and willing to undergo pre-treatment and during/post-treatment biopsies. The planned biopsy should not, in the investigator's judgment, significantly increase the patient's risk of complications. Every effort will be made to biopsi the same lesion in repeat biopsies. If the patient is eligible according to all other criteria but refuses to consent to a biopsy or has other medical reasons for excluding a biopsy, this will be discussed with the sponsor.

11. Measurable disease according to RECIST v1.1. Note that the lesion to be biopsied should not be a target lesion.

12. Adequate hematological and end-organ function, defined by the following laboratory results obtained prior to the first dose of the study drug, provided that no anticancer treatment was administered during the last 7 days:

a. Neutrophil count ≥1 x ^10⁹ /L

b. Platelet count ≥100 x ^10⁹ /L; Hepatocellular carcinoma (HCC) of Part 1 ≥50 x ^10⁹ /L

c. Hemoglobin ≥9.0 g/dL if no infusion was administered in the previous week.

d. Creatinine ≤ 1.5x ULN; or eGFR > 50 mg/mmol

e. Aspartate aminotransferase (AST) ≤ 3x ULN (≤ 5x ULN when HCC or liver metastasis is present)

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

g. Bilirubin ≤1.5xULN (bilirubin ≤3.0xULN in patients with known Gilbert's disease)

h. Albumin ≥3.0 g/dL

i. International Normalized Ratio (INR) and Partial Thromboplastin Time (PTT) ≤ 1.5 x ULN, unless the patient is receiving anticoagulation therapy.

13. There is no evidence of active, serious infection or infection requiring parenteral antibiotics.

14. Women of childbearing potential must have a negative pregnancy test within 72 hours prior to the start of treatment. For women of childbearing potential: consent to abstinence (avoidance of heterosexual intercourse) or use a contraceptive method with an annual failure rate of <1% during treatment and for at least 180 days after the last study treatment.

A woman is fertile if she is postmenopausal (≥12 consecutive months of amenorrhea with no definite cause other than menopause) and has not undergone surgical sterilization (removal of the ovaries and/or uterus).

Examples of contraceptive methods with an annual failure rate of <1% include bilateral tubal ligation, male sterilization, ovulation-suppressing hormonal contraceptives, hormone-releasing intrauterine devices (IUDs), and copper IUDs. The reliability of sexual abstinence should be evaluated based on the duration of the clinical trial, as well as the patient's preferences and habitual lifestyle. Regular abstinence (e.g., calendar, ovulation-based, symptom-thermal, or post-ovulation methods) and withdrawal are unacceptable contraceptive methods. Men of fertility must use effective contraception during the study period unless they have a history of infertility.

15. Four (4) weeks or five half-lives (whichever is shorter) from the last anticancer therapy prior to the first administration of G9.2-17 (IgG4).

16. Bisphosphonate therapy (e.g., zoledronic acid) or denosumab is permitted if it was previously used before the start of a clinical trial.

17. Patient:

a. Patients who have received at least one prior line of systemic therapy for metastatic or locally advanced disease, and/or

b. Having a type of tumor for which there are no standard care options available.

18. Patients who have not previously received a regimen containing gemcitabine.

Exclusion criteria

1. Patients who are unwilling or unable to comply with the protocol requirements

2. Patients diagnosed with metastatic cancer of unknown primary origin.

3. Current addiction to illicit drugs

4. Any patient with clinically significant, active, uncontrolled bleeding, and any patient with a bleeding predisposition (e.g., active peptic ulcer disease). Preventive or therapeutic use of anticoagulants is permitted.

5. Pregnant and/or breastfeeding women

6. If a patient receives any other investigational drug or participates in any other clinical trial involving the treatment of solid tumors with another investigational drug within 3 weeks or 5 half-lives (whichever is shorter) prior to the first dose of the investigational drug, or undergoes major surgery or planned surgery (including dental surgery) within 4 weeks prior to the first dose of the investigational drug.

7. Receive radiotherapy within 4 weeks of the first dose of the study drug, except for limited-field palliative radiotherapy, such as for the treatment of bone pain or focal pain tumor masses, and without compromising the measurable lesions required for response assessment (RECIST v1.1).

8. Patients with fungal tumor masses

9. May obscure test results and interfere with patient experience.

Any history or current evidence of any disease, treatment, active infection, or laboratory abnormality that the investigator believes is not in the best interest of the patient during the entire trial period.

10. Grade 4 immune-mediated toxicity resulting from prior checkpoint inhibitors. 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, alopecia, and vitiligo resulting from prior treatment.

11. History of other prior or concomitant malignancies requiring further aggressive treatment.

12. Patients with active brain, carcinomatous meningitis, or leptomeningeal metastases. Patients with brain metastases are eligible if they show clinically and radiographically stable disease for at least 4 weeks after definitive therapy and have not used steroids (>10 mg/day of prednisone or equivalent) for at least 4 weeks prior to the first dose of the study drug.

13. Severe or uncontrolled systemic disease, congestive heart failure > New York Heart Association (NYHA) Class 2, myocardial infarction (MI) within the past 6 months, or evidence of laboratory results that the investigators believe do not wish the patient to participate in the trial.

14. Any medical condition that the investigator believes seriously impairs patient safety or undermines the interpretation of the LYT-200 toxicity assessment.

15. Severe, unhealed wounds, active ulcers, or untreated fractures, unless, for example, a rib fracture (which does not warrant treatment).

16. Uncontrolled pleural effusion, pericardial effusion, or ascites requiring repeated drainage procedures. For the purposes of this study, “recurrence” is defined as ³⁺ 3 drainage within the last 30 days.

17. History of severe allergic, anaphylactic, or other hypersensitivity reactions to chimeric or humanized antibodies or fusion proteins.

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

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

20. Active autoimmune diseases (excluding type I/II diabetes, hypothyroidism requiring hormone replacement only, vitiligo, psoriasis or alopecia areata).

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 immunosuppressive drugs (e.g., ≤10 mg/day of prednisone or its equivalent) are eligible for enrollment. Replacement therapies (e.g., thyroxine, insulin, physiological corticosteroid replacement therapy [e.g., ≤10 mg/day of prednisone equivalent for adrenal or pituitary insufficiency]) are not considered a form of systemic therapy. Inhaled corticosteroids and mineralocorticoids (e.g., fludrocortisone), topical steroids, intranasal steroids, intra-articular steroids, and ophthalmic steroids are permitted.

22. Severe tumor-related pain (Grade 3, CTCAE) v.5.0 unresponsive to extensive analgesic interventions (oral and/or patch).

23. Despite the use of bisphosphonates, hypercalcemia persists (defined as grade ³ according to CTCAE v 5.0).

24. Any other disease, metabolic disorder, physical examination finding, or clinical laboratory finding that reasonably suggests a disease or condition is a contraindication to the use of the investigational drug, or may affect the interpretation of results or expose the patient to a high risk of treatment complications.

25. Receiving an organ transplant

26. Patients undergoing dialysis

27. For Part 1, patients with metastatic castration-resistant prostate cancer are permitted to continue hormone androgen deprivation therapy.

28. Any ablation therapy (radiofrequency ablation or percutaneous ethanol injection) of HCC within 6 weeks prior to trial entry.

29. Hepatic encephalopathy or severe hepatic adenoma

30. Child-Pugh score ≥ 7

Research drugs and other interventions

A research intervention is defined as any investigational agent, marketed product, placebo, or medical device intended to be administered to/used by a research participant in accordance with the research protocol.

Drugs administered in combination with G9.2-17 IgG4

Tislelizumab

Tislelizumab is a mAb drug that inhibits PD-1 and is being developed for the treatment of cancer. Tislelizumab is formulated in disposable glass vials (20R glass, USPI type) with rubber stoppers for intravenous injection, containing a total of 100 mg of tislelizumab mAb in 10 mL of buffered isotonic solution. Tislelizumab is administered as an intravenous infusion of 300 mg every 4 weeks over approximately 30 minutes during a 28-day cycle (unless otherwise instructed).

The active ingredient of tislelizumab is a humanized IgG4 variant mAb targeting PD-1, binding to the ECD of human PD-1 with high specificity and affinity (KD = 0.15 nM). The excipients for tislelizumab include: sodium citrate dihydrate, citrate monohydrate, L-histidine hydrochloride monohydrate, L-histidine, trehalose dihydrate, polysorbate-20, and WFI. Tislelizumab competitively blocks the binding of PD-L1 and PD-L2, inhibits PD-1-mediated negative signaling, and enhances functional activity in T cells in cell-based in vitro assays. Furthermore, tislelizumab has demonstrated antitumor activity in several human cancer allogeneic xenograft models and human PD-1 transgenic mouse models.

In vitro assays showed that the IgG4 variant antibody exhibited very low binding affinity for FcγRIIIA and C1q, indicating low or no ADCC and CDC effects in humans. Unlike the native IgG4 antibody, tislelizumab showed no observable Fab arm exchange activity in vitro, predicting that the antibody is stable in vivo and unlikely to form bispecific antibodies.

In various advanced solid tumors, the exposure-response (E-R) relationship between tislelizumab exposure and efficacy supports the 300 mg Q4W regimen. The 300 mg Q4W regimen is not expected to have clinically significant differences in safety or efficacy outcomes compared to the 200 mg Q3W regimen.

Tislelizumab's safety profile is consistent with that of drugs with a relatively low incidence of treatment-related grade 3 or higher toxicities.

Tislelizumab adverse events (AEs) are listed in Table 4 below according to their frequency. Reported AEs potentially related to IMAR are summarized in Table 5.

Table 4. Adverse events of tislelizumab reported by frequency (non-IMAR related)

By disrupting PD-1-mediated signaling, tislelizumab can restore anti-tumor immunity and halt tumor growth progression. This restoration of immune system activity may lead to immune-related adverse events involving one or more body systems, which in rare cases can be life-threatening or fatal. While these events typically become apparent during tislelizumab treatment, they can also occur after discontinuation of tislelizumab treatment.

Table 5. Reported adverse events of tislelizumab considered as IMAR*

For management of IMA caused by the combination of G9.2-17 (IgG4) and tislelizumab, please refer to Table 6 below.

Table 6: Management of Immune-Mediated Adverse Reactions (IMARs) Caused by G9.2-17 IgG4 + Tislelizumab Combination Therapy

For non-IMAR, hematologic, and non-hematologic adverse events occurring in the combined arm, assess causality as follows:

○ If it is related to G9.2-17 IgG4, then follow the AE management guidelines for G9.2-17 IgG4.

○ If it is related to combination drugs (tislelizumab), follow the management instructions in Table 7 for tislelizumab.

Dosage adjustment

The next dose level for G9.2-17 IgG4 in Part 1 can be determined based on safety, tolerability, and preliminary PK data obtained in at least two patients at the previous dose level.

The dosing schedule can also be adjusted based on the obtained PK data. Detailed dosage adjustment instructions are available as shown in Tables 7-9:

Table 7: Recommended dose adjustment of tislelizumab for adverse events (excluding IMAR)

Toxicity is classified according to NCI CTCAE V5.

Table 8. G9.2-17 Management of Immune-Mediated Adverse Reactions (IMARs) Caused by IgG4

Table 9: Recommended dose adjustment for G9.2-17 IgG4 (AEs outside the DLT window and excluding IMAR)

Dosage administration and dose delay

If an infusion-related reaction occurs, interrupt the infusion and administer the relevant medication (e.g., antihistamines, antiemetics, steroids, antipyretics, beta-blockers, etc.) if there is a clinical indication. If it is deemed appropriate to resume the infusion, do so at a slower infusion rate.

For subsequent cycles of the same patient, administer appropriate preoperative medications (as clinically indicated, such as antihistamines, antiemetics, steroids, antipyretics, beta-blockers, etc.) and consider using a slower infusion rate.

If any clinically significant adverse event (AE) of grade 3 or higher occurs that may be related to one or more investigational drugs, it will be discussed with the medical monitor before continuing dosing. For AEs of grade 3 or higher, a dose delay may be necessary.

dose reduction

For any patient being evaluated for DLT (within the 28-day DLT window), dose reduction is not permitted. If dose reduction is necessary, the study intervention will be administered as follows:

For Parts 1 and 2, patients with G9.2-17 IgG4 alone: Dose reduction may be permitted when assessment shows clinical benefit and can continue to be achieved under dose-reduction conditions, see Table 8 (for IMAR) or Table 9 (for other AEs).

For the Part 2 combination therapy group of G9.2-17 IgG4 or G9.2-17 IgG4 tislelizumab, experience from clinical trials of the approved drug (as summarized in the approved product label) will inform the management of adverse events, including guidance on delaying, reducing and/or completely discontinuing tislelizumab, see Tables 6-7 and 9.

Dosage adjustment of specific adverse events associated with tislelizumab administration

Recommendations for tislelizumab modulation based on specific AEs are provided in Table 6 (for IMAR) and Table 7 (for other AEs).

Dosage adjustment for IMAR

If an IMAR occurs, refer to Tables 8 and 6 for dosage management guidelines for G9.2-17 IgG4 and/or tislelizumab. Perform all relevant medical examinations/tests to confirm the adverse event as an IMAR.

The cessation of research intervention

In rare cases, it may be necessary for a patient to permanently discontinue the study intervention. If the study intervention is permanently discontinued for reasons other than disease progression, and the patient is not receiving other anticancer therapies, disease progression will continue to be assessed for up to 2 years. See the assessment timeline for information on data collected at the time of intervention discontinuation and follow-up, as well as any further evaluations required.

Researchers must make every effort to keep patients on study treatment until one of the reasons for termination of study treatment is met (disease progression, toxicity related to the study drug, or withdrawal of consent). If a patient has radiographic progression but no clear clinical progression and has not started alternative therapy, the patient may continue study treatment. However, if a patient has clear clinical progression but no radiographic progression, study treatment should be discontinued, and available treatment options should be recommended to the patient.

Patients may discontinue treatment before disease progression for one of the following reasons:

• DLT as defined in this article

• AEs requiring discontinuation of one or more investigational treatments that occur/relapse outside the DLT window

• IMAR occurrence/relapse requiring discontinuation of one or more investigational treatments

• Concurrent diseases or medical conditions that could hinder further treatment or endanger patient safety if the investigational treatment continues.

·Pregnant

• Use of non-protocol cancer therapy

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

• Patients exhibited significant deviations from the treatment plan (including lack of adherence).

Companion therapy

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

·Reasons for use

• Application date, including start and end dates

• Dosage information, including dosage and frequency

Approved drugs

The following concomitant medications are permitted:

• Any standard preoperative medications for patients receiving combination therapy regimens.

For bone metastases that have been stable for at least 6 months prior to treatment (C1D1), continue bisphosphonate therapy (e.g., zolamronic acid) or denosumab.

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

• Preventive or therapeutic uses of anticoagulants

• Vaccination for COVID-19, common influenza, and/or other common clinically required indications (such as tetanus, pneumococcal, HBV, etc.) is permitted before or during the study period. The timing and type of vaccination must be recorded.

Prohibited drugs

The following drugs are not permitted in this study:

• Administer other investigational agents concurrently for any indication, except for G9.2-17 IGG4.

• Systemic immunosuppressive therapy, including but not limited to cyclophosphamide, azathioprine, methotrexate, thalidomide, and anti-TNF agents. However, patients are permitted to take acute, low-dose systemic immunosuppressive drugs (e.g., ≤10 mg/day of prednisone or equivalent).

Replacement therapies (e.g., thyroid hormone, insulin, or physiological corticosteroid replacement therapy for adrenal or pituitary insufficiency [e.g., prednisone equivalents ≤10 mg/day]) are not considered forms of systemic treatment.

Efficacy evaluation

The evaluation timeline table provides the planned time points for all efficacy evaluations.

RECIST v1.1 Tumor Assessment Criteria

During tumor screening and evaluation, tumor lesions/lymph nodes are classified as measurable or non-measurable, with measurable lesions recorded based on the longest diameter in the measurement plane (except for pathological lymph nodes, which are measured on the shortest axis). When more than one measurable lesion is present at screening, all lesions representing a maximum of five lesions across all affected organs (and 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 can be calculated and reported as the baseline sum of diameters.

All other lesions (or disease sites), including pathological lymph nodes, should be identified as non-target lesions and documented during screening. Measurements are not required, and these lesions should be tracked as "present," "absent," or "clearly progressing."

According to the RECIST v1.1 guidelines (Eisenhauer et al., 2009), the following disease response measures are used to assess tumor target lesions.

• Complete response (CR): All target lesions disappear. The short axis of any pathological lymph node (whether target or non-target) must be reduced to <10 mm.

Partial response (PR): The total diameter of the target lesion is reduced by at least 30% relative to the total diameter of the baseline.

• Stable disease (SD): In the study, the minimum total diameter was used as a reference, with neither sufficient contraction to meet PR nor sufficient increase to meet PD.

• Progressive disease: The total diameter of the target lesions must increase by at least 20%, using the minimum total in the study as a reference (or including the baseline total if the minimum is in the study). In addition to a relative increase of 20%, the total must also demonstrate an absolute increase of at least 5 mm. (Note: The appearance of one or more new lesions is also considered progressive).

The following guidelines can be used to evaluate non-target lesions. See also Table 10 below.

• Complete response (CR): All non-target lesions disappear and tumor marker levels normalize. All lymph nodes must be of non-pathological size (<10 mm short axis).

• Non-CR/Non-PD: One or more non-target lesions persist and/or tumor marker levels remain above the normal range.

• Progressive disease (PD): Clear progression of existing non-target lesions. (Note: The appearance of one or more new lesions is also considered progression).

The disease response measures at different time points will allow for the calculation of the following:

• Disease control rate (DCR) is defined as the percentage of patients who achieve CR, PR, and SD.

• Objective response rate (ORR) is defined as the proportion of patients whose tumor size shrinks to the predetermined amount (tumor shrinkage ≥30%).

• Progression-free survival (PFS) is defined as the time from the start of investigational drug treatment to disease progression (tumor growth ≥30%).

• Duration of response (DoR) is defined as the length of time a tumor continues to respond to treatment without cancer growth or spread.

• Overall survival (OS) is defined as the time from the start of investigational drug treatment to death from any cause.

Table 10. Evaluation of total time-point responses in patients with measurable disease at baseline

Target lesions Non-target lesions New lesions Overall Response CR CR no CR CR Non-CR/Non-PD no PR CR NE no PR PR Non-PD or NE no PR SD Non-PD or NE no SD Not all reviews Non-PD no NE Progressive diseases any Yes or No Progressive diseases any Progressive diseases Yes or No Progressive diseases any any yes Progressive diseases

CR: Complete response; Non-PD: Non-progressive disease; PR: Partial response; SD: Stable disease; NE: Not evaluable.

*When target lesions show SD/PR and certain subsets of non-target lesions are not evaluable, a careful decision must be made as to whether the overall response at this time point should be referred to as SD/PR or NE. This is based on whether the non-evaluable lesions (if they show growth) will contribute to an overall response to progressive disease in the context of responses to other observed lesions. If non-evaluable non-target lesions represent a large proportion of the overall disease burden, then the appropriate time point response is NE.

Adverse event management

AEs may not have been recorded prior to the administration of the first dose of the study drug. AEs that begin after administration of the study drug or medically relevant symptoms that worsen after administration of the study drug will be recorded. AEs should be followed up until they resolve, return to baseline, or are identified as stable or chronic. All SAEs should be collected up to 30 days after the last dose of the study drug.

Immune-mediated adverse reactions

Identify immune-mediated adverse reactions (IMARs) of tislelizumab.

The specific IMAR mentioned is:

Immune-mediated hepatitis

Immune-mediated nephritis

Immune-mediated pneumonia

Immune-mediated pneumonia

• Immune-mediated colitis and diarrhea; immune-mediated endocrine disorders

• Immune-mediated skin response

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

The monitoring program aims to limit the severity and duration of IMAs occurring during the development of the combined drug and includes: physical examination, vital signs, and safety laboratory assessments (including hematology and biochemistry) at scheduled visits; assessment of endocrine function on Day 1 of each new dosing cycle (before dosing); assessment of coagulation status; and urinalysis. The assessment schedule (see Example 1) also covers assessment of ejection fraction every three months and regular ECGs.

All relevant medical examinations/tests will be conducted to confirm that the adverse event is an IMAR.

Instructions for the management of these IMAs are included in Table 8 App4 (for G9.2-17 IgG4 alone) and Table 6 (for combination therapy of G9.2-17 IgG4 + tislelizumab).

Equivalent solution

From the above description, those skilled in the art can readily identify the basic 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.

While several inventive embodiments have been described and illustrated herein, those skilled in the art will readily conceive of a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more advantages described herein, and each such variation and/or modification is considered to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and constructions described herein are meant to be exemplary, and actual parameters, dimensions, materials, and/or constructions depend on one or more specific applications using the teachings of this invention. Those skilled in the art recognize or are able to determine many equivalents of the specific inventive embodiments described herein using only conventional experimentation. Therefore, it should be understood that the foregoing embodiments are presented by way of example only, and that 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 is included within the scope of this disclosure if such features, systems, articles, materials, kits, and/or methods do not contradict each other.

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.

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

Unless otherwise expressly indicated to the contrary, the indefinite articles “a” and “an” as used herein in the specification and claims shall be understood to mean “at least one”.

As used herein in the specification and claims, the phrase “and/or” should be understood to mean “any one or both” of the connected elements, i.e., elements that are combined in some cases and separate in others. Multiple elements listed with “and/or” should be interpreted in the same way, i.e., “one or more” elements so combined. In addition to the elements specifically identified by the “and/or” clause, other elements may optionally be present, whether related to or unrelated to those specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended terms such as “comprising/including,” may in one embodiment refer only to A (optionally including elements other than B); in another embodiment refer only to B (optionally including elements other than A); in yet another embodiment refer to A and B (optionally including other elements); and so on.

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, that is, including at least one, but also including more than one, a plurality, or a series of elements, as well as optional additional unlisted items. Only when the opposite term is explicitly specified, such as “only one of…” or “exact one of…” or, when used in a claim, “consisting of…” will refer to including multiple elements or exactly one element from a list of elements. In general, when the term “or” is preceded by an exclusive term (such as “any one,” “one of…,” “only one of…,” or “exact one of…”), it should only be interpreted as an alternative to exclusivity (i.e., “one or the other, but not two”). “Substantially consisting of…”, when used in a claim, should have its ordinary meaning as used in the field of patent law.

As used herein in the specification and claims, the phrase “at least one” relating 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 does not necessarily include every and 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 related to or unrelated to those specifically identified elements. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B”, or equivalently, “at least one of A and/or B”) may in one embodiment mean at least one (optionally including more than one) A without the presence of B (and optionally including elements other than B); in another embodiment mean at least one (optionally including more than one) B without the presence of A (and optionally including elements other than A); in yet another embodiment mean 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.

It should also be understood that, unless expressly indicated to the contrary, the order of steps or actions in any method claimed herein that includes more than one step or action is not necessarily limited to the order in which the steps or actions of the method are described.

Claims (30)

1. A method for treating a solid tumor, the method comprising administering to a subject in need of treatment... (a) An effective amount of antibody binding to human galactoglobulin-9 (anti-galactoglobulin-9 antibody), and (b) An effective amount of tislelizumab The anti-galactoglobulin-9 antibody comprises: (i) a light chain variable region (V _L ), comprising a light chain complementarity determination region 1 (CDR1) as shown in SEQ ID NO:1, a light chain complementarity determination region 2 (CDR2) as shown in SEQ ID NO:2, and a light chain complementarity determination region 3 (CDR3) as shown in SEQ ID NO:3, and (ii) a heavy chain variable region comprising heavy chain complementarity determination region 1 (CDR1) as shown in SEQ ID NO:4, heavy chain complementarity determination region 2 (CDR2) as shown in SEQ ID NO:5, and heavy chain complementarity determination region 3 (CDR3) as shown in SEQ ID NO:6, and The anti-galactoglobulin-9 antibody was administered to the subject at a dose of about 0.2 mg/kg to about 18 mg/kg. 2. The method of claim 1, wherein the solid tumor is head and neck cancer, urothelial carcinoma, gastric and esophageal cancer, or non-small cell lung cancer. 3. The method of claim 1 or claim 2, wherein the solid tumor is a metastatic tumor. 4. The method of any one of claims 1-3, wherein the anti-galactoglobulin-9 antibody is administered to the subject at a dose of about 4 mg/kg to about 18 mg/kg. 5. The method of claim 4, wherein the anti-galactoglobulin-9 antibody is administered to the subject at a dose of about 4 mg/kg, about 6.3 mg/kg, about 10 mg/kg, about 12 mg/kg, about 14 mg/kg, about 16 mg/kg, or about 18 mg/kg, optionally wherein the dose of the anti-galactoglobulin-9 antibody is about 6.3 mg/kg, about 10 mg/kg, or about 16 mg/kg. 6. The method of any one of claims 1-5, wherein the anti-galactoglobulin-9 antibody is administered to the subject once weekly. 7. The method of claim 6, wherein the anti-galactoglobulin-9 antibody is administered to the subject at a dose of about 6.3 mg/kg, about 10 mg/kg once weekly, or about 16 mg/kg. 8. The method of any one of claims 1-7, wherein the anti-galactoglobulin-9 antibody is administered to the subject via intravenous infusion. 9. The method of any one of claims 1-8, wherein the tislelizumab is administered to the subject at a dose of about 200 mg every 3 weeks, at a dose of about 300 mg every 4 weeks, or at a dose of about 400 mg every 6 weeks. 10. The method of claim 9, wherein the tislelizumab is administered to the subject at a dose of about 300 mg every 4 weeks. 11. The method of claim 9 or claim 10, wherein the tislelizumab is administered to the subject via intravenous infusion. 12. The method of any one of claims 1-4, wherein the anti-galactoglobulin-9 antibody is administered to the subject by intravenous infusion at a dose of about 6.3 mg/kg once weekly, and the tislelizumab is administered to the subject by intravenous infusion at a dose of about 300 mg once every 4 weeks. 13. The method of any one of claims 1-4, wherein the anti-galactoglobulin-9 antibody is administered to the subject by intravenous infusion at a dose of about 10 mg/kg once weekly, and the tislelizumab is administered to the subject by intravenous infusion at a dose of about 300 mg once every 4 weeks. 14. The method of any one of claims 1-4, wherein the anti-galactoglobulin-9 antibody is administered to the subject by intravenous infusion at a dose of about 16 mg/kg once weekly, and the tislelizumab is administered to the subject by intravenous infusion at a dose of about 300 mg once every 4 weeks. 15. The method of any one of claims 1-14, wherein the tislelizumab is administered to the subject on the same day the subject receives the anti-galactoglobulin 9 antibody, or wherein the administration of the tislelizumab and the administration of the anti-galactoglobulin 9 antibody are performed on two consecutive days. 16. The method of any one of claims 1-14, wherein the administration of tislelizumab is performed prior to the administration of the anti-galactoglobulin 9 antibody. 17. The method of any one of claims 1-16, wherein the subject is a human patient suffering from the solid tumor. 18. The method of any one of claims 1-17, wherein the V _L of the anti-galactoglobulin-9 antibody comprises the amino acid sequence of SEQ ID NO:8, and wherein the V _H of the anti-galactoglobulin-9 antibody comprises the amino acid sequence of SEQ ID NO:7. 19. The method of claim 18, wherein the anti-galactoglobulin-9 antibody is an IgG1 or IgG4 molecule. 20. The method of claim 19, wherein the anti-galactoglobulin-9 antibody is an IgG4 molecule having a modified Fc region of human IgG4. 21. The method of claim 20, wherein the modified Fc region of the human IgG4 comprises the amino acid sequence of SEQ ID NO:14. 22. The method of any one of claims 1-17, wherein the anti-galactoglobulin-9 antibody comprises 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. 23. The method of any one of claims 1-22, wherein the subject has undergone one or more prior anticancer therapies. 24. The method of claim 23, wherein the one or more prior anticancer therapies comprise chemotherapy, immunotherapy, radiotherapy, therapies involving biological agents, or combinations thereof. 25. The method of claim 23 or claim 24, wherein the subject's disease progresses or becomes resistant to the one or more prior anticancer therapies. 26. The method of any one of claims 1-25, wherein the subject is a human patient with elevated galactoglucan-9 levels relative to a control. 27. The method of claim 26, wherein the human patient has an elevated serum or plasma galactolectin-9 level relative to the control value. 28. The method of any one of claims 1-27, wherein the human patient has cancer cells expressing galectin-9, immune cells expressing galectin-9, or both. 29. The method of any one of claims 1-28, further comprising monitoring the occurrence of side effects in the subject. 30. The method of claim 29, further comprising reducing the dose of the galactolectin-9 antibody, the dose of the tislelizumab, or both, in the event of an adverse reaction.