Treatment of hematological malignancies with antibodies that inhibit galectin-9

Abstract

Disclosed herein are methods of treating hematological malignancies (e.g., acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or acute lymphoblastic leukemia (ALL)) using an antigalectin-9 antibody (e.g., G9.2-17 (IgG4)). In some embodiments, the anti-galectin-9 antibody is used as a monotherapy.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of the filing dates of U.S. Provisional Application No. 63 / 356,840 filed on June 9, 2022, U.S. Provisional Application No. 63 / 394,506 filed on August 2, 2022, U.S. Provisional Application No. 3 / 394,507 filed on August 2, 2022, and U.S. Provisional Application No. 63 / 425,986 filed on November 16, 2022, the entire contents of each of which are incorporated herein by reference.

[0003] References to sequence listings

[0004] This application contains a sequence listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy created on June 21, 2023 is named 112174-0259-NP013WO00 and is 40,880 bytes in size. Background Art

[0005] Galectin-9 is a tandem repeat lectin consisting of two carbohydrate recognition domains (CRDs) and was first discovered and described in 1997 in patients with Hodgkin lymphoma (HL) (Tureci et al., J. Biol. Chem. 1997, 272, 6416–6422). There are three isoforms, and they can be located intracellularly or extracellularly. Elevated levels of galectin-9 have been observed in a variety of cancers, including melanoma, Hodgkin lymphoma, hepatocellular carcinoma, pancreatic cancer, gastric cancer, colon cancer, and clear cell renal cell carcinoma (Wdowiak et al., Int. J. Mol. Sci. 2018, 19, 210). In renal cancer, patients with high galectin-9 expression show more advanced disease progression and larger tumor size (Kawashima et al.; BJU Int. 2014; 113: 320–332). In melanoma, galectin-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). Many studies have shown the utility of galectin-9 as a prognostic marker and, more recently, as a potential new drug target (Enninga et al., 2016; Kawashima et al., BJU Int 2014;113:320–332; Kageshita et al., Int J Cancer. 2002 Jun 20;99(6):809-16, and references therein).

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

[0007] Galectin-9 has also been found to play a role in polarizing T cell differentiation toward a tumor suppressor phenotype and promoting tolerogenic macrophage programming and adaptive immunosuppression (Daley et al., Nat Med., 2017, 23, 556-567). In a mouse model of pancreatic ductal adenocarcinoma (PDAC), it has been shown that blocking the checkpoint interaction between galectin-9 and the receptor Dectin-1 found on innate immune cells in the tumor microenvironment (TME) can increase anti-tumor immune responses in the TME and slow tumor progression (Daley et al., Nat Med., 2017, 23, 556-567). Galectin-9 was also found to bind to CD206, a surface marker of M2 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

[0008] The present disclosure is based, at least in part, on the development of treatment regimens for hematological malignancies, such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or acute lymphoblastic leukemia (ALL).

[0009] Therefore, a method for treating a hematological malignancy is provided herein, comprising administering to a subject in need thereof a pharmaceutical composition comprising an antibody that binds to human galectin-9 (anti-Gal9 antibody), wherein the anti-Gal9 antibody is administered to the subject at a dose of about 2 mg / kg to about 32 mg / kg. In some embodiments, the anti-Gal9 antibody is administered to the subject once a week to once every 6 weeks (e.g., once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks). For example, the anti-Gal9 antibody can be administered to the subject at 2 mg / kg, 4 mg / kg, 6.3 mg / kg, 7.5 mg / kg, 10 mg / kg, 12 mg / kg, 16 mg / kg or 32 mg / kg once a week to once every six weeks (e.g., once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks). In some embodiments, the anti-Gal9 antibody is administered once a week. In some embodiments, the anti-Gal9 antibody is administered once every two weeks. In some embodiments, any of the anti-Galectin-9 antibodies disclosed herein can be administered to a subject by intravenous infusion.

[0010] In some embodiments, an anti-Gal9 antibody may comprise: (a) a light chain comprising a light chain variable region (V L ), which comprises a light chain (LC) complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1, a LC complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 2, and a LC complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3; and (b) a heavy chain comprising a heavy chain variable region (V H ), which comprises a heavy chain (HC) complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 4, a HC complementary determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 5, and a HC complementary determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 6. In some examples, the anti-Gal9 antibody comprises V L , which comprises the amino acid sequence of SEQ ID NO: 8; and V H , which comprises the amino acid sequence of SEQ ID NO:7.

[0011] Any anti-Gal9 antibody used in the methods disclosed herein can be an IgG1 molecule. Alternatively, the antibody can be an IgG4 molecule. In some embodiments, the anti-Gal9 antibody can be an IgG4 molecule (e.g., a human IgG4 molecule) comprising a modified Fc region relative to a wild-type IgG4 counterpart (e.g., a wild-type human IgG4 counterpart). In some instances, the modified Fc region comprises the amino acid sequence of SEQ ID NO: 14. In one example, the anti-Gal9 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and a light chain comprising the amino acid sequence of SEQ ID NO: 15. Such an anti-Gal9 antibody can be G9.2-17 (IgG4).

[0012] Any anti-Gal9 antibody disclosed herein (e.g., G9.2-17 (IgG4)) can be administered to a subject at a dose of 2 mg / kg, 4 mg / kg, 6.3 mg / kg, 7.5 mg / kg, 10 mg / kg, 12 mg / kg, 16 mg / kg, or 32 mg / kg. In some embodiments, the anti-Gal9 antibody is administered to a subject once a week to once every 6 weeks (e.g., once a week, once every two weeks, once every three weeks, once every 4 weeks, once every five weeks, once every six weeks). For example, the anti-Gal9 antibody can be administered to a subject at 2 mg / kg, 4 mg / kg, 6.3 mg / kg, 7.5 mg / kg, 10 mg / kg, 12 mg / kg, 16 mg / kg, or 32 mg / kg once a week to once every six weeks (e.g., once a week, once every two weeks, once every three weeks, once every 4 weeks, once every five weeks, once every six weeks). In some embodiments, the anti-Gal9 antibody is administered once a week. In some embodiments, the anti-Gal9 antibody is administered once every two weeks to once every four weeks. In some examples, the anti-galectin-9 antibody can be administered to the subject once every two weeks. In a specific embodiment, the anti-galectin-9 antibody (e.g., G9.2-17 (IgG4)) is administered to the subject once a week at a dose of 6.3 mg / kg, 7.5 mg / kg, 10 mg / kg, 12 mg / kg, or 16 mg / kg. In a specific embodiment, the anti-galectin-9 antibody (e.g., G9.2-17 (IgG4)) is administered to the subject once every two weeks to once every four weeks (e.g., once every two weeks) at a dose of 6.3 mg / kg, 7.5 mg / kg, 10 mg / kg, 12 mg / kg, or 16 mg / kg.

[0013] Alternatively, an anti-Gal-9 antibody (such as G9.2-17 (IgG4)) can be administered to a subject at a dose of about 410 mg to about 2056 mg (e.g., about 410 mg to about 650 mg, about 410 mg to about 1040 mg, or about 410 mg to about 1280 mg) once every 2-6 weeks (e.g., every 2-4 weeks), for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, or once every 6 weeks. In some examples, an anti-Gal-9 antibody is administered to a subject at a dose of about 410 mg to about 505 mg (e.g., about 410 mg to about 450 mg or about 410 mg to about 480 mg) once every 2-6 weeks (e.g., every 2 weeks), for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, or once every 6 weeks. In other examples, an anti-Gal-9 antibody is administered to a subject at a dose of about 650 mg to about 800 mg. mg (e.g., about 650 mg to about 700 mg) is administered to a subject once every 2-6 weeks (e.g., every 2-4 weeks), such as once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, or once every 6 weeks. In other examples, the anti-Gal-9 antibody is administered to a subject at a dose of about 1040 mg to about 1280 mg (e.g., about 1040 mg to about 1120 mg) every 2-6 weeks (e.g., every 2-4 weeks), such as once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, or once every 6 weeks. In yet other examples, the anti-Gal-9 antibody is administered to a subject at a dose of about 2080 mg to about 2560 mg (e.g., about 2080 mg to about 2400 mg) every 2-6 weeks (e.g., every 2 weeks), such as once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, or once every 6 weeks.

[0014] In other embodiments, any anti-Gal9 antibody disclosed herein (e.g., G9.2-17 (IgG4)) can be administered to a subject once a week at a dose of 2 mg / kg, 4 mg / kg, 6.3 mg / kg, 7.5 mg / kg, 10 mg / kg, 12 mg / kg, 16 mg / kg, or 32 mg / kg. In specific embodiments, an anti-galectin-9 antibody (e.g., G9.2-17 (IgG4)) is administered to a subject once a week at a dose of 4 mg / kg, 6.3 mg / kg, 7.5 mg / kg, 10 mg / kg, 12 mg / kg, or 16 mg / kg.

[0015] Alternatively, an anti-Gal-9 antibody (such as G9.2-17 (IgG4)) can be administered to a subject once a week at a dose of about 410 mg to about 2560 mg (e.g., about 410 mg to about 1040 mg or about 410 mg to about 650 mg). In some examples, the anti-Gal-9 antibody is administered to a subject once a week at a dose of about 410 mg to about 505 mg (e.g., about 410 mg to about 450 mg). In other examples, the anti-Gal-9 antibody is administered to a subject once a week at a dose of about 650 mg to about 800 mg (e.g., about 650 mg to about 700 mg). In yet other examples, the anti-Gal-9 antibody is administered to a subject once a week at a dose of about 1040 mg to about 1280 mg (e.g., about 1040 mg to about 1120 mg). In yet other examples, the anti-Gal-9 antibody is administered to a subject once a week at a dose of about 2080 mg to about 2560 mg (eg, about 2080 mg to about 2400 mg).

[0016] In one example, an anti-Gal9 antibody disclosed herein (e.g., G9.2-17 (IgG4)) can be administered to a subject once a week at a dose of 2 mg / kg. In another example, an anti-Gal9 antibody disclosed herein (e.g., G9.2-17 (IgG4)) can be administered to a subject once a week at a dose of 4 mg / kg. In still another example, an anti-Gal9 antibody disclosed herein (e.g., G9.2-17 (IgG4)) can be administered to a subject once a week at a dose of 7.5 mg / kg. In yet another example, an anti-Gal9 antibody disclosed herein (e.g., G9.2-17 (IgG4)) can be administered to a subject once a week at a dose of 10.0 mg / kg. In yet another example, an anti-Gal9 antibody disclosed herein (e.g., G9.2-17 (IgG4)) can be administered to a subject once a week at a dose of 12.0 mg / kg. In another example, an anti-Gal9 antibody disclosed herein (eg, G9.2-17 (IgG4)) can be administered to a subject once a week at a dose of 16.0 mg / kg.

[0017] In some embodiments, the subject treated by any method disclosed herein is a human patient suffering from leukemia or lymphoma. In a specific example, the human patient suffers from acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) or acute lymphoblastic leukemia (ALL, such as T cell ALL or B cell ALL). Alternatively or in addition, hematological malignancies are refractory and / or recurrent. In some cases, the human patient suffers from recurrent and / or refractory AML after at least one previous treatment line. In other cases, the human patient suffers from recurrent and / or refractory MDS after at least one previous treatment line. Alternatively or in addition, the human patient does not suffer from acute promyelocytic leukemia (APL).

[0018] In some embodiments, the human patient has undergone or is undergoing chemotherapy for the treatment of a hematological malignancy. In other embodiments, the human patient has not undergone or is not undergoing chemotherapy for the treatment of a hematological malignancy.

[0019] Any of the methods disclosed herein may be effective in reducing or eliminating Gal9+ blood cancer cells, such as leukemia or lymphoma cells.Alternatively or additionally, the methods disclosed herein may be effective in modulating an immune response targeting cancer cells.

[0020] In some cases, any of the methods disclosed herein includes at least one treatment cycle consisting of 28 days, during which the anti-Gal9 antibody is administered to the subject once a week. For example, treatment may include one cycle, two cycles, three cycles, four cycles, five cycles, six cycles or more cycles.

[0021] In some cases, the methods disclosed herein comprise an anti-Gal9 antibody as the only active agent (monotherapy) for treating a hematological malignancy.

[0022] Any method disclosed herein may also include monitoring the occurrence of adverse events during treatment. In some cases, when adverse events occur, the dose of the anti-Gal9 antibody may be reduced.

[0023] Also provided herein are anti-Gal9 antibodies (e.g., G9.2-17 (IgG4)) for use in treating hematological cancers (e.g., those disclosed herein) in any of the methods disclosed herein and the use of such anti-Gal9 antibodies in the manufacture of a medicament for use in the therapeutic methods disclosed herein.

[0024] The details of one or more embodiments of the 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 and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a diagram depicting a schematic diagram of a study using the anti-Gal9 antibody G9.2-17 (IgG4) to treat hematological malignancies such as AML and high-risk MDS. RP2D = recommended phase 2 dose. PK = pharmacokinetics. PD = pharmacodynamics.

[0026] Figure 2 is a diagram depicting the timeline of clinical trial evaluations. A maximum of 40 patients will be enrolled in Phase 1. Each patient will receive clinical visits for pre-Cycle 1 dose 1 administration (preC1) as well as C1D1 administration (Cycle 1 day 1, etc.) and QW thereafter. Please note that whenever possible a bone marrow aspirate / biopsy will be performed simultaneously, MRD will be assessed from both the bone marrow and blood. Bone marrow analysis will be performed every 3 months after 4 months, provided that the patient remains on study drug. Blood MRD assessments will continue monthly.

[0027] Figure 3 It is a diagram showing markers used for examination. DETAILED DESCRIPTION

[0028] Galectin-9 (a tandem repeat lectin) is a β-galactoside binding protein that has been shown to play a role in regulating cell-cell and cell-matrix interactions. It has been found to be strongly overexpressed in Hodgkin's disease tissue and other pathological states. It can also be found circulating in the tumor microenvironment (TME).

[0029] It was found that galectin-9 can interact with TIM-3 (a type I cell surface glycoprotein expressed on the surface of leukemic stem cells in all types of acute myeloid leukemia (except M3 (acute promyelocytic leukemia)), but not in normal human hematopoietic stem cells (HSC). TIM-3 signaling generated by galectin-9 ligation 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-a (TNF-a), causing monocytes to mature into dendritic cells, leading to inflammation caused by innate immunity (Kuchroo et al., Nat Rev Immunol., 2008, 8: 577-580). It has been found that additional galectin-9 / TIM-3 signaling co-activates NF-KB and catenin signaling (two pathways that promote LSC self-renewal) (Kikushige et al., Cell Stem Cell, 2015, 17(3):341-352). Anti-galectin-9 antibodies that interfere with galectin-9 / TIM-3 binding may have therapeutic effects, particularly for leukemia and other hematological malignancies.

[0030] In addition, galectin-9 is a multi-effect immunomodulator that affects the innate and adaptive immunity of multiple cell types. Gal-9 promotes inflammation, induces M2 macrophages and monocyte-derived dendritic cells (mDC), promotes the differentiation of regulatory T cells, and inhibits NK cell killing ability. There are multiple transcriptional and functional changes induced by galectin-9 independent of Tim-3. It is also found that Gal9 expression is higher in AML patients who have failed chemotherapy. See, for example, OncoImmunology, 5 (7): 00-00 DOI: 10.1080 / 2162402X.2016.1195535. It was found that anti-Gal9 antibodies significantly inhibited the reconstruction of AML and LSC. Kikushige et al., Cell Stem Cell 17 (3): 341-352 (2015).

[0031] Anti-Gal9 antibodies disclosed herein (such as G9.2-17 (IgG4)) can directly kill Gal9+ blood cancer cells via inducing cytotoxicity. In addition, galectin-9 has been found to affect multiple immunosuppressive pathways. The immunological effects make anti-Gal9 antibodies (such as G9.2-17 (IgG4)) a potential single-agent therapeutic efficacy.

[0032] Thus, provided herein are methods of treating hematological malignancies (e.g., acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or acute lymphoblastic leukemia (ALL, such as T-cell ALL or B-cell ALL)) using anti-Galectin-9 (e.g., G9.2-17 (IgG4)) at a dose of 2 mg / kg to about 32 mg / kg (or about 130 mg to about 2,560 mg) once a week to once every 6 weeks (e.g., once a week to once every 4 weeks), e.g., once a week or once every 2 weeks.

[0033] I. Antibodies that bind to Galectin-9

[0034] The present disclosure provides anti-Galectin-9 antibodies (such as G9.2-17) and functional variants thereof for use in the therapeutic methods disclosed herein.

[0035] Antibodies (used interchangeably in the plural) are immunoglobulin molecules that are capable of specifically binding to targets (such as carbohydrates, polynucleotides, lipids, polypeptides, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" (e.g., anti-Galectin-9 antibody) encompasses not only complete (e.g., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chains (scFv), mutants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configurations of immunoglobulin molecules comprising antigen recognition sites of desired specificity, including antibody glycosylation variants, antibody amino acid sequence variants, and covalently modified antibodies. Antibodies (e.g., anti-galectin-9 antibodies) include antibodies of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and the antibodies need not belong to any particular class. Depending on the amino acid sequence of the constant domain of the heavy chain of the antibody, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0036] A typical antibody molecule contains a heavy chain variable region (V H ) and light chain variable region (V L ). H and V L The V regions can be further subdivided into regions of hypervariability, also called "complementarity determining regions" ("CDRs"), interspersed with regions that are more conserved, called "framework regions" ("FRs"). H and V LIt is usually composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Methods known in the art can be used (e.g., by Kabat definition, Chothia definition, AbM definition, EU definition, "Contact" numbering scheme, "IMGT" numbering scheme, "AHo" numbering scheme and / or contact definition, all of which are well known in the art) to accurately identify the scope of the framework region and CDR. See, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US 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; Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948; Edelman et al., Proc Natl Acad Sci US A. 1969 May;63(1):78-85; and Almagro, J. Mol. Recognit. 17:132-143 (2004); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Lefranc MP et al., Dev Comp Immunol, 2003 Jan;27(1):55-77; and Honegger A and Pluckthun A, J Mol Biol, 2001 Jun 8;309(3):657-70. See also hgmp.mrc.ac.uk andbioinf.org.uk / abs).

[0037] In some embodiments, the anti-galectin-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-galectin-9 antibody may be an antigen-binding fragment of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of a full-length antibody include (i) a Fab fragment, which consists of a V L 、V H , C L and C H1 domain; (ii) F(ab')2 fragment, which is a bivalent fragment consisting of two Fab fragments connected by a disulfide bond at the hinge region; (iii) H and C H 1 domain; (iv) a V fragment consisting of a single arm of the antibody L and V H (v) dAb fragments (Ward et al., (1989) Nature 341:544-546), which are composed of V H domain composition; and (vi) separate complementary determining regions (CDRs) that retain functionality. In addition, although the two domains V L and V H Encoded by separate genes, but they can be joined using recombinant methods with synthetic linkers to enable them to be made into a single protein chain, where V L and V H The domains pair to form monovalent molecules, termed single-chain Fvs (scFvs). See, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883.

[0038] Any of the antibodies described herein (e.g., anti-galectin-9 antibodies) can be monoclonal or polyclonal. "Monoclonal antibody" refers to a homogeneous antibody population, and "polyclonal antibody" refers to a heterogeneous antibody population. These two terms do not limit the source of the antibody or how it is made.

[0039] Reference antibody G9.2-17 refers to an antibody capable of binding to human galectin-9 and comprises a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO: 8, both of which are provided below. In some embodiments, the anti-galectin-9 antibody used in the methods disclosed herein is a G9.2-17 antibody. In some embodiments, the anti-galectin-9 antibody used in the methods disclosed herein is an antibody having the same heavy chain complementary determining region (CDR) as the reference antibody G9.2-17 and / or the same light chain complementary determining region as the reference antibody G9.2-17. H and / or V L CDRs of two antibodies means that their CDRs are identical when determined by the same method (eg, the Kabat method, Chothia method, AbM method, Contact method or IMGT method as known in the art. See, eg, bioinf.org.uk / abs / ).

[0040] The heavy and light chain CDRs of the reference antibody G9.2-17 are provided in Table 1 below (determined using the Kabat method):

[0041] Table 1. Heavy and light chain CDRs of G9.2-17

[0042]

[0043] In some examples, the anti-galectin-9 antibody used in the methods disclosed herein may comprise (according to the Kabat scheme) a heavy chain complementary determining region 1 (CDR1) as shown in SEQ ID NO:4, a heavy chain complementary determining region 2 (CDR2) as shown in SEQ ID NO:5, and a heavy chain complementary determining region 3 (CDR3) as shown in SEQ ID NO:6, and / or may comprise a light chain complementary determining region 1 (CDR1) as shown in SEQ ID NO:1, a light chain complementary determining region 2 (CDR2) as shown in SEQ ID NO:2, and a light chain complementary determining region 3 (CDR3) as shown in SEQ ID NO:3. Anti-galectin-9 antibodies (including reference antibody G9.2-17) may be in any form as disclosed herein, such as a full-length antibody or Fab. The term "G9.2-17 (IgG4)" used herein refers to the G9.2-17 antibody as an IgG4 molecule. Similarly, the term "G9.2-17 (Fab)" refers to the G9.2-17 antibody as a Fab molecule.

[0044] In some embodiments, the anti-galectin-9 antibody or binding portion thereof comprises heavy and light chain variable regions, wherein the light chain variable region CDR1, CDR2 and CDR3 amino acid sequences have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and any increments therein) sequence identity to the light chain variable region CDR1, CDR2 and CDR3 amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively. In some embodiments, the anti-Galectin-9 antibody, or binding portion thereof, comprises heavy and light chain variable regions, wherein the heavy chain variable region CDR1, CDR2, and CDR3 amino acid sequences have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and any increments therein) sequence identity to the heavy chain variable region CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.

[0045] Additional Galectin-9 antibodies (e.g., that bind to the CRD1 and / or CRD2 regions of Galectin-9) are described in U.S. Pat. No. 10,344,091 and co-owned, co-pending U.S. patent applications 16 / 173,970 and WO2020 / 198390, the relevant disclosures of each of which are incorporated herein by reference for the subject matter and purposes cited herein.

[0046] In some embodiments, the anti-Galectin-9 antibodies disclosed herein comprise light chain CDRs that are identical to corresponding V sequences of reference antibody G9.2-17. L The CDRs may be individually or collectively at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and any increments thereof) sequence identity compared to the reference antibody G9.2-17. Alternatively or additionally, in some embodiments, the anti-Galectin-9 antibody comprises a heavy chain CDR that is identical to the corresponding V CDR of the reference antibody G9.2-17. H The CDRs may individually or collectively have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and any increments therein) sequence identity when compared.

[0047] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, as modified in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program (score = 50, wordlength = 3) to obtain amino acid sequences homologous to protein molecules of the invention. When gaps exist between the two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0048] In other embodiments, the anti-Galectin-9 antibodies described herein comprise V H, the VH comprises HC CDR1, HC CDR2 and HC CDR3 that collectively contain up to 8 amino acid residue variations (8, 7, 6, 5, 4, 3, 2 or 1 variation, including additions, deletions and / or substitutions) relative to the HC CDR1, HC CDR2 and HC CDR3 of the reference antibody G9.2-17. Alternatively or additionally, in some embodiments, the anti-galectin-9 antibodies described herein comprise V H , the VH comprises LC CDR1, LC CDR2 and LC CDR3 that collectively contain up to 8 amino acid residue variations (8, 7, 6, 5, 4, 3, 2 or 1 variations, including additions, deletions and / or substitutions) relative to the LC CDR1, LC CDR2 and LC CDR3 of the reference antibody G9.2-17.

[0049] In one example, the amino acid residue variation is a conservative amino acid residue substitution. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein to which the amino acid substitution is made. Variants can be prepared according to methods known to those of ordinary skill in the art for changing polypeptide sequences, such as methods found in reference materials for compiling such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook et al., ed., 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FM Ausubel et al., ed., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0050] In some embodiments, the anti-Galectin-9 antibodies disclosed herein having heavy chain CDRs disclosed herein contain a germline V H The framework region of the fragment subclass. This type of germline V HRegions are well known in the art. See, for example, the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php. Examples include the IGHV1 subfamily (e.g., IGHV1-2, IGHV1-3, IGHV1-8, IGHV1-18, IGHV1-24, IGHV1-45, IGHV1-46, IGHV1-58, and IGHV1-69), the IGHV2 subfamily (e.g., IGHV2-5, IGHV2-26, and IGHV2-70), the IGHV3 subfamily (e.g., IGHV3-7, IGHV3-9, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-20, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-33, IGHV3-43, IGHV3-48, IGHV3-49, IGHV3-53, IGHV3-64, IGHV3-66, IGHV3-72 and IGHV3-73, IGHV3-74), 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).

[0051] Alternatively or additionally, in some embodiments, the anti-galectin-9 antibodies having light chain CDRs disclosed herein contain framework regions derived from germline Vκ fragments. Examples include IGKV1 frameworks (e.g., IGKV1-05, IGKV1-12, IGKV1-27, IGKV1-33, or IGKV1-39), IGKV2 frameworks (e.g., IGKV2-28), IGKV3 frameworks (e.g., IGKV3-11, IGKV3-15, or IGKV3-20), and IGKV4 frameworks (e.g., IGKV4-1). In other cases, the anti-galectin-9 antibodies comprise a light chain variable region containing a framework derived from a 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), the IGλ3 framework (e.g., IGλV3-1, IGλV3-9, IGλV3-10, IGλV3-12, IGλV3-16, IGλV3-19, IGλV3-21, IGλV3- -25, IGλV3-27), IGλ4 framework (e.g., IGλV4-3, IGλV4-60, IGλV4-69), IGλ5 framework (e.g., IGλV5-39, IGλV5-45), IGλ6 framework (e.g., IGλV6-57), IGλ7 framework (e.g., IGλV7-43, IGλV7-46), IGλ8 framework (e.g., IGλV8-61), IGλ9 framework (e.g., IGλV9-49) or IGλ10 framework (e.g., IGλV10-54).

[0052] In some embodiments, the anti-galectin-9 antibody used in the methods disclosed herein may be a reference antibody G9.2-17 having the same heavy chain variable region (V H ) and / or the same light chain variable region (V L ) of the antibody, the V H and V L The amino acid sequence of the region is provided below:

[0053] V H :

[0054]

[0055] V L :

[0056]

[0057] In some embodiments, the anti-Galectin-9 antibody has at least 80% sequence identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to the heavy chain variable region of SEQ ID NO: 7. Alternatively or additionally, the anti-Galectin-9 antibody has at least 80% sequence identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to the light chain variable region of SEQ ID NO: 8.

[0058] In some cases, the anti-galectin-9 antibodies disclosed herein are functional variants of the reference antibody G9.2-17. The functional variants can be structurally similar to the reference antibody (e.g., comprising a limited number of amino acid residue variations in one or more heavy and / or light chain CDRs of G9.2-17 as disclosed herein, or 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 as disclosed herein), and have substantially similar binding affinity to human galectin-9 (e.g., having a KD value of the same order).

[0059] In some embodiments, an anti-Galectin-9 antibody as described herein can bind to Galectin-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 therein). The apparent inhibition constant (Ki) provides a measure of the potency of the inhibitor. app or K i,app ) is related to the inhibitor concentration required to reduce enzyme activity and is independent of enzyme concentration. The inhibitory activity of the anti-Galectin-9 antibodies described herein can be determined by conventional methods known in the art.

[0060] Antibody K i, app The Ki value can be determined by measuring the inhibitory effect of different concentrations of the antibody on the extent of a reaction (e.g., enzyme activity); fitting the change in the pseudo-first-order rate constant (v) as a function of inhibitor concentration to the modified Morrison equation (Equation 1) to obtain an estimate of the apparent Ki value. For competitive inhibitors, Ki app It can be extracted from K i, app The y-intercept was obtained from the linear regression analysis of the plot of the relationship between the concentration of the substrate and the concentration of the substrate.

[0061]

[0062] Where A is equal to v o / E, the initial rate of the enzymatic reaction in the absence of an inhibitor (I) (v o ) divided by the total enzyme concentration (E). In some embodiments, the anti-galectin-9 antibodies described herein have a Ki of 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 pM or less for the target antigen or antigenic epitope. app In some embodiments, the anti-galectin-9 antibody has a lower Ki for a first target (e.g., CRD2 of galectin-9) relative to a second target (e.g., CRD1 of galectin-9). app . Ki app The difference (e.g., specificity or other comparison) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000, or 10 5 In some examples, the anti-Galectin-9 antibody inhibits the first antigen (e.g., the first protein or its mimetic in the first conformation) to a greater extent than the second antigen (e.g., the same first protein or its mimetic in the second conformation; or the second protein). In some embodiments, any of the anti-Galectin-9 antibodies are further affinity matured to reduce the Ki of the antibody to the target antigen or its antigenic epitope. app .

[0063] In some embodiments, the anti-Galectin-9 antibodies described herein have a suitable binding affinity for the target antigen (e.g., Galectin-9) or an antigenic epitope thereof. As used herein, "binding affinity" refers to the apparent association constant or K A . K A is the dissociation constant (K D ). The anti-galectin-9 antibodies described herein may have at least 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 M or lower binding affinity (K D ). An increase in binding affinity corresponds to a K DReduced. 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 measurement). Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20).

[0064] These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. Under certain conditions, the fractional concentration of bound binding protein ([bound] / [total]) is generally related to the concentration of total target protein ([target]) by the following equation:

[0065] [Bound] / [Total] = [Target] / (Kd+[Target])

[0066] However, it is not always necessary to determine K precisely. A , because sometimes quantitative measurements of affinity are obtained (e.g., using methods such as ELISA or FACS analysis, as opposed to K A It is sufficient to measure the affinity of an antibody to determine whether it is proportional to the affinity of the antibody and therefore can be used for comparison, such as determining whether the higher affinity is, for example, 2-fold higher), to obtain a qualitative measurement of affinity, or to obtain an inferred result of affinity (e.g., by activity in a functional assay (e.g., in vitro or in vivo assay)). In some cases, an in vitro binding assay is indicative of in vivo activity. In other cases, an in vitro binding assay is not necessarily indicative of in vivo activity. In some cases, tight binding is beneficial, but in other cases, tight binding is not ideal in vivo, and antibodies with lower binding affinity are more ideal.

[0067] In some embodiments, the heavy chain of any of the anti-galectin-9 antibodies described herein further comprises a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region can be of any suitable origin, such as human, mouse, rat, or rabbit. In a specific example, the heavy chain constant region is from human IgG (γ heavy chain) of any IgG subfamily as described herein.

[0068] In some embodiments, the heavy chain constant region of the antibodies described herein comprises a single domain (e.g., CH1, CH2 or CH3) of a constant region (e.g., SEQ ID NO: 4, 5, 6) or a combination of any single domains. In some embodiments, the light chain constant region of the antibodies described herein comprises a single domain (e.g., CL) of a constant region. Exemplary light and heavy chain sequences are listed below. Exemplary light and heavy chain sequences are listed below. The hIgG1 LALA sequence includes two mutations (L234A and L235A (EU numbering), which inhibit FcgR binding) and a P329G mutation (EU numbering) to eliminate complement C1q binding, thereby eliminating all immune effector functions. The hIgG4 Fab arm exchange mutation sequence includes a mutation (S228P; EU numbering) that inhibits Fab arm exchange. The IL2 signal sequence (MYRMQLLSCIALSLALVTNS; SEQ ID NO: 9) can be located at the N-terminus of the variable region. It is used in an expression vector, which is cut during secretion and is therefore not in a mature antibody molecule. The heavy chain of the mature protein (after secretion) begins with "EVQ" and the light chain begins with "DIM". The amino acid sequence of an exemplary heavy chain constant region is provided below:

[0069] hIgG1 heavy chain constant region (SEQ ID NO: 10)

[0070]

[0071] hIgG1 LALA heavy chain constant region (SEQ ID NO: 12)

[0072]

[0073] hIgG4 heavy chain constant region (SEQ ID NO: 13)

[0074]

[0075] hIgG4 heavy chain constant region (SEQ ID NO: 20)

[0076]

[0077] hIgG4 mutant heavy chain constant region (SEQ ID NO: 14)

[0078]

[0079] hIgG4 mutant heavy chain constant region (SEQ ID NO: 21)

[0080]

[0081] In some cases, the heavy chain constant region in the anti-Galectin-9 antibodies disclosed herein (e.g., G9.2-17) may have the C-terminal lysine (K) residue removed, for example, for manufacturing purposes. The corresponding amino acid sequences of those without the terminal K residue are provided below:

[0082] hIgG1 heavy chain constant region without C-terminal lysine (SEQ ID NO: 24)

[0083]

[0084] hIgG1 LALA heavy chain constant region without C-terminal lysine (SEQ ID NO: 25)

[0085]

[0086] hIgG4 heavy chain constant region without C-terminal lysine (SEQ ID NO: 26)

[0087]

[0088] hIgG4 heavy chain constant region without C-terminal lysine (SEQ ID NO: 27)

[0089]

[0090] hIgG4 mut heavy chain constant region without C-terminal lysine (SEQ ID NO: 28)

[0091]

[0092] hIgG4 mut heavy chain constant region without C-terminal lysine (SEQ ID NO: 29)

[0093]

[0094] In some embodiments, an anti-Galectin-9 antibody having any of the above heavy chain constant regions is paired with a light chain having the following light chain constant regions:

[0095] Light chain constant region (SEQ ID NO: 11)

[0096]

[0097] Exemplary full-length anti-Galectin-9 antibodies are provided below:

[0098] G9.2-17hIgG1 heavy chain (SEQ ID NO: 16)

[0099]

[0100] G9.2-17hIgG1 heavy chain without C-terminal lysine residue (SEQ ID NO: 30)

[0101]

[0102] G9.2-17hIgG1 LALA heavy chain (SEQ ID NO: 17)

[0103]

[0104] G9.2-17hIgG1 LALA heavy chain without C-terminal lysine residue (SEQ ID NO: 31)

[0105]

[0106] G9.2-17hIgG4 heavy chain (SEQ ID NO: 18)

[0107]

[0108] G9.2-17hIgG4 heavy chain without C-terminal lysine residue (SEQ ID NO: 32)

[0109]

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

[0111]

[0112] G9.2-17hIgG4 heavy chain without C-terminal lysine residue (SEQ ID NO: 33)

[0113]

[0114] G9.2-17hIgG4 Fab arm exchange mutant heavy chain (SEQ ID NO: 19)

[0115]

[0116] G9.2-17hIgG4 Fab arm-swap mut heavy chain without C-terminal lysine residue (SEQ ID NO: 34)

[0117]

[0118] G9.2-17hIgG4 Fab arm exchange mutant heavy chain (SEQ ID NO: 23)

[0119]

[0120] G9.2-17hIgG4 Fab arm-swap mut heavy chain without C-terminal lysine residue (SEQ ID NO: 35)

[0121]

[0122] Any of the above heavy chains can be paired with the light chain shown below (SEQ ID NO: 15):

[0123]

[0124] In some embodiments, the anti-Galectin-9 antibody comprises a heavy chain IgG1 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments therein) sequence identity to SEQ ID NO: 10. In one embodiment, the constant region of the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO: 10. In one embodiment, the constant region of the anti-Galectin-9 antibody comprises a heavy chain IgG1 constant region consisting of SEQ ID NO: 10.

[0125] In some embodiments, the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments therein) sequence identity to SEQ ID NO: 20. In one embodiment, the constant region of the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO: 20. In one embodiment, the constant region of the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO: 20.

[0126] In some embodiments, the constant region is from human IgG4. In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and any increments thereof) sequence identity to SEQ ID NO: 13. In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO: 13. In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO: 13.

[0127] In some embodiments, the constant region is from human IgG4. In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and any increments thereof) sequence identity to SEQ ID NO: 20. In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO: 20. In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO: 20.

[0128] In any of these embodiments, the anti-Galectin-9 antibody comprises a light chain constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments therein) sequence identity to SEQ ID NO: 11. In some embodiments, the anti-Galectin-9 antibody comprises a light chain constant region comprising SEQ ID NO: 11. In some embodiments, the anti-Galectin-9 antibody comprises a light chain constant region consisting of SEQ ID NO: 11.

[0129] In some embodiments, the IgG is a mutant with minimal Fc receptor engagement. In one example, the constant region is from human IgG1 LALA. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG1 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and any increments therein) sequence identity to SEQ ID NO: 12. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG1 constant region comprising SEQ ID NO: 12. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG1 constant region consisting of SEQ ID NO: 12.

[0130] In some embodiments, the anti-galectin-9 antibody comprises a modified constant region. In some embodiments, the anti-galectin-9 antibody comprises an immunologically inert modified constant region, for example, complement-mediated lysis is not triggered, or antibody-dependent cell-mediated cytotoxicity (ADCC) is not stimulated. ADCC activity can be assessed using the method 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, WO / 1999 / 058572 and / or British Patent Application No. 9809951.8. In some embodiments, the IgG4 constant region is a mutant with reduced heavy chain exchange. In some embodiments, the constant region is from human IgG4 Fab arm exchange mutant S228P.

[0131] In one embodiment, the constant region of the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and any increments therein) sequence identity to SEQ ID NO: 14. In one embodiment, the constant region of the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO: 14. In one embodiment, the constant region of the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO: 14.

[0132] In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and any increments therein) sequence identity to SEQ ID NO: 21. In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO: 21. In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO: 21.

[0133] In some embodiments, the light chain of the anti-Galectin-9 antibody has a chain corresponding to SEQ ID NO: 15; and the amino acid sequence of an exemplary heavy chain corresponds to SEQ ID NO: 10 (hIgG1); 12 (hIgG1 LALA); 13 (hIgG4); 20 (hIgG4); 14 (hIgG4mut); and 21 (hIgG4 mut).

[0134] In some embodiments, the anti-Galectin-9 antibody has a light chain comprising, consisting essentially of, or consisting of SEQ ID NO: 15. In some embodiments, the anti-Galectin-9 antibody has a heavy chain comprising, consisting essentially of, or consisting of any one of the sequences selected from the group consisting of SEQ ID NOs: 16-19, 22, and 23. In some embodiments, the anti-Galectin-9 antibody has a light chain comprising, consisting essentially of, or consisting of SEQ ID NO: 15 and a heavy chain comprising, consisting essentially of, or consisting of any one of the sequences selected from the group consisting of SEQ ID NOs: 16-19. In some embodiments, the anti-Galectin-9 antibody has a light chain comprising SEQ ID NO: 15 and a heavy chain comprising any one of the sequences selected from the group consisting of SEQ ID NOs: 16-19, 22, and 23. In some embodiments, the anti-Galectin-9 antibody has a light chain consisting 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 NOs: 16-19, 22, and 23. In some embodiments, the anti-Galectin-9 antibody has a light chain consisting essentially of SEQ ID NO: 15 and a heavy chain consisting essentially of any one of the sequences selected from the group consisting of SEQ ID NOs: 16-19, 22, and 23. In a specific embodiment, the anti-Galectin-9 antibody has a light chain consisting essentially of SEQ ID NO: 15 and a heavy chain consisting essentially of SEQ ID NO: 19. In another specific embodiment, the anti-Galectin-9 antibody has a light chain consisting essentially of SEQ ID NO: 15 and a heavy chain consisting essentially of SEQ ID NO: 20.

[0135] In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and any increments therein) sequence identity to SEQ ID NO: 16. In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO: 16. In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 16.

[0136] In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and any increments therein) sequence identity to SEQ ID NO: 17. In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO: 17. In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 17.

[0137] In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and any increments therein) sequence identity to SEQ ID NO: 18. In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO: 18. In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 18.

[0138] In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and any increments therein) sequence identity to SEQ ID NO: 22. In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO: 22. In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 22.

[0139] In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and any increments therein) sequence identity to SEQ ID NO: 19. In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO: 19. In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 19.

[0140] In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and any increments therein) sequence identity to SEQ ID NO: 23. In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO: 23. In one embodiment, the anti-Galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO: 23.

[0141] In any of these embodiments, the anti-Galectin-9 antibody comprises a light chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments therein) sequence identity to SEQ ID NO: 15. In some embodiments, the anti-Galectin-9 antibody comprises a light chain sequence comprising SEQ ID NO: 15. In some embodiments, the anti-Galectin-9 antibody comprises a light chain sequence consisting of SEQ ID NO: 15.

[0142] In a specific example, the anti-Galectin-9 antibody used in the treatment methods disclosed herein has a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 15. In some embodiments, the anti-Galectin-9 antibody used in the treatment methods disclosed herein is G9.2-17IgG4. In some examples, such anti-Galectin-9 antibodies do not have a C-terminal lysine residue in their heavy chain.

[0143] II. Preparation of anti-galectin-9 antibody

[0144] Antibodies capable of binding to Galectin-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.

[0145] The nucleic acid encoding the heavy and light chains of the anti-galectin-9 antibody as described herein can be cloned into an expression vector, and each nucleotide sequence is operably linked to a suitable promoter. In one example, each of the nucleotide sequences encoding the heavy and light chains is operably linked to a different promoter. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operably linked to a single promoter so 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.

[0146] In some examples, the nucleotide sequences encoding the two chains of the antibody are cloned into two vectors, which can be introduced into the same or different cells. When the two chains are expressed in different cells, each of them can be isolated from the host cell expressing it, and the isolated heavy and light chains can be mixed and incubated under appropriate conditions that allow the formation of antibodies.

[0147] Generally speaking, methods known in the art can be used to clone the nucleic acid sequence of one or all chains of the encoding antibody into a suitable expression vector operably connected to a suitable promoter. For example, the nucleotide sequence and the carrier can be contacted with a restriction enzyme under suitable conditions to produce complementary ends that can be paired with each other and connected together with a ligase on each molecule. Alternatively, a synthetic nucleic acid adapter can be connected to the end of the gene. These synthetic adapters contain the nucleic acid sequence corresponding to a specific restriction site in the carrier. The selection of the expression vector / promoter will depend on the type of host cell used to produce the antibody.

[0148] 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 (E. coli) lac UV5 promoter, and herpes simplex tk virus promoter.

[0149] Regulatable promoters can also be used. Such regulatable promoters include those that use the lac repressor from E. coli as a transcriptional regulator to regulate transcription of mammalian cell promoters with the lac operon [Brown, M. et al., Cell, 49:603-612 (1987)], those that use the tetracycline repressor (tetR) [Gossen, M. and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P. et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimers, VP16, or p65 using estradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech, and Ariad.

[0150] Regulatable promoters including repressors with operons can be used. In one embodiment, the lac repressor from Escherichia coli can act as a transcriptional regulator to regulate the 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)), tetracycline repressor (tetR) is combined with transcription activator (VP 16) to produce tetR-mammalian cell transcription activator fusion protein tTa (tetR-VP 16), wherein the minimal promoter with tetO is derived from the human cytomegalovirus (hCMV) major immediate early promoter to produce tetR-tet operon gene system, thereby controlling gene expression in mammalian cells. In one embodiment, a tetracycline inducible switch is used. When the tetracycline operator is correctly positioned downstream of the TATA element of the CMVIE promoter, the tetracycline repressor (tetR) alone, rather than tetR-mammalian cell transcription factor fusion derivatives, can act as a potent trans-regulator to regulate gene expression in mammalian cells (Yao et al., Human Gene Therapy, 10 (16): 1392-1399 (2003)). A particular advantage of this tetracycline-inducible switch is that it does not require the use of tetracycline repressor-mammalian cell transactivator or repressor fusion proteins (which may be toxic to cells in some cases) (Gossen et al., Natl. Acad. Sci. USA, 89: 5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92: 6522-6526 (1995)) to achieve its regulatable effect.

[0151] In addition, the vector may contain, for example, some or all of the following: a selection marker gene, such as a neomycin gene for selecting stable or transient transfectants in mammalian cells; an enhancer / promoter sequence for high-level transcription from the immediate early gene of human CMV; transcription termination and RNA processing signals for mRNA stability from SV40; SV40 polyoma virus replication origin and ColE1 for proper episomal gene replication; an internal ribosome binding site (IRES); 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.

[0152] Examples of polyadenylation signals that can be used to practice the methods described herein include, but are not limited to, the human collagen I polyadenylation signal, the human collagen II polyadenylation signal, and the SV40 polyadenylation signal.

[0153] One or more vectors (e.g., expression vectors) comprising any one of the nucleic acids encoding the antibody can be introduced into a suitable host cell to produce the antibody. 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 by cultured cells (e.g., from cells or culture supernatants) via conventional methods (e.g., affinity purification). If desired, the polypeptide chains of the antibody can be incubated under suitable conditions to allow the production of the antibody for a suitable period of time.

[0154] In some embodiments, the method for preparing the antibodies described herein involves recombinant expression vectors encoding the heavy and light chains of the anti-galectin-9 antibodies also described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods (e.g., calcium phosphate-mediated transfection). Positive transformant host cells can be selected and cultured under suitable conditions that allow the expression of the two polypeptide chains that form the antibody, and the antibody can 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 the antibody to form.

[0155] In one example, two recombinant expression vectors are provided, one encoding the heavy chain of an anti-galectin-9 antibody and the other encoding the light chain of an anti-galectin-9 antibody. Both recombinant expression vectors can be introduced into suitable host cells (e.g., dhfr-CHO cells) by conventional methods (e.g., calcium phosphate-mediated transfection). Alternatively, each of the expression vectors can be introduced into a suitable host cell. Positive transformants can be selected and cultured under suitable conditions that allow the expression of the polypeptide chain of the antibody. When the two expression vectors are introduced into the same host cell, the antibodies produced therein can be recovered from the host cell or culture medium. If necessary, the polypeptide chains can be recovered from the host cell or culture medium and then cultured under suitable conditions that allow 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.

[0156] 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 protein A or protein G coupled matrix.

[0157] Any of nucleic acids encoding the heavy chain, light chain, or both of the anti-Galectin-9 antibodies described herein, vectors (eg, expression vectors) containing the nucleic acids, and host cells comprising the vectors are within the scope of the present disclosure.

[0158] The anti-Galectin-9 antibodies thus prepared can be characterized using methods known in the art, thereby detecting and / or measuring the reduction, improvement or neutralization of Galectin-9 biological activity. For example, in some embodiments, ELISA-type assays are suitable for qualitative or quantitative measurement of Galectin-9 inhibition of Dectin-1 or TIM-3 signaling.

[0159] The biological activity of the anti-galectin-9 antibody can be verified by incubating the candidate antibody with Dectin-1 and galectin-9 and monitoring any one or more of the following features: (a) binding between Dectin-1 and galectin-9 and inhibition of signal transduction mediated by the binding; (b) prevention, improvement or treatment of any aspect of hematological malignancies; (c) blocking or reducing Dectin-1 activation; (d) inhibiting (reducing) the synthesis, production or release of galectin-9. Alternatively, TIM-3 can be used to verify the biological activity of the anti-galectin-9 antibody using the above protocol. Alternatively, CD206 can be used to verify the biological activity of the anti-galectin-9 antibody using the above protocol.

[0160] In some embodiments, biological activity or efficacy is assessed in a subject, for example, by measuring peripheral and intratumoral T cell ratios, T cell activation, or by macrophage profiling.

[0161] Other assays to determine the biological activity of anti-galectin-9 antibodies include measuring CD8+ and CD4+ (conventional) T cell activation (in in vitro or in vivo assays, e.g., by measuring levels of inflammatory cytokines, e.g., IFNγ, TNFα, CD44, ICOS granzyme B, perforin, IL2 (upregulated); CD26L and IL-10 (downregulated)); measuring reprogramming of macrophages, e.g., from M2 to M1 phenotype (in vitro or in vivo) (e.g., increased MHCII, decreased CD206, increased TNFα and iNOS). Alternatively, the level of ADCC can be assessed, e.g., in an in vitro assay as described herein.

[0162] III. Treatment

[0163] The present disclosure provides a method for treating a hematological malignancy, which is a cancer that begins in blood-forming tissues (such as cells of the bone marrow or immune system). Hematological malignancies include acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome and myeloproliferative tumors, such as essential thrombocythemia, polycythemia vera and myelofibrosis. Subjects suffering from hematological malignancies can be identified by routine medical examinations, such as laboratory tests, organ function tests, CT scans or ultrasound.

[0164] A. Pharmaceutical Composition

[0165] Any anti-Gal9 antibody disclosed herein (e.g., G9.2-17 (IgG4)) and encoding nucleic acid or nucleic acid group, vector comprising the same can be mixed with a pharmaceutically acceptable carrier (excipient) to form a pharmaceutical composition for treating a target disease. "Acceptable" means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and harmless to the subject to be treated. Pharmaceutically acceptable excipients (carriers) include buffers well known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Edition (2000) Lippincott Williams and Wilkins, editor KE Hoover.

[0166] The pharmaceutical composition used in the method of the present invention may include a pharmaceutically acceptable carrier, excipient or stabilizer in the form of a lyophilized formulation or aqueous solution. (Remington: The Science and Practice of Pharmacy 20th Edition (2000) Lippincott Williams and Wilkins, editor KE Hoover). Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dosage and concentration used, and may include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN TM 、PLURONICS TM or polyethylene glycol (PEG).

[0167] In some examples, the pharmaceutical compositions described herein include liposomes containing antibodies (or encoding nucleic acids), which can be prepared by methods known in the art, such as Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980); and described in U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with extended circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be produced by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter of limited pore size to produce liposomes with a desired diameter.

[0168] Antibodies or encoding nucleic acids can also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions, respectively. Such techniques are known in the art, see, for example, Remington, The Science and Practice of Pharmacy 20th edition Mack Publishing (2000).

[0169] Pharmaceutical compositions for in vivo administration must be sterile. This is easily achieved, for example, by filtration through a sterile filtration membrane. The therapeutic antibody composition is usually placed in a container with a sterile access port, such as an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle.

[0170] In some instances, the pharmaceutical composition is an injectable preparation. Injectable preparations 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 by a drip method, thereby infusing a pharmaceutical preparation containing an antibody and a physiologically acceptable excipient. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients. The intramuscular preparation of the antibody (e.g., a sterile preparation in a suitable soluble salt form) may be dissolved in a pharmaceutical excipient (such as water for injection, 0.9% saline or 5% glucose solution) and administered.

[0171] (B) Treatment of hematological malignancies

[0172] In some embodiments, the present disclosure provides methods of treating hematological malignancies (e.g., hematological malignancies involving Gal9+ cancer cells) using an effective amount of an anti-Galectin-9 antibody described herein, including but not limited to G9.2-17IgG4 (having a heavy chain of SEQ ID NO: 19 and a light chain of SEQ ID NO: 15).

[0173] Exemplary Targeted Hematological Malignancies

[0174] In some instances, the methods disclosed herein are applicable to human patients with leukemia (e.g., acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL), including T cell ALL and B cell ALL). In other instances, the methods disclosed herein are applicable to human patients with lymphomas (e.g., those disclosed herein). In yet another instance, the methods disclosed herein are applicable to human patients with myelodysplastic syndrome (MDS).

[0175] Acute myeloid leukemia (AML) is a blood cancer that starts in the blood and bone marrow and can progress quickly. AML involves abnormal white blood cells produced by the bone marrow that fill the blood and bone marrow, leaving less room for healthy cells. Symptoms associated with AML include bleeding and anemia. Untreated AML can be life-threatening.

[0176] Acute lymphoblastic leukemia (ALL) is a cancer of the bone marrow that spreads quickly to blood cells. ALL causes the bone marrow to produce too many immature white blood cells (lymphoblasts). These abnormal cells exclude healthy red and white blood cells and platelets in the blood and bone marrow, making it difficult for the body to fight infection and disease. As the most common childhood leukemia and cancer, ALL usually occurs before the age of 15. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of the bone marrow and accounts for about 20% of all ALL cases. B-cell acute lymphoblastic leukemia (B-ALL) is the most common type of cancer in children, usually affecting the patient's B cells.

[0177] Myelodysplastic syndrome (MDS) is a condition that can occur when the blood-forming cells in the bone marrow become abnormal. This results in a low number of one or more types of blood cells. In MDS, some cells in the bone marrow are abnormal (dysplastic) and have problems making new blood cells. Many of the blood cells formed from these bone marrow cells are defective. The defective cells often die earlier than normal cells, and the body also destroys some of the abnormal blood cells, leaving the patient without enough normal blood cells. Although the most common finding in MDS is a lack of red blood cells (anemia), different cell types may be affected.

[0178] In some cases, the subject may be a human patient with a refractory disease (e.g., refractory AML, refractory ALL, or refractory MDS). As used herein, "refractory" refers to a tumor that is not responsive to treatment or becomes resistant. In some cases, the subject may be a human patient with a recurrent disease (e.g., recurrent AML, recurrent ALL, recurrent MDS, or recurrent cholangiocarcinoma). As used herein, "recurrent" or "relapse" refers to a tumor that reappears or progresses after a period of treatment improvement (e.g., partial or complete response).

[0179] In some cases, the subject is a human patient with elevated levels of galectin-9, such as relative to a control level. The level of galectin-9 may be the plasma or serum level of galectin-9 in a human patient. In other examples, the level of galectin-9 is the level of galectin-9 of cancer cells within a tumor. In other examples, the level of galectin-9 is the level of galectin-9 of immune cells within a tumor. In other examples, the level of galectin-9 may be the level of cell surface galectin-9, such as the level of galectin-9 on cancer cells. In one example, the level of galectin-9 may 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. In some examples, the control level represents the level of galectin-9 in a healthy subject. In some embodiments, the control level may be a baseline level before treatment.

[0180] To identify such subjects, a suitable biological sample can be obtained from a subject suspected of having a hematological malignancy, and the biological sample can be analyzed using conventional methods (e.g., ELISA or FACS) to determine the level of galectin-9 (e.g., free, cell surface expressed, or total) contained therein. In some embodiments, for example, organoid cultures are prepared as described herein and used to assess the level of galectin-9 in a subject. Single cells obtained from certain fractions obtained as part of the organoid preparation process are also suitable for assessing the level of galectin-9 in a subject. In some cases, an assay for measuring the level of galectin-9 in free form or expressed on the cell surface involves the use of an antibody that specifically binds to galectin-9 (e.g., specifically binds to 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, the antibodies described herein (e.g., G9.2-17 antibodies) can be used in assays such as. In some embodiments, the antibodies are described in U.S. Pat. No. 10,344,091 and WO2019 / 084553, the relevant disclosures of each of which are incorporated by reference for the purposes and subject matter cited herein. In some instances, the anti-Galectin-9 antibody is a Fab molecule. Assays for determining Galectin-9 levels as disclosed herein are also within the scope of the present disclosure.

[0181] In some embodiments, a human patient to be treated by any of the methods disclosed herein may meet one or more of the inclusion and exclusion criteria listed below in Example 1. For example, such a human patient may meet all of the inclusion and exclusion criteria listed below in Example 1.

[0182] Exemplary Treatment Conditions

[0183] In some embodiments, a pharmaceutical composition comprising an effective amount of an anti-Gal9 antibody disclosed herein (e.g., G9.2-17IgG4) can be administered to a subject (e.g., a human patient, which can be an adult or a child) in need of treatment, for example, at a suitable dose and a suitable frequency of administration. For example, the antibody can be administered to a subject via a suitable route (e.g., intravenous infusion) at a dose of about 2 mg / kg (e.g., about 4 mg / kg) to about 32 mg / kg once a week to once every 6 weeks. See also the disclosure herein.

[0184] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within an acceptable standard deviation. Alternatively, "about" can mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and more preferably up to ±1% of a given value. Alternatively, particularly for biological systems or processes, the term can mean within an order of magnitude of a value, preferably within 2 times. Where specific values ​​are described in the present application and claims, the term "about" is implicit, unless otherwise stated, and in this context means within an acceptable error range for a particular value.

[0185] An effective amount of a pharmaceutical composition described herein can be administered systemically or topically to a subject (e.g., a human) in need of treatment via an appropriate route. In some embodiments, the anti-galectin-9 antibody is administered by intravenous administration (e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarterial, intraarticular, intrasynovial, intrathecal, intratumoral, suburothelial, oral, inhaled, or topical routes). In one embodiment, the anti-galectin-9 antibody is administered to a subject by intravenous infusion. In one embodiment, the anti-galectin-9 antibody is administered to a subject intraperitoneally.

[0186] As used herein, "effective amount" refers to the amount of each active agent required to impart a therapeutic effect to a subject alone or in combination with one or more other active agents. In some embodiments, the therapeutic effect is to reduce the activity and / or amount / expression of galectin 9 in the tumor microenvironment, reduce Dectin-1 signaling, reduce TIM-3 signaling, reduce CD206 signaling, and / or increase anti-tumor immune response. Non-limiting examples of increasing anti-tumor response include increasing the activation level of effector T cells or converting TAM from an M2 phenotype to an M1 phenotype. In some cases, the anti-tumor response includes increasing the ADCC response. It is obvious to those skilled in the art to determine whether a certain amount of antibody achieves a therapeutic effect. As recognized by those skilled in the art, the effective amount varies according to the specific illness being treated, the severity of the illness, individual patient parameters (including age, physical condition, body shape, sex, and weight), the duration of treatment, the nature of concurrent treatment (if any), the specific route of administration, and similar factors within the scope of health practitioners' knowledge and expertise.

[0187] Empirical considerations (such as half-life) generally help determine dosage. For example, antibodies compatible with the human immune system (such as humanized antibodies or fully human antibodies) are used to extend the half-life of the antibody and prevent the antibody from being attacked by the host immune system in some cases. The frequency of administration can be determined and adjusted during the course of treatment, and is usually but not necessarily based on the treatment and / or suppression and / or improvement and / or delay of the target disease / disorder. Alternatively, the sustained continuous release formulation of the antibody may be suitable. Various preparations and devices for achieving sustained release are known in the art.

[0188] As used herein, the term "treatment" refers to the application or administration of a composition comprising one or more active agents to a subject suffering from a target disease or condition, a symptom of a disease / condition, or a predisposition to a disease / condition, with the purpose of curing, healing, alleviating, mitigating, altering, remedying, alleviating, improving, or affecting the condition, symptom of a disease or condition, or a predisposition to a disease or condition. Non-limiting examples of treatment include reducing tumor size, delaying tumor growth, alleviating lesions, and / or delaying the development of lesions, and / or prolonging the survival of human patients suffering from target hematological malignancies such as AML, MDS, or ALL (T-cell or B-cell ALL), who may be refractory and / or relapsed.

[0189] Alleviating the target disease / disorder includes delaying the development or progression of the disease or reducing the severity of the disease or prolonging survival. Alleviating the disease or prolonging survival does not necessarily require a curative outcome. As used herein, "delaying" the development of the target disease or condition refers to postponing, hindering, slowing down, delaying, stabilizing and / or slowing the progression of the disease. This delay can be a different length of time, depending on the history of the disease and / or the individual being treated. A method of "delaying" or alleviating the development of a disease or delaying the onset of a disease is a method that reduces the likelihood of one or more symptoms of the disease occurring within a given time frame and / or alleviates the degree of symptoms within a given time frame compared to not using the method. Such comparisons are typically based on clinical studies, and the number of subjects used is sufficient to produce statistically significant results.

[0190] "Development" or "progression" of a disease refers to the initial manifestation of the disease and / or the subsequent progression. 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 not be detected. For the purposes of this disclosure, development or progression refers to the biological course of symptoms. "Development" includes occurrence, recurrence, and onset. As used herein, "onset" or "occurrence" of a target disease or disorder includes initial onset and / or recurrence.

[0191] In one example, the dosage of an antibody as described herein is determined empirically in an individual who has been given one or more administrations of the antibody. Increasing doses of the antibody antagonist are administered to the individual. In order to assess the efficacy of the antibody antagonist, an indicator of the disease / disorder, such as any disclosed indicator, can be followed.

[0192] Monotherapy

[0193] In some embodiments, any anti-Gal9 antibody disclosed herein (such as G9.2-17 (IgG4)) can be used as a monotherapy (the only active agent) for treating a target hematological malignancy, such as AML, MDS or ALL. In view of the direct cell killing effect of the antibody against Gal9+ cancer cells, it is expected that the anti-Gal9 antibodies disclosed herein are effective as monotherapy in treating hematological cancers involving Gal9+ cancer cells.

[0194] In some embodiments, the anti-Gal9 antibody can be administered to the patient once a week to once every six weeks, for example, via intravenous infusion. In some instances, the antibody can be administered once every 1-4 weeks (e.g., every 2-4 weeks). In one example, the antibody can be administered once a week. In another example, the antibody can be administered once every 2 weeks.

[0195] In some embodiments, the anti-galectin-9 antibodies disclosed herein (e.g., G9.2-17IgG4) are administered intravenously over an infusion period of 30 minutes to 6 hours. In some instances, the intravenous infusion of the anti-galectin-9 antibodies can be performed for 30 minutes to 2 hours. In other instances, the anti-galectin-9 antibodies can be administered over a long infusion period (e.g., about 2-6 hours, such as about 2-4 hours or about 4-6 hours). In specific instances, exemplary anti-galectin-9 antibodies can be intravenously infused over a period of about 3 hours, about 4 hours, about 5 hours, or about 6 hours.

[0196] In some embodiments, the anti-galectin-9 antibodies (e.g., G9.2-17 (IgG4)) disclosed herein for use in treating hematological malignancies (e.g., those disclosed herein, such as AML, MDS, or ALL) can be administered to a subject at a dose of about 0.2 mg / kg to about 32 mg / kg, for example, the dose can be selected from 0.2 mg / kg, 0.63 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 6.3 mg / kg, 7.5 mg / kg, 8 mg / kg, 10 mg / kg, 12 mg / kg, 16 mg / kg, 32 mg / kg or higher dose levels. In some embodiments, the anti-galectin-9 antibodies can be administered to a subject at a dose of about 1 mg / kg to about 32 mg / kg, for example, the dose can be selected from 2 mg / kg, 4 mg / kg, 8 mg / kg, 12 mg / kg, 16 mg / kg, 32 mg / kg or higher dose levels. In some examples, the anti-galectin-9 antibody can be administered to a subject at a dose of about 0.2 mg / kg to about 32 mg / kg, for example, the dose can be selected from 0.2 mg / kg, 0.63 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 6.3 mg / kg, 7.5 mg / kg, 10 mg / kg, 12 mg / kg, 16 mg / kg, 32 mg / kg or higher dose levels.

[0197] In one example, an anti-galectin-9 antibody disclosed herein (e.g., G9.2-17 (IgG4)) is administered to a subject at a dose of 2 mg / kg per week. In another example, an anti-galectin-9 antibody disclosed herein (e.g., G9.2-17 (IgG4)) is administered to a subject at a dose of 4 mg / kg per week. In yet another example, an anti-galectin-9 antibody disclosed herein (e.g., G9.2-17 (IgG4)) is administered to a subject at a dose of 6.3 mg / kg per week. In yet another example, an anti-galectin-9 antibody disclosed herein (e.g., G9.2-17 (IgG4)) is administered to a subject at a dose of 7.5 mg / kg per week. In still another example, an anti-galectin-9 antibody disclosed herein (e.g., G9.2-17 (IgG4)) is administered to a subject at a dose of 10 mg / kg per week. In yet another example, an anti-galectin-9 antibody disclosed herein (e.g., G9.2-17 (IgG4)) is administered to a subject at a dose of 12 mg / kg per week. In another example, an anti-galectin-9 antibody disclosed herein (e.g., G9.2-17 (IgG4)) is administered to a subject at a dose of 16 mg / kg per week.

[0198] In one example, an anti-Gal9 antibody disclosed herein (e.g., G9.2-17 (IgG4)) can be administered to a human patient with a target hematological malignancy (such as AML, MDS, or ALL) at 4 mg / kg (e.g., by iv) once a week or once every two weeks. In another example, the anti-Gal9 antibody can be administered to a human patient at 7.5 mg / kg (e.g., by iv) once a week or once every two weeks. In yet another example, the anti-Gal9 antibody can be administered to a human patient at 12 mg / kg (e.g., by iv) once a week or once every two weeks. In still another example, the anti-Gal9 antibody can be administered to a human patient at 16 mg / kg (e.g., by iv) once a week or once every two weeks.

[0199] In some cases, a steady dosing schedule can be used in any of the treatment methods disclosed herein. For example, an anti-Gal-9 antibody disclosed herein (such as G9.2-17 (IgG4)) can be administered to a subject at a steady dose (e.g., about 410 mg to about 1120 mg) once a week to once every 4 weeks (e.g., once a week or once every two weeks). In some instances, an anti-Gal-9 antibody is administered to a subject at about 410 mg to about 505 mg (e.g., about 410 mg to about 450 mg) once a week. In some instances, an anti-Gal-9 antibody is administered to a subject at about 410 mg to about 505 mg (e.g., about 410 mg to about 450 mg) once every two weeks. In some instances, an anti-Gal-9 antibody is administered to a subject at about 650 mg to about 800 mg (e.g., about 650 mg to about 700 mg) once a week. In some instances, the anti-Gal-9 antibody is administered to a subject at about 650 mg to about 800 mg (e.g., about 650 mg to about 700 mg) once every two weeks. In some instances, the anti-Gal-9 antibody is administered to a subject at about 1040 mg to about 1,280 mg (e.g., about 1040 mg to about 1120 mg) once a week. In some instances, the anti-Gal-9 antibody is administered to a subject at about 1040 mg to about 1,280 mg (e.g., about 1040 mg to about 1120 mg) once every two weeks. In some instances, the anti-Gal-9 antibody is administered to a subject at about 2080 mg to about 2560 mg (e.g., about 2080 mg to about 2400 mg) once a week. In some instances, the anti-Gal-9 antibody is administered to a subject at about 2080 mg to about 2560 mg (e.g., about 2080 mg to about 2400 mg) once every two weeks.

[0200] In other cases, the stable dosing schedule may include once a week to once every six weeks (e.g., every 1 week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks or every 6 weeks) about 130 mg to about 320 mg. For example, the stable dosing schedule may include about 130 mg to about 160 mg once a week. Alternatively, the stable dosing schedule may include about 130 mg to about 160 mg once every two weeks. In another example, the stable dosing schedule may include about 260 mg to about 320 mg once a week. Alternatively, the stable dosing schedule may include about 260 mg to about 320 mg once every two weeks.

[0201] In some cases, the treatment period can be 6 months to 12 months. In other cases, the treatment period can be 12 months to 24 months. In other cases, the treatment period can be greater than 24 months.

[0202] In some embodiments, the dosage is adjusted based on the patient's response to treatment. In some embodiments, the dosage is changed between treatment intervals. In some embodiments, treatment may be temporarily stopped. In some embodiments, treatment may be temporarily stopped. In some embodiments, anti-galectin-9 therapy is temporarily stopped. In some embodiments, checkpoint inhibitor therapy used in combination with anti-galectin-9 antibodies is temporarily stopped. In some embodiments, both are temporarily stopped.

[0203] In some cases, a human patient may be started at a low dose (e.g., 4 mg / kg, 6.3 mg / kg, or 7.5 mg / kg) of an anti-galectin-9 antibody disclosed herein, such as G9.2-17 (IgG4). When applicable, the dose may be increased to, for example, 10 mg / kg, 12 mg / kg, 16 mg / kg, or 32 mg / kg, as determined by a medical practitioner. Similarly, a human patient may be started at a long dosing frequency (e.g., every two weeks) and adjusted to a short dosing frequency (e.g., weekly), or vice versa, when applicable.

[0204] Combination therapy

[0205] In some embodiments, any anti-Gal9 antibody described herein (e.g., a G9.2-17 antibody as disclosed herein, such as G9.2-17 (IgG4)) can be used in combination with a second therapeutic agent (e.g., a chemotherapeutic agent) for treating a target hematological malignancy as disclosed herein (e.g., AML, MDS, or ALL). The selection of an appropriate chemotherapeutic agent or other combination agent and treatment modality will depend on various factors, including the target hematological malignancy, severity of the disease, age, sex, treatment history, resistance to previous treatment, etc., which are within the knowledge of medical practitioners.

[0206] For example, anti-Gal9 antibodies (such as G9.2-17 (IgG4)) can be used in combination with suitable chemotherapeutic agents for the treatment of AML. Exemplary chemotherapeutic agents include, but are not limited to, Azacitidine, Cerubidine (Daunorubicin Hydrochloride), Cyclophosphamide, Cytarabine, Daunorubicin Hydrochloride and Cytarabine Liposomal, Daurismo (Glasdegib Maleate), Dexamethasone, Doxorubicin Hydrochloride, Enasidenib Mesylate, Gemtuzumab Ozogamicin, Gilteritinib Fumarate, Glagib Maleate, Idarubicin PFS (Idarubicin Hydrochloride), and 1,2-dihydroquinone. Hydrochloride), Idarubicin Hydrochloride, Idhifa (Ensipine Mesylate), Ivosidenib, Midostaurin, Mitoxantrone Hydrochloride, Mylotarg (Gemtuzumab Ozomicin), Onureg (Azacitidine), Prednisone, Rubidomycin (Daunorubicin Hydrochloride), Rydapt (Midostaurin), Tabloid (Thioguanine), Thioguanine, Tibsovo (Ivosidenib), Trisenox (Arsenic Trioxide), Venclexta (Venetoclax), Venclexta, Vincristine Sulfate, Vyxeos (Daunorubicin Hydrochloride and Cytarabine Liposomal), Xo spata (gilteritinib fumarate), Sabatolimab, or a combination thereof. In some cases, the second therapeutic agent used in conjunction with an anti-Gal9 antibody for the treatment of AML may be a PD-1 inhibitor.Examples include, but are not limited to, anti-PD-1 antibodies (e.g., pembrolizumab, nivolumab, tiselliz umab, dostarlimab, and cemiplimab) or anti-PD-L1 antibodies (e.g., durvalumab, avelumab, and atezolizumab).

[0207] In other examples, anti-Gal9 antibodies (such as G9.2-17 (IgG4)) can be used in combination with a suitable chemotherapeutic agent to treat MDS. Exemplary chemotherapeutic agents include, but are not limited to, azacitidine (e.g., for low-risk and high-risk patients with all MDS subtypes); Decitabine (e.g., for low-risk and high-risk patients with all MDS subtypes); Lenalidomide (e.g., for transfusion-dependent MDS patients with isolated del(5q) and an IPSS score of low risk or intermediate-1 risk); Luspatercept-aamt (e.g., for adult MDS patients with ring sideroblasts (MDS-RS) or myelodysplastic / myeloproliferative neoplasms with ring sideroblasts and thrombocytosis (MDS / MPN-RS-T), which in some cases have been patients with MDS or CMML who have failed erythropoiesis stimulating agents (ESAs) or are unlikely to respond to ESAs and require 2 or more red blood cell (RBC) transfusions within 8 weeks); ASTX727 (e.g., for adult patients with MDS or CMML who are intermediate-1, intermediate-2, and high-risk IPSS groups, including previously treated and untreated, primary and secondary MDS, including patients with refractory anemia, refractory anemia with ring sideroblasts, refractory anemia with excess blasts, and CMML); Luspatercept, Decitabine / Cedazuridine, Pevonedistat, Magrolimab, or a combination thereof.

[0208] In some embodiments, anti-Gal9 antibodies can be administered simultaneously with any second therapeutic agent disclosed herein. In some embodiments, anti-Gal9 antibodies can be administered before the second therapeutic agent. Alternatively, anti-Gal9 antibodies can be administered after the second therapeutic agent. In some cases, the second therapeutic agent is systemically administered. In other cases, the second therapeutic agent is topically administered. In some instances, the second therapeutic agent is administered intravenously (e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarterial, intraarticular, intravesical, intrasynovial, intrathecal, intratumoral or suburothelial route). In one embodiment, the second therapeutic agent is administered to the subject by intravenous infusion.

[0209] Monitoring treatment response

[0210] The efficacy of a treatment as disclosed herein on a target hematological malignancy can be assessed by methods well known in the art.

[0211] In some embodiments, the anti-tumor activity of any treatment method disclosed herein can be monitored by conventional methods. For example, before, during and after the administration of the antibody and optionally the second therapeutic agent, cancer cells and / or biomarkers in the subject can be measured in a biological sample (such as blood, serum, plasma, urine, peritoneal fluid and / or a biopsy from a tissue or organ). The results thus obtained can be analyzed to evaluate the efficacy of the treatment. For example, the level of Gal9+ cancer cells can be measured before, during and after treatment to evaluate the direct killing effect of anti-Gal9 antibodies (such as G9.2-17 (IgG4)).

[0212] Alternatively or in addition, the response to treatment can also be characterized (e.g., to assess immunomodulatory effects) by one or more of the following: immune phenotype in blood and tumor, cytokine profile (serum), soluble Galectin-9 levels in blood (serum or plasma), Galectin-9 tumor tissue expression levels and expression patterns by immunohistochemistry (tumor, stroma, immune cells), tumor mutation burden (TMB), mismatch repair status, or disease-associated tumor markers (e.g., as measured at about 3, 6, or 12 months, or later, or at any other clinically indicated time point).

[0213] In some instances, changes in immune cells and immune cell marker levels in blood or tumors, such as immune activation, can be measured before and after treatment. Such changes can be measured in patient blood and tissue samples using methods known in the art (such as multiple flow cytometry and multiple immunohistochemistry). For example, a group of phenotypes and functional PBMC immune markers can be evaluated at baseline before the start of treatment and at different time points during treatment. Table 2 lists non-limiting examples of markers that can be used for these evaluation methods. Flow cytometry (FC) is a fast and informative preferred technical method for analyzing cell phenotype and function, and has occupied a prominent position in immunophenotypic monitoring. It allows characterization of many cell subpopulations in complex mixtures (such as blood), including rare subpopulations, and represents a fast method for obtaining a large amount of data. The advantages of FC are fast speed, high sensitivity and strong specificity. Standardized antibody groups and procedures can be used to analyze and classify immune cell subtypes. Multiple IHC is a powerful research tool that provides objective quantitative data describing tumor immune background in terms of immune subset quantity and location, and allows multiple markers to be evaluated on a single tissue section. Computer algorithms can be used to quantify IHC-based biomarker content in whole-section images of patient biopsies, thereby integrating chromogenic IHC methods and staining with digital pathology approaches.

[0214] Table 2. PBMC typing markers

[0215]

[0216]

[0217] Subjects treated with any anti-Gal9 antibody as disclosed herein (e.g., G9.2-17 (IgG4)) alone or in combination with a second therapeutic agent as disclosed herein (e.g., a chemotherapeutic agent) may be monitored for the occurrence of adverse effects (e.g., severe adverse effects). Exemplary adverse effects to be monitored are provided in Example 1 below. If an adverse effect is observed, the treatment conditions for that subject may be changed. For example, the dose of the anti-galectin-9 antibody may be reduced and / or the dosing interval may be extended. The appropriateness and extent of the reduction may be assessed by a qualified clinician. In some embodiments, one or more dose reductions of about 10% to about 80% of the previous dose level may be implemented. In some embodiments, one or more dose reductions of about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, or about 70% to about 80% of the previous dose level may be implemented. In some embodiments, one or more doses can be implemented 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 can be implemented 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 embodiments, one or more doses are implemented to reduce the previous dose level by 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%. Alternatively or in addition, the dosage of the second therapeutic agent can be reduced and / or the dosing interval of the checkpoint inhibitor can be extended. In some cases (for example, life-threatening adverse effects occur), treatment can be terminated.

[0218] Any of the methods disclosed herein may involve reducing the dosage of the anti-Gal9 antibody when one or more adverse events (eg, those disclosed herein) are observed during treatment.

[0219] IV. Kit for treating diseases associated with galectin-9

[0220] The present disclosure also provides kits for treating or alleviating hematological malignancies such as those disclosed herein (e.g., AML, MDS, or ALL). Such kits may include one or more containers comprising an anti-Gal9 antibody, such as any of those described herein (e.g., G9.2-17 (IgG4)), and optionally a second therapeutic agent to be used in conjunction with the anti-Gal9 antibody also described herein (e.g., a chemotherapeutic agent as disclosed herein).

[0221] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include a description of administering an anti-Gal9 antibody and optionally a second therapeutic agent to treat, delay onset, or alleviate those target diseases as described herein. In some embodiments, the kit also includes a description of selecting an individual suitable for treatment based on identifying whether the individual suffers from the target disease (e.g., using a diagnostic method as described herein). In yet other embodiments, the instructions include a description of administering the antibody to an individual at risk of the target disease.

[0222] Instructions related to the use of anti-Gal9 antibodies typically include information about the dosage, dosing schedule, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., multi-dose package), or subunit dose. The instructions provided in the kit of the present invention are typically written instructions on a label or package insert (e.g., a sheet of paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic disk or optical storage disk) are also acceptable.

[0223] The label or package insert indicates that the composition is used to treat, delay onset, and / or ameliorate the target hematological malignancy.In some embodiments, instructions for practicing any of the methods described herein are provided.

[0224] The kit of the present invention is in a suitable package. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. It is also contemplated that packaging is used in combination with a specific device (such as an inhaler, a nasal administration device (e.g., a nebulizer), or an infusion device (such as a mini pump). In some embodiments, the kit has a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle). In some embodiments, the container also has a sterile access port (e.g., the container is an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle). At least one active agent in the composition is an anti-Gal9 antibody described herein.

[0225] The kit may optionally provide additional components, such as buffer and interpretative information. Typically, the kit includes a container and a label or package insert on or associated with the container. In some embodiments, the invention provides an article comprising the contents of the above-described kit.

[0226] General techniques

[0227] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture (JP Thayer and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths and DG Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Weir and CC Blackwell, eds.); Cells (JM Miller and MP Calos, 1987); Current Protocols in Molecular Biology (FM Ausubel et al., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., 1994); Current Protocols in Immunology (JE Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: a practical approach (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 antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (edited by M. Zanetti and JD Capra, Harwood Academic Publishers, 1995).

[0228] Without further elaboration, it is believed that those skilled in the art can make the most of the present invention based on the above description. Therefore, the following specific embodiments should be interpreted as being merely illustrative and not limiting the remainder of the present disclosure in any way. For the purposes or themes cited herein, all publications cited herein are incorporated by reference.

[0229] Example

[0230] Example 1: Phase 1 open-label, multicenter study of the safety, pharmacokinetics (PK), and antitumor activity of G9.2-17 (IgG4) in patients with relapsed / refractory acute myeloid leukemia (AML) or relapsed / refractory high-risk myelodysplastic syndrome (MDS)

[0231] G9.2-17 (IgG4) is a fully human immunoglobulin gamma (IgG) 4 monoclonal antibody (mAb) targeting the carbohydrate recognition domain 2 (CRD2) of galectin-9 (gal-9) protein, and is being developed for the treatment of patients with recurrent and refractory solid tumors and hematological malignancies. Gal-9 plays a key regulatory role in anti-tumor immune responses. Overexpressed and / or secreted Gal-9 in many cancer types (solid tumors and hematological malignancies) acts as an immunosuppressant, granting tumor cells immune privileges and disabling immune-mediated cancer attacks by regulating macrophages, T cells, natural killer (NK) cells, and bone marrow-derived suppressor cells (MDSCs), as well as hindering the susceptibility of cancer cells to cytotoxic T cell-induced death. G9.2-17 (IgG4) has been designed to induce gal-9 blocking and interfere with the immunosuppressive function of gal-9, which can lead to immune reactivation and inhibition / control of tumor growth.

[0232] G9.2-17 (IgG4) is expected to be beneficial in the treatment of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), such as relapsed and / or refractory AML or MDS, at least in the following aspects:

[0233] ·Good safety profile.

[0234] Potential immune modulation in the periphery and bone marrow to be monitored by PD and efficacy assessments. Clinical improvement of patients.

[0235] The research schematic is shown in Figure 1 A summary of the study objectives and endpoints is shown in Table 3 below.

[0236] Table 3. Study objectives and endpoints

[0237]

[0238]

[0239] I. Research objectives

[0240] Main Objectives

[0241] Determine the safety and tolerability of G9.2-17 (IgG4) as a single agent

[0242] Confirm the dose of G9.2-17 (IgG4) in Phase 2 (R2PD)

[0243] Determine the incidence of DLTs at each dose level during Cycle 1 (28 days)

[0244] Secondary Objectives

[0245] Determine the preliminary efficacy of G9.2-17 (IgG4) as a monotherapy

[0246] Characterization of the PK profile of G9.2-17 (IgG4)

[0247] Exploratory goals

[0248] Study pharmacodynamic markers, including immunological and molecular changes in peripheral blood, bone marrow and serum Gal-9

[0249] ·Evaluation of the immunogenicity of G9.2-17 (IgG4)

[0250] II. Study End Points

[0251] Primary End Point

[0252] Safety parameter evaluation, including adverse events, vital sign measurements, clinical safety laboratory tests, 12-lead electrocardiogram (ECG), echocardiogram / cardiac ultrasound (ECHO), physical examination

[0253] Determination of the biologically effective dose of G9.2-17 (IgG4)

[0254] Determination of the maximum tolerated dose (MTD) of G9.2-17 (IgG4)

[0255] Determination of one or more RP2D doses for G9.2-17 (IgG4)

[0256] Evaluation of DLT during the first cycle

[0257] Secondary End Points

[0258] Secondary endpoints to determine primary efficacy (disease response) are defined in Tables 4 and 5 below:

[0259] Table 4. AML Efficacy Endpoints and Definitions

[0260]

[0261]

[0262] Table 5. MDS Efficacy Endpoints and Definitions

[0263]

[0264]

[0265] PK endpoint

[0266] Secondary PK endpoints included evaluation of PK parameters of G9.2-17 (IgG4) including but not limited to the area under the curve [AUC 0-168h ], maximum observed serum concentration [C max ] and implement C max The time [T max ]) and trough serum concentration [C trough ].

[0267] Exploratory End Points

[0268] The exploratory endpoints are shown in Tables 6 and 7 below.

[0269] Table 6. Exploratory Efficacy Endpoints for AML

[0270]

[0271] Table 7. Exploratory Efficacy Endpoints for MDS

[0272]

[0273] Exploratory immune and molecular changes in peripheral blood and bone marrow endpoints

[0274] Serum / plasma cytokine multiplexing

[0275] Flow cytometry analysis of whole blood immune profiles

[0276] Gene array from whole blood

[0277] ELISA method for determination of Gal-9 in serum

[0278] Exploratory immunogenicity endpoints

[0279] ·Evaluation of ADA

[0280] III. Study Design

[0281] This is an open label, non-randomized, multicenter, Phase 1, dose escalation study for patients with AML whose disease is relapsed / refractory to at least one prior line of treatment with or without allogeneic stem cell transplantation, or for patients with documented relapsed / refractory, high-risk MDS diagnosis after at least one line of treatment, and no standard therapy that can provide clinical benefit is available for the patient. A dose escalation design based on a 4+2 algorithm (Lin et al., Biostatistics, 2001; Wheeler et al., PLoS One, 2016) is used to establish DLT and help identify RP2D. This study was conducted at approximately 15 sites in the United States. The estimated study duration is 24 months. Survival follow-up continues for up to 2 years.

[0282] Study design Figure 1 The visit schedule and planned assessments for each visit are detailed in Table 8.

[0283] Dose escalation and de-escalation

[0284] Treatment period

[0285] This study covers the following time periods:

[0286] Screening period : Up to 2 weeks before first dose (Day -14 to Day -1)

[0287] Treatment period : 28-day treatment cycle as presented in the Schedule of Assessments (SoA) (Table 8) Late treatment : 30 days after last treatment (end of treatment visit / early termination visit)

[0288] Follow-up period : Long-term follow-up up to 2 years (every 3 months)

[0289] Dose escalation

[0290] A dose escalation design based on the 4+2 algorithm (Lin et al., Biostatistics, 2001; Wheeler et al., PLoS One, 2016) was used to determine the MTD or a biologically active dose that can be used to help identify the RP2D dose. The biologically effective dose is defined as the dose experienced by 0-100% of the 6 patients in the cohort:

[0291] For AML: molecular residual disease (MRD)-negative status or reduction of bone marrow blasts to ≤5% with absence of circulating blasts with Auer bodies.

[0292] For MDS: Minimum duration of transfusion independence of 8 weeks or decrease in bone marrow myeloblasts to ≤5%.

[0293] Up to 6 patients per treatment cohort 1-5 received weekly (QW) 60-minute intravenous (IV) infusions of G9.2-17 (IgG4) on Days 1, 8, 15, and 22 of each 28-day cycle, starting at a dose of 2.0 mg / kg.

[0294] Patients assigned to a specific dose-escalation cohort received the corresponding study dose for that cohort. They received study drug until disease progression, unacceptable toxicity, or withdrawal from the study for other reasons. Patients who withdrew for reasons other than toxicity or tolerability issues during the first treatment cycle were replaced.

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303] A total of 5 dose levels were evaluated in a 4+2 design, starting at 2.0 mg / kg QW:

[0304] Dose escalation cohort 1 = 2.0 mg / kg QW (maximum n = 6)

[0305] Dose escalation cohort 2 = 4.0 mg / kg QW (maximum n = 6)

[0306] Dose escalation cohort 3 = 7.5 mg / kg QW (maximum n = 6)

[0307] Dose escalation cohort 4 = 12.0 mg / kg QW (maximum n = 6)

[0308] Dose escalation cohort 5 = 16.0 mg / kg QW (maximum n = 6)

[0309] One cycle (28 days) is 4 doses of G9.2-17 (IgG4) administered.

[0310] Based on analysis of patient safety data regarding the occurrence of DLTs, other relevant safety and available PK data from previous cohorts, the next higher dose cohort will be opened. Skipping of dose levels will not be allowed.

[0311] Patients treated before the identification of RP2D are allowed to gradually increase the dose to the highest dose level once they have spent the 28-day DLT period in the group they were initially selected for. Dose escalation may not occur in the middle of the cycle. Patients can continue to increase the dose to the highest approved dose level until they are discontinued due to toxicity or disease progression or for other reasons (e.g., the patient chooses to discontinue the study). They may only be increased after completing a full cycle of their current dose level rather than an intermediate cycle.

[0312] In the absence of confirmed clinical benefit, the administration time of the study drug is limited to 6 cycles, i.e. 6 months. If the patient does not achieve the minimum value of PR as a clinical benefit, the patient will be stopped from studying the drug even in the absence of unacceptable toxicity. If the patient has demonstrated at least PR with clinical benefit within 6 cycles (6 months) of starting the study drug, the patient can continue to be administered for more than 6 months until disease progression, patient refusal or unacceptable toxicity.

[0313] If a DLT occurred in any patient during the first 28 days of treatment, study drug administration to that patient was permanently discontinued.

[0314] For patients who develop toxicity (including IMARs) outside the DLT window, dose reductions are allowed only if the investigator assesses that clinical benefit has been achieved and may continue to be achieved at a lower dose level of G9.2-17 (IgG4). The dose of G9.2-17 (IgG4) is initially reduced by 50% and may be further reduced by 50% as defined by the dose modification guidelines provided in the protocol. No further dose reductions are allowed. In the event that a patient withdraws from the first treatment cycle only for reasons other than toxicity or tolerability issues, they will be replaced until the MTD has been identified.

[0315] Dose reduction (Cohort-1 only)

[0316] In Cycle 1 of Cohort 1 (2.0 mg / kg G9.2-17(IgG4) only), a new Cohort 1 may be opened with a lower dose of G9.2-17(IgG4) as follows:

[0317] If 2 or more patients achieve DLT, a lower dose cohort-1 may be opened at a lower dose.

[0318] If any signs of clinical benefit are observed in Cohort 1 based on secondary exploratory endpoints or reductions in other disease activity markers (such as a ≥50% reduction in bone marrow blasts compared to pre-treatment), a lower dose Cohort 1 may be added for exploring the dose response relationship, which will be incorporated into dose selection for further development. The selection of a lower dose will be based on available PK, PD data, and disease activity markers.

[0319] Complete dose escalation based on the 4+2 algorithm

[0320] Dose escalation based on the 4+2 algorithm was completed when one or more of the evaluated G9.2-17 (IgG4) doses was identified as one or more of the RP2D doses.

[0321] Dose-limiting toxicity criteria

[0322] DLTs evaluated in this trial were defined as clinically significant hematologic and / or non-hematologic AEs or abnormal laboratory values ​​that were not clearly due to underlying leukemia or external causes occurring during the first cycle (28 days) of the study. Any patient who experienced a DLT during the first 28 days of treatment was permanently discontinued from study drug.

[0323] A DLT is a toxicity that meets any of the following criteria:

[0324] Any death not clearly due to the underlying leukemia or external causes

[0325] Indications for potential drug-induced liver injury (Hy's Law cases), as follows:

[0326] ○ ALT or AST > 3x upper limit of normal (ULN) with confirmation by repeat testing 24 hours later, and

[0327] o Serum total bilirubin (TBL) > 2×ULN, with confirmation by repeat testing after 24 hours.

[0328] ○ No alternative explanation can be found for the elevated TBL and / or aspartate aminotransferase (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 syndrome, disease progression, or other medications that could cause the observed effects

[0329] All grade ≥4 hematologic toxicities that were not clearly attributable to the underlying leukemia or external causes and lasted for >7 days. Events beyond the 28-day DLT monitoring period were also counted. All grade 3-5 non-hematologic toxicities that were not clearly attributable to the underlying leukemia or external causes, except for the following:

[0330] Grade 3 fatigue, asthenia, pyrexia, anorexia (Grade 3 anorexia can be ruled out only if it does not lead to hospitalization, tube feeding, or use of total parenteral nutrition), or constipation.

[0331] Grade 3 nausea, vomiting, or diarrhea not requiring tube feeding, total parenteral nutrition, or requiring or prolonging hospitalization.

[0332] ■Infection, bleeding, or other expected immediate complications of cytopenias due to active underlying leukemia.

[0333] Grade 3 infusion reactions, including cytokine release syndrome (CRS), if successfully managed and resolved within 72 hours.

[0334] Grade 3 or 4 TLS if it is clinically successfully managed and resolves within 7 days without end-organ damage.

[0335] ■Grade 3 or 4 isolated electrolyte abnormalities lasting < 72 hours will not be considered a DLT.

[0336] End of study

[0337] The end of the study was defined as the point at which the RP2D or dose had been identified, all patients had received treatment with G9.2-17(IgG4) until confirmed disease progression, or withdrawal from treatment for other reasons, and all patients had completed OS follow-up.

[0338] Patients were followed up for OS up to 2 years after the last dose of G9.2-17 (IgG4).

[0339] The end of the study was defined as the date of the last patient's last visit.

[0340] Trial stopping rules

[0341] When one dose or intermittent dose in groups 1 to 5 is determined as RP2D, the dose escalation phase of the 4+2 design is completed. The RP2D is determined based on MTD, DLT, PK, PD and additional safety data observed at each dose level and any other factors that the sponsor may consider. If the lower limit of the Agresti and Coull binomial CI of the lowest study dose level exceeds the target DLT rate, the trial is stopped for safety reasons (Agresti et al., The American Statistician, 1998).

[0342] IV. Study Group

[0343] Inclusion criteria

[0344] 1. Patients aged ≥18 years old when obtaining informed consent.

[0345] 2. Patients with morphologically documented primary or secondary AML whose disease is relapsed / refractory to at least one prior line of treatment with or without allogeneic stem cell transplantation according to World Health Organization (WHO) criteria (Arber et al., Blood, 2016) and for whom no standard therapy is available that provides clinical benefit or for whom the available standard of care is reduced.

[0346] 3. Patients diagnosed with high-risk myelodysplastic syndrome (MDS) based on the Revised International Prognostic Scoring System (IPSS-R) (Greenberg et al., Blood, 2012) after at least one line of treatment (whose disease is relapsed / refractory) and for whom there is no standard therapy that provides clinical benefit.

[0347] 4. The patient is able to understand, sign, and date the written informed consent at the Screening Visit prior to any protocol-specific procedures.

[0348] 5. The patient is able and willing to comply with study procedures according to the protocol, including bone marrow biopsy.

[0349] 6. Patients have an ECOG performance status of ≤2.

[0350] 7. The patient interval from prior treatment to the time of study drug administration was at least 2 weeks for cytotoxic agents (except for hydroxyurea given for cytoreduction) or at least 5 half-lives for prior experimental or non-cytotoxic agents. A washout period shorter than the specified washout period may be considered after discussion with the medical supervisor if the patient has recovered from any clinically relevant safety issues and has recovered to ≤ Grade 1 toxicity from prior treatment.

[0351] 8. Patients must meet the following criteria as shown in clinical laboratory tests:

[0352] a. WBC count <25,000 / uL at first dose.

[0353] b. Aspartate aminotransferase or alanine aminotransferase ≤3×ULN (≤5.0×ULN if considered to be due to leukemic involvement).

[0354] c. Total bilirubin ≤2×ULN (≤3×ULN if thought to be due to leukemic involvement or Gilbert's syndrome).

[0355] d. Creatinine clearance ≥60mL / min.

[0356] 9. The patient's minimum life expectancy is ≥ 3 months.

[0357] 10. Female patients must be: i) infertile: postmenopausal (defined as no menstruation for at least 1 year) before screening, or have surgically proven infertility or post-hysterectomy status (at least 1 month before screening); ii) if of childbearing potential, must have a negative serum or urine pregnancy test at screening, and must use 2 forms of birth control (at least one of which must be a barrier method) at the start of screening and throughout the study and within 90 days after the final study drug administration.

[0358] 11. Female patients should not breastfeed during screening, during the study, and within 90 days after the final study drug administration.

[0359] 12. Female patients are not allowed to donate eggs during screening and throughout the study and within 90 days after the final study drug administration.

[0360] 13. Male patients of childbearing potential and their female spouses / partners must be using highly effective contraception consisting of 2 forms of birth control (one of which must be a barrier method) at the time of screening.

[0361] Exclusion criteria

[0362] Subjects who met any of the following exclusion criteria were not eligible for inclusion in the study:

[0363] 1. The patient was diagnosed with acute promyelocytic leukemia (APL).

[0364] 2. Patients with active malignancies other than AML. Participants with a history of adequately treated malignancies do not undergo or require anticancer systemic therapy (i.e., chemotherapy, immunotherapy, small molecule inhibitors, radiotherapy, or surgery) during the course of the study. Participants receiving adjuvant therapy (such as hormone therapy or zoledronic acid or denosumab) are eligible.

[0365] 3. Patients have persistent non-hematologic toxicity of ≥ Grade 2 (CTCAE v5.0), symptoms and objective findings from previous AML treatment (including chemotherapy, kinase inhibitors, immunotherapy, experimental drugs, radiation, hematopoietic stem cell transplantation [HSCT], or surgery).

[0366] 4. Patients who have received HSCT and meet any of the following criteria: HSCT within 6 months prior to the first study dose; persistent non-hematological toxicity of grade ≥ 2 related to transplant donor lymphocyte infusion.

[0367] 5. The patient has active graft-versus-host disease (GVHD) and is receiving immunosuppressive therapy for GVHD. Calcineurin inhibitors must be discontinued for at least 4 weeks before screening.

[0368] 6. Patients with symptomatic central nervous system (CNS) involvement from leukemia or other CNS diseases associated with underlying and secondary effects of malignancy.

[0369] 7. The patient suffers from disseminated intravascular coagulation (DIC).

[0370] 8. Patients underwent major surgery within 4 weeks before the first study dose.

[0371] 9. Patients have received radiotherapy within 4 weeks before receiving the first study dose.

[0372] 10. Patients with New York Heart Association (NYHA) grade 3 or 4 congestive heart failure, or patients with a history of NYHA grade 3 or 4 congestive heart failure, unless echocardiography or multi-gated acquisition (MUGA) scan performed within 3 months before the start of the study showed a left ventricular ejection fraction (LVEF) ≥ 45%.

[0373] 11. Patients have any clinically important abnormalities in: i) rhythm; ii) conduction; iii) morphology on resting ECG (e.g., complete left fascicular block, third-degree heart block, second-degree heart block, PR interval >250 msec); and / or iv) any factors that increase the risk of QTc prolongation or arrhythmic events, such as congenital long QT syndrome or family history of long QT syndrome, which, in the investigator's judgment, are not medically managed.

[0374] 12. The patient is known to have symptomatic active infection, including any established active COVID-19 infection.

[0375] 13. The patient is known to have human immunodeficiency virus infection.

[0376] 14. Patients have known active hepatitis B or C, or other active liver disease.

[0377] 15. The researcher believes that the patient has any condition that makes him or her unsuitable for participating in the study.

[0378] 16. Any other medical, psychological, or social condition that the investigator believes may interfere with study participation or compliance or jeopardize patient safety.

[0379] 17. The patient is unwilling or unable to comply with the regimen.

[0380] 18. Live vaccines administered within 30 days prior to starting treatment.

[0381] 19. History of severe allergic reaction to any monoclonal antibody (mAb) and / or its excipients.

[0382] 20. Known inherited or acquired bleeding disorders.

[0383] 21. Active bleeding.

[0384] 22. Patients with new-onset thrombosis, embolism, cerebral hemorrhage or other diseases or medical history within one year before enrollment.

[0385] 23. Drug abuse or chronic alcohol abuse that may affect the assessment results.

[0386] 24. Active autoimmune disease requiring systemic treatment (except autoimmune hypothyroidism which can be treated with synthetic thyroid hormone).

[0387] V. Description of the study evaluation

[0388] Demographic and other screening assessments

[0389] At Screening, patient demographic data will be collected. These data include age, gender, race, and ethnicity. Safety assessments as part of the Screening assessments are described below.

[0390] Medical history

[0391] Medical history included oncology history, surgery / transplant history, radiation therapy history, and COVID-19 history and testing.

[0392] Personal medical history, including previous treatments / surgeries, records of any in situ implants or past implants, previous and / or current use of medical devices, concomitant medications (name, indication, dose, route, start and end dates, dose modifications (if any) and reason), pre-existing symptoms and AEs), at-risk genetic diseases based on family history and a complete family history best known to the patient.

[0393] Records of any dental treatment carried out in the past 12 months

[0394] For patients who had previously had a resected pancreatic adenocarcinoma, record whether the primary tumor was located in the head, body, or tail of the pancreas.

[0395] Bowel habits / typical frequency and consistency

[0396] Record any dietary requirements or preferences (e.g., following a specific diet: intermittent fasting, ketogenic diet, etc.)

[0397] Record past and present allergies (allergens, severity).

[0398] ECHO / MUGA

[0399] ECHO and / or MUGA will be obtained at the time points indicated in the SoA (Table 8). Assessments will be repeated every 3 months if clinically indicated.

[0400] ECOG

[0401] ECOG performance status was assessed at the time points indicated in the SoA (Table 8) using the following ratings:

[0402] Level 0: Fully active, able to perform all pre-disease manifestations without restriction

[0403] Level 1: Vigorous physical activity is limited, but the patient can walk and perform light or sedentary work, such as light housework or office work

[0404] Level 2: Ambulatory and able to take care of themselves, but unable to perform any work activities. Able to get up and walk, awake more than 50% of the time

[0405] Level 3: Limited self-care, confined to bed or chair, awake more than 50% of the time

[0406] Level 4: Total disability. Unable to perform any self-care. Totally confined to bed or chair

[0407] Level 5: Death

[0408] Efficacy evaluation

[0409] Efficacy evaluation (secondary)

[0410] Efficacy assessments were derived from updated International Working Group (IWG) and key opinion leader recommendations for AML and MDS. Efficacy endpoints and definitions for AML and MDS are provided here. All endpoints for AML and MDS were derived from 4 types of assessments collected according to the SoA (Table 8):

[0411] Hematology (routine component of clinical laboratory evaluation; see below)

[0412] ·MRD

[0413] MRD included central multiparameter flow cytometry (MFC) in blood for immunophenotypic markers and was performed according to SoA (Table 8).

[0414] Bone marrow biopsy / aspiration

[0415] Bone marrow aspirates were subjected to MRD analysis by RT-qPCR / NGS molecular genetic analysis according to SoA (Table 8), and also included MFC. Bone marrow biopsies were evaluated locally to assess the extent and nature of bone marrow cellularity.

[0416] After cycle 3, it was permitted not to complete a bone marrow biopsy / aspiration if it was medically not feasible.

[0417] Hematology blood smear

[0418] Using hematologic blood films (myeloperoxidase [MPO], Sudan black, alpha-naphthyl acetate esterase [NAE], and periodic acid Schiff [PAS] staining) to measure:

[0419] ■% of mother cells

[0420] ■Morphology of blasts with particular attention to the presence of Auer bodies (typical in AML)

[0421] ■Hemocyte dysplasia

[0422] Hematology Blood films will be performed locally according to SoA (Table 8).

[0423] Safety Assessment

[0424] Adverse Events

[0425] See the disclosure below.

[0426] Clinical laboratory evaluation

[0427] Blood samples were collected from patients for routine clinical laboratory tests (approximately 5 mL at each time point) according to the SoA (Table 8); additional tests could be performed at any time during the study if necessary based on the investigator's needs or local regulatory requirements. The protocol-specific requirements for inclusion or exclusion of patients are described in detail above.

[0428] Clinical laboratory parameters were analyzed in the local laboratory at the study center. Laboratory evaluations to be completed included hematology and serum chemistry and were defined as follows:

[0429] Serum chemistry: including glucose, total protein, albumin, electrolytes [sodium, potassium, chloride, magnesium, phosphorus], calcium, bilirubin (total, direct), SGPT (ALT) or SGOT (AST), alkaline phosphatase, gamma glutamyl transferase (γGT), lactate dehydrogenase (LDH), creatinine, hemoglobin A1c (HgbA1c) (only if there is a history of type 1 or type 2 diabetes), blood urea nitrogen, creatine phosphokinase (CPK)

[0430] ○ In addition, thyroid stimulating hormone (TSH), fT4, lipase, amylase, PTH, FSH, luteinizing hormone (LH), free cortisol at designated visits

[0431] ○ Fasting blood glucose will be assessed only when clinically indicated

[0432] Hematology: including differential complete blood count, platelets, hemoglobin

[0433] Coagulation: including PT and PTT, activated partial thromboplastin time (APTT) and INR (if anticoagulants are allowed), C-reactive protein (CRP), and troponin

[0434] Urinalysis: A urine sample will be collected from the patient for routine urinalysis. The urinalysis will include color, appearance, and specific gravity test strips, protein, leukocyte esterase, glucose, ketones, urobilinogen, nitrites, WBC, RBC, and pH, as well as a urine culture if the patient is clinically symptomatic.

[0435] Abnormal laboratory findings related to the underlying disease were not considered clinically significant unless judged to be more severe than expected for the participant's condition.

[0436] All laboratory tests considered to have clinically significant abnormal values ​​should be repeated during study participation or within 30 days after the last dose of study drug until such values ​​return to normal or baseline or are no longer considered clinically significant.

[0437] If clinically significant values ​​do not return to normal / baseline or Grade 1 within a period of time, the etiology should be determined.

[0438] All protocol-required laboratory tests must be performed according to the laboratory manual and SoA (Table 8).

[0439] If laboratory values ​​for non-protocol-mandated laboratory tests performed at the institution's local laboratory require a change in the participant's management or are considered clinically significant (e.g., SAE or AE or dose modification), these results must be recorded.

[0440] 12-lead ECG

[0441] A 12-lead ECG was obtained as outlined in the SoA (Table 8) using an ECG machine that automatically calculated heart rate and measured heart rate, PR interval, QRS duration, the time distance from the start of the QRS complex to the end of the T wave (QT) interval on the ECG trace, and the QTcF interval.

[0442] Vital signs

[0443] Vital signs will be measured in the supine position after 5 minutes of rest according to the SoA (Table 8) and will include temperature, blood pressure (systolic and diastolic), heart rate, and respiratory rate.

[0444] To monitor reactions to the G9.2-17 (IgG4) infusion on Day 1 of Cycle 1, vital signs must be recorded every 15 minutes during the 1-hour infusion of the antibody and every 30 minutes for 1 hour after the infusion, and every hour thereafter, if applicable.

[0445] Physical examination

[0446] Medical and physical examinations must be performed by a qualified physician, nurse practitioner, or physician assistant and should include a thorough review of all body systems. In addition, height will be measured only at screening to determine body surface area. Weight will be included at all scheduled examination times. Neurological examinations will only be performed in patients with stable and / or pre-treated brain metastases.

[0447] Monitoring and treatment of tumor lysis syndrome

[0448] Implement the following preventive measures to monitor for and treat potential TLS:

[0449] To adequately monitor TLS-related laboratory parameters during the first week of G9.2-17(IgG4) treatment, a second blood draw will be collected on Cycle 1 Day 2 in addition to the scheduled blood draw on Cycle D1 Day 1.

[0450] Prior to each G9.2-17(IgG4) infusion, patients may be assessed for risk of TLS and, if clinically indicated (e.g., TLS experienced on a prior treatment regimen), treated according to institutional standards of care, such as with appropriate hydration and uric acid stabilizers, prior to initiating G9.2-17(IgG4) infusion.

[0451] • In cases where subjects have a high disease burden and higher risk of TLS, it may also be appropriate to keep / admit them for additional monitoring after G9.2-17 (IgG4) administration.

[0452] Pharmacokinetics

[0453] If possible, the following serum PK parameters will be calculated for G9.2-17 (IgG4):

[0454] AUC 0-168h

[0455] ■C max

[0456] ■T max

[0457] ■ Serum concentration vs. time curve

[0458] Approximately 5 mL of blood samples were collected and processed to serum at each time point as specified in the SoA (Table 8). A complete list of PK parameters will be provided in the statistical analysis plan.

[0459] Pharmacodynamic biomarkers

[0460] Planned time points for biomarker assessments are provided in the SoA (Table 8); after 6 months of treatment, sampling can be reduced to every 3 cycles. The following samples are required for biomarker studies and will be collected from all participants in this study as specified in the SoA:

[0461] • Blood samples, collected prior to study drug administration (approximately 15 mL pre-dose).

[0462] • Samples will be tested for PD biomarkers (by flow cytometry, ELISA, IHC or multiplex phenotyping) to evaluate their association with observed clinical responses to G9.2-17 (IgG4) using validated assays.

[0463] The following biomarkers are planned to be evaluated in this study (note that the following list includes prospective biomarkers but is not limited to these):

[0464] Serum / plasma cytokine multiplexing

[0465] Flow cytometry analysis of whole blood immune profiles

[0466] Gene array from whole blood

[0467] ELISA method for determination of Gal-9 in serum

[0468] Immunogenicity assessment

[0469] Blood samples (approximately 3 mL) were collected from all participants and processed to serum according to the SoA (Table 8). In addition, serum samples were collected from patients who discontinued study drug or withdrew from the study at the end-of-treatment / early termination visit.

[0470] VI. treat

[0471] Preparation, handling and storage

[0472] G9.2-17 (IgG4) is provided in single-use vials. G9.2-17 (IgG4) liquid drug product is diluted to the target dose prior to administration. All dilutions should be performed in a controlled and sterile environment (preparation of patient doses and delivery via approximately 60-minute IV infusion).

[0473] Dosage and Administration

[0474] All patients received G9.2-17 (IgG4). G9.2-17 (IgG4) was administered via IV infusion over approximately 60 minutes, QW. Infusion was started and infusion duration was recorded.

[0475] According to a 4+2 design, patients received G9.2-17 (IgG4) as a single agent, with doses increasing sequentially starting from 2.0 mg / kg.

[0476] Patients who experienced a DLT in Part 1 did not resume treatment.

[0477] Dosage Administration

[0478] If an infusion-related reaction is encountered, interrupt the infusion and administer relevant medications (e.g., antihistamines, antiemetics, steroids, antipyretics, beta-blockers, etc.) if clinically indicated. If resumption of the infusion is deemed appropriate, resume at a slower infusion rate. When the infusion is restarted, the new rate and the stop and restart times of the infusion will be recorded.

[0479] For subsequent cycles in the same patient, use appropriate premedication (antihistamines, antiemetics, steroids, antipyretics, beta-blockers, etc. as clinically indicated) and consider using a slower infusion rate.

[0480] Dosage adjustment

[0481] The decision to proceed to the next dose level of G9.2-17 (IgG4) is based on safety, tolerability and potential preliminary PK data obtained in at least 4 patients at the previous dose level. The dosing schedule can also be adjusted based on the PK data obtained.

[0482] Detailed dosage modification instructions can be found in Tables 9 and 10:

[0483] Table 9. Management of immune-mediated adverse reactions (IMARs) caused by G9.2-17 (IgG4)

[0484]

[0485]

[0486]

[0487]

[0488] Table 10. Recommended dose modifications for G9.2-17 (IgG4) (AEs outside the DLT window and other than IMARs)

[0489]

[0490]

[0491] Dose delay

[0492] If any clinically significant AE of Grade ≥ 3 that may be related to or attributable to one or more study drugs occurs, it will be discussed prior to continuing dosing. Dose delays may be required for Grade ≥ 3 AEs, if judged appropriate.

[0493] Dose reduction

[0494] No dose reductions were permitted for any patient being evaluated for DLT (within the 28-day DLT window).

[0495] Dose reductions were permitted only if clinical benefit was achieved and was likely to continue to be achieved with the dose reduction, see Table 9 (for IMARs) or Table 10 (for other AEs).

[0496] Dose Adjustment for IMAR

[0497] If IMAR occurs, the dosing guidelines for G9.2-17(IgG4) are shown in Table 9 (IMAR).

[0498] All relevant medical investigations / tests should be performed to confirm that the adverse event is an IMAR.

[0499] Discontinuation of study drug

[0500] In rare cases, it may be necessary for a patient to permanently discontinue study drug. If study drug is permanently discontinued for reasons other than disease progression, and the patient is not treated with other anticancer therapy, continue to evaluate the patient for disease progression for up to 2 years. See Table 8 (SoA) for data to be collected at discontinuation of study drug and follow-up and any further evaluations to be completed.

[0501] Investigators must make every effort to keep patients on study treatment until one of the reasons for discontinuation of study treatment is met (disease progression, development of study drug-related toxicity, withdrawal of consent). If a patient has radiographic progression but no clear clinical progression and no alternative treatment is started, the patient may continue to receive study treatment at the investigator's discretion. However, if a patient has clear clinical progression without radiographic progression, study treatment should be discontinued at the investigator's discretion and the patient should be advised of available treatment options.

[0502] Patients may stop before disease progression for one of the following reasons:

[0503] DLT

[0504] AEs requiring discontinuation of one or more study treatments occurred / recurred outside the DLT window

[0505] · Occurrence / recurrence of IMAR requiring discontinuation of one or more study treatments

[0506] PureTech Health, LLC terminates study

[0507] Concurrent illness or medical condition that would preclude further administration of treatment or that could jeopardize the patient's safety if the study treatment were continued

[0508] Due to the patient or investigator's decision

[0509] ·Pregnant

[0510] Use of non-protocol anticancer therapy

[0511] Significant deviations from the regimen on the part of the patient (including lack of compliance)

[0512] The researcher seriously violated the protocol

[0513] The reason for a patient to discontinue study treatment should be recorded in the CRF. If a patient discontinues study treatment due to toxicity, "dose limiting toxicity" or "adverse event" should be recorded as the primary reason for withdrawal. If a patient discontinues the study prematurely at any time due to an AE or SAE, the patient must be followed until it resolves to Grade 2 or less, unless improvement is unlikely due to the underlying disease.

[0514] Concomitant therapy

[0515] Each patient's previous and concomitant medications, including vaccines and complementary therapies / supplements, will be recorded at each scheduled visit (Table 8).

[0516] Any medications or vaccines that the participant is taking at the time of enrollment or receives during the study (including over-the-counter or prescription medications, recreational drugs, vitamins, and / or herbal supplements) must be recorded along with the following information:

[0517] Reasons for use

[0518] Date of application, including start and end dates

[0519] Dosage information, including dose and frequency

[0520] Permitted medications / therapies

[0521] The following concomitant medications are permitted:

[0522] In patients on study with WBC counts greater than 25,000 / uL, hydroxyurea was allowed for the first 2 cycles, concurrently with G9.2-17 (IgG4), according to standard institutional practice.

[0523] Investigators should follow standard institutional practice when addressing any of the following:

[0524] Infusion reactions

[0525] Tumor lysis syndrome

[0526] ■Cytokine release syndrome.

[0527] Continue bisphosphonate therapy (e.g., zoledronic acid) or denosumab to treat bone metastases that have been stable for at least 6 months prior to treatment (C1D1).

[0528] Use inhaled corticosteroids and mineralocorticoids (e.g., fludrocortisone), topical steroids, intranasal steroids, intra-articular and ophthalmic steroids.

[0529] · Prophylactic or therapeutic use of anticoagulants.

[0530] Vaccination for COVID-19, common influenza, and / or other common clinically needed indications (e.g., tetanus, pneumococcal, HBV, etc.) prior to or during the study period is permitted. The timing and type of vaccination must be recorded.

[0531] Closely monitor sensitive narrow therapeutic index CYP3A4 substrates such as, but not limited to, alfentanil, dihydroergotamine, ergotamine, fentanyl, pimozide, and quinidine, and adjust dose as needed, provided that G9.2-17 (IgG4) can effectively inhibit TGF-β and IL-10 in vitro. Reducing cytokine levels from patient baseline levels may alter CYP expression / activity and exposure to sensitive CYP3A4 substrates. Note: Immunosuppressive CYP3A4 substrates (such as tacrolimus, cyclosporine, everolimus, and sirolimus) are contraindicated.

[0532] Prohibited medications / therapies

[0533] The following drugs were not allowed to be used in this study:

[0534] • Concomitant administration of study agent for any indication, except G9.2-17 (IgG4).

[0535] Systemic immunosuppressive therapy, including but not limited to cyclophosphamide, azathioprine, methotrexate, thalidomide, tacrolimus, cyclosporine, everolimus, sirolimus, and anti-TNF agents. However, patients were allowed to take acute, low-dose systemic immunosuppressive drugs (e.g., ≤10 mg / day of prednisone or equivalent).

[0536] Replacement therapy (eg, thyroxine, insulin, physiologic corticosteroid replacement [eg, ≤10 mg / day prednisone equivalents for adrenal or pituitary insufficiency]) is not considered a form of systemic treatment.

[0537] VII. Adverse Events

[0538] definition

[0539] Adverse Events

[0540] An AE is any event, side effect, or other untoward medical occurrence in humans associated with the use of a medicinal product, whether or not considered to be causally related to such treatment. Thus, an AE can be any unfavorable and unexpected sign (and can include clinically significant abnormal laboratory findings), symptom, or disease temporally associated with the use of a medicinal product, whether or not considered to be related to the medicinal product.

[0541] Events that meet the definition of AE include:

[0542] Exacerbation of a chronic or intermittent pre-existing condition, including an increase in the frequency and / or intensity of the condition

[0543] New conditions detected or diagnosed during the reporting period after study drug administration, even if the condition may have existed before the start of the study

[0544] Signs, symptoms, or clinical sequelae of suspected interactions

[0545] Suspected signs, symptoms, or clinical sequelae of overdose of study drug or concomitant medication (overdose itself will not be reported as an AE / SAE)

[0546] Events that do not meet the definition of an AE include:

[0547] Medical or surgical procedures (e.g., endoscopy, appendectomy); the condition leading to the procedure should be reported as an AE if the AE criteria are met

[0548] · Situation in which no adverse medical events occurred (e.g., social and / or convenience of hospital admission)

[0549] Expected day-to-day fluctuations in pre-existing diseases or conditions that were present or detected at study entry and not exacerbated

[0550] If evidence of an AE is found through reports or observations, the Investigator or designee will further evaluate and record the following information:

[0551] Onset time and wear-off time

[0552] Severity

[0553] Causality / relationship to study treatment

[0554] Actions taken on study drugs

[0555] ·result

[0556] Serious adverse events

[0557] An SAE is any event that meets any of the following criteria:

[0558] Causes death.

[0559] Life-threatening.

[0560] · Requires hospitalization or prolongation of existing hospitalization.

[0561] · Resulting in persistent or severe disability / incapacity.

[0562] Congenital anomalies / birth defects.

[0563] An important medical event that is not likely to result in death, be life-threatening, or require hospitalization. An event is considered a SAE when, based on appropriate medical and scientific judgment, the event could endanger the subject and may require medical or surgical intervention to prevent one of the above outcomes. Examples of such events include: intensive treatment of allergic bronchospasm, blood dyscrasias, or seizures in the emergency room or at home that does not result in hospitalization, or the development of drug dependence or drug abuse.

[0564] Suspected unexpected serious adverse reactions

[0565] Suspected, unexpected serious adverse reactions (SUSARs) are adverse events that are both unexpected (i.e., not listed in the safety reference information for the study drug) and meet the definition of a serious adverse drug reaction, but whose specificity or severity is inconsistent with that noted in the safety reference information (i.e., the IB for the study drug).

[0566] Clinical laboratory abnormalities and other abnormalities evaluation

[0567] Abnormal laboratory results (e.g., clinical chemistry, hematology, and urinalysis) or other abnormal assessments (e.g., ECG or vital signs) judged to be clinically significant will be recorded as AEs and SAEs if they meet the definition of an AE or SAE. Clinically significant abnormal laboratory findings or other abnormal assessments detected during the study or present at screening and significantly worsened after the start of the study will be reported as AEs or SAEs. However, clinically significant abnormal laboratory findings or other abnormal assessments related to the disease being studied (unless judged by the investigator to be more severe than expected for the patient's condition) or present or detected at the start of the study and not worsened will not be reported as AEs or SAEs.

[0568] Laboratory measurements that deviate clinically significantly from previous measurements (as determined by the investigator) may be repeated. Additional or more frequent examinations than specified in the protocol should be performed if necessary to provide adequate documentation of AEs and resolution of AEs.

[0569] The investigator uses his or her medical and scientific judgment to decide whether an abnormal laboratory finding or other abnormal evaluation is clinically significant.

[0570] Adverse Events of Special Interest

[0571] AESIs are AEs of scientific and medical concern to the sponsor's product or program that require additional monitoring. Such events may warrant further investigation to better characterize and understand it.

[0572] For this study, IMARs and IRRs were considered AESIs if the event severity was grade 3 or higher:

[0573] ·IMAR

[0574] IRR

[0575] ·CRS

[0576] TLS

[0577] AESIs must be reported to the PureTech Health Pharmacovigilance Department or its designee within the same time period applicable to SAEs.

[0578] Adverse event assessment

[0579] Severity

[0580] All AEs were graded according to severity using CTCAE version 5.0. If an AE is not listed in the CTCAE criteria, the investigator should grade it accordingly based on their best medical judgment as follows:

[0581] Mild (Grade 1): Asymptomatic or mildly symptomatic; clinical or diagnostic observations only; intervention not indicated

[0582] Moderate (Grade 2): Minimally invasive, local, or noninvasive interventions indicated; limited age-appropriate instrumental activities of daily living (ADL)

[0583] Severe (Grade 3): Medically significant but not immediately life-threatening; requiring hospitalization or prolonged hospitalization; disabling; or limiting self-care ADLs

[0584] Life-threatening (Grade 4): Life-threatening consequences; urgent intervention indicated

[0585] Death (Grade 5): Death related to AE

[0586] The terms "serious" and "serious" are not synonymous. Severity is a measure of intensity (as characterized above), whereas severity as defined herein defines the requirements of reporting obligations.

[0587] VIII. statistics

[0588] Sample size

[0589] This dose escalation study will establish the MTD and / or the lowest safe and effective biologic dose to determine the RP2D dose of G9.2-17(IgG4) using a dose escalation design based on a 4+2 algorithm. With the possibility of including patient backfill, a total sample size of up to 50 patients is expected to be enrolled in this study.

[0590] Analyze groups

[0591] The intention to treat (ITT) population will be defined as patients who receive at least one dose of G9.2-17 (IgG4). Efficacy analysis will be performed on ITT. Patient distribution will be performed for ITT. The efficacy population will be defined as all patients in the ITT and with at least one measurable overall response at 1 month. This population will be used for sensitivity analysis.

[0592] The per-protocol (PP) population included any patient who received at least one complete cycle of G9.2-17 (IgG4) and had no major protocol deviations.

[0593] The safety population (SAF) is defined as all patients who received at least one dose of G9.2-17 (IgG4). Safety analyses will be performed on the SAF.

[0594] The PK population included any patient who received at least one dose of G9.2-17 (IgG4) and had at least one evaluable post-dose concentration.

[0595] The pharmacodynamic (PD) population was defined as those patients who had received at least one complete cycle of G9.2-17 (IgG4) and had at least one post-treatment PD assessment as well as a baseline PD assessment, respectively.

[0596] General Statistics Program

[0597] The database was locked and the primary analysis was performed after the last patient had experienced their primary endpoint event. The final study analysis was performed after the study was completed. All analyses were descriptive.

[0598] Unless otherwise stated, all continuous endpoints were summarized using descriptive statistics, including the number of patients (n), mean, standard deviation, median, minimum, and maximum. All categorical endpoints were summarized using frequencies and percentages with 95% CIs. Screening measurements were the last values ​​on or before the date of first study treatment. Survival curves for PFS and OS were generated from the day of treatment initiation by the Kaplan-Meier method, but there were no comparative analyses between study groups in any part of the trial. Waterfall plots and Swimmers plots were used to graphically present the ORR and DoR for all patients in each study group in each tumor type evaluated in Part 2. The ORR and DoR for all patients in each study group were graphically presented using the All statistical analyses were performed using SAS version 9.2 (SAS, Cary, NC).

[0599] Security Analysis

[0600] Unless otherwise stated, all safety analyses were performed on SAF and were analyzed using descriptive statistics.

[0601] Efficacy analysis

[0602] For ITT and PP, the assessed disease responses were summarized descriptively. Sensitivity analyses were performed using the efficacy population.

[0603] Pharmacokinetics, pharmacodynamics, and immunogenicity

[0604] A descriptive summary of PK, PD, and immunogenicity for the PK / PD population.

[0605] G9.2-17 (IgG4) has a rapid clearance rate in human subjects compared to conventional antibody therapies. Once the safety of the anti-galectin-9 antibody was established, a treatment regimen was developed that included a once-weekly dosing schedule to maintain consistent systemic exposure levels of the anti-galectin-9 antibody.

[0606] The treatment methods provided herein, including administration of an anti-Galectin-9 antibody, such as G9.2-17 (IgG4), at specific doses and dosing schedules provided herein (e.g., those used in the clinical trials provided herein), are expected to produce one or more positive clinical outcomes, e.g., reduction in tumor size, delay in tumor growth, palliation of lesions and / or delay in progression of lesions and / or prolongation of survival in human patients with target hematological malignancies, such as AML, MDS or ALL (T-cell or B-cell ALL), who may be refractory and / or relapsed.

[0607] Example 2: Analysis of AML patients treated with anti-galectin-9 antibodies

[0608] Biological samples (such as blood samples) were collected from AML patients participating in the clinical trial described in Example 1 above. To study the immunological and molecular changes in peripheral blood and bone marrow, the pharmacokinetic (PK), immunogenicity and pharmacodynamic (PD) characteristics of anti-galectin-9 antibody (G9.2-17 (IgG4)) were studied. The clinical trial schedule for the evaluation is provided in Figure 2 middle.

[0609] The following assay was used to evaluate the PD profile of anti-galectin-9 antibodies.

[0610] Analytical determination:

[0611] 1. Luminex

[0612] 2. PBMC Immunophenotyping Process

[0613] 3. CyTOF (Cytometry by Time of Flight)

[0614] 4. MRD Assessment by PCR

[0615] 5. Gene array: beneficial for immune-tumor gene expression determination

[0616] 6. Total G9.2-17 (IgG4) (PK) LC / MS / MS analysis - used in G9.2-17 (IgG4) solid tumor studies

[0617] 7. Anti-G9.2-17 (IgG4) MSD-ECL - has been used in G9.2-17 (IgG4) solid tumor research

[0618] 8.Galectin-9 serum ELISA - used in G9.2-17 (IgG4) solid tumor studies

[0619] Total G9.2-17 (IgG4) (PK) LC / MS / MS analysis, anti-G9.2-17 (IgG4) MSD-ECL, and Galectin-9 serum ELISA have been performed in the G9.2-17 (IgG4) solid tumor assay at PPD Central Laboratories using methods developed and validated.

[0620] Luminex :Luminex is an important tool for interrogating the cytokine environment during physiological and pathological processes because it can evaluate the pattern of cytokines, thereby providing a more comprehensive description of complex cell signaling networks than previously possible. Compared with traditional methods, it provides more robust data delivery at a higher throughput and density, but with accuracy comparable to ELISA. Compared with ELISA, Luminex requires significantly lower sample volume and shorter assay time, and has revolutionized our ability to quickly assess the relative concentrations of various soluble factors. Luminex is unique in that it provides both high density (100 tests per sample multiplexed) and high throughput (up to 1,000 samples can be tested per day). It facilitates the simultaneous evaluation of multiple immune mediators with the advantages of high throughput, small sample volume, and low cost. The markers reviewed were: IFN-γ, IL-10, IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-6, IL-8, TNF-α, MIP 1b, MCP-1, MIP1a, IL-17a, IL-5, TGF-β.

[0621] Immunophenotyping PBMC workflow:Multiparameter flow cytometry technology is used to comprehensively monitor the immune response to immunotherapy from a single tube of blood, maximizing the information from the often limited patient material. The immune markers covered in the PBMC immunophenotype do not overlap with those in the CyTOF panel, and in addition to standard immune cell class markers (e.g., CD3, CD4, CD8, CD11b / c The present invention relates to a novel class of immune checkpoint molecules (i.e., gal-9, TIM-3, TIGIT, PD-1, PD-L1, CTLA-4, CD47, CD73, CD123, KI67) that allows the expression of immune checkpoint molecules and other immune targets in AML to be stratified among these immune cell populations, allowing their potential changes / regulation to be dynamically tracked after exposure to anti-galectin-9 antibodies. Granulocytes will be excluded because PBMCs rather than whole blood will be analyzed. These markers can also indicate potential combination therapies by flow cytometry, which may be valuable for clinical exploration with G9.2-17 (IgG4), and some such as anti-CD47, anti-CD123, anti-TIGIT, anti-CD73, anti-TIM-3 are already in clinical trials.

[0622] CyTOF : Cytometry by time of flight is an application of mass cytometry for quantification of labeled targets on the surface and inside of single cells. CyTOF exploits low cell numbers and antibodies conjugated to rare heavy metal isotopes that are not normally present in biological samples. CyTOF allows simultaneous quantification of multiple cellular components using an ICP-MS detector. Use the most comprehensive Clinical trial samples are processed with the Human Immune Monitoring Panel, which contains 30 intracellular and extracellular parameters / markers, which allows highly multiplexed analysis of patient samples, reliably quantifying 37 immune cell populations in human whole blood or PBMCs. Due to the preciousness of clinical trial samples, this method was chosen because it only requires 270 μl of whole blood. This method was used because flow cytometry is limited by the number of parameters that can be analyzed simultaneously, severely limiting its practicality. The markers to be examined are provided in Figure 3 Additional markers for examination are provided above.

[0623] MRD assessment by RT-qPCR:Monitoring minimal residual disease (MRD) is routine clinical practice for hematological cancers. MRD technology needs to be sensitive, widely applicable, accurate, reliable, rapid and affordable. MRD has several important functions. Clinicians use this prognostic marker to evaluate the response to treatment, predict the possibility of relapse, and identify whether the patient is in remission or suffering from relapse. The sensitivity of the test indicates its effectiveness in detecting MRD, and the primary outcome is the MRD cell level. The cutoff level is 0.01% MRD cells, or 1 MRD cell per 10,000 cells. 1 Measurements above this value indicate a higher risk of relapse compared to results below 0.01%, and as the number increases (e.g., MRD>1%), the risk of relapse also increases, while the chance of survival decreases. To date, flow cytometry and polymerase chain reaction (PCR) analysis are the preferred methods. In clinical trials, MRD assessments by flow cytometry are performed by local laboratories, and samples are sent for PCR analysis. Peripheral blood can be used for both methods, but MRD levels are 10 times lower than bone marrow. Therefore, whenever possible, bone marrow samples were also collected. PCR has a one-log higher sensitivity compared to flow cytometry, which is why this modality is used to measure MRD in addition to flow cytometry.

[0624] Gene Array : The Immuno-Oncology Gene Expression Assay for Translational and Clinical Research consists of 170 highly informative genes involved in immune cell recognition, immune and cancer cell function, immune regulation and cell fate, and response to checkpoint inhibitors (and therefore immunotherapy agents). It is designed for use with Biomark TMUsed together with the HD system, the Advanta Immuno-Oncology Gene Expression Assay sensitively detects these gene expression markers, which are analyzed in defined cell subpopulations. This assay can enable and accelerate the development of immunotherapy by identifying predictive biomarker signatures of treatment response. Tumor gene expression profiling has been shown to be effective in measuring immune response and treatment response during cancer progression. However, previous approaches using large preconfigured panels containing hundreds of genes or transcriptome analysis are expensive and time-consuming. Large preconfigured panels are also difficult to customize to specific experimental needs. The Advanta Immuno-Oncology Gene Expression Assay was developed in collaboration with leading researchers from academia and the biopharmaceutical community to provide the right balance of biomarker breadth, assay flexibility, and workflow efficiency. The Advanta Immuno-Oncology Gene Expression Assay is available as a dual-group setup. The first group includes 91 key markers of tumor immune response previously shown in multicenter international clinical trials to inform tumor progression and immunotherapy response. The second group includes 74 additional highly informative immuno-oncology markers and 17 open assay entries for additional customization. Both groups contain the same five reference genes. Combined with Fluidigm microfluidics technology, this Advanta assay uniquely offers significant workflow efficiencies over traditional gene expression profiling methods. Each reaction is miniaturized to nanoliter volumes, meaning clinical trial blood samples are used in the most efficient manner and controlled using precise automation to enable accurate and cost-effective qPCR testing over a large dynamic range.

[0625] Galectin-9 ELISA : Patient blood was collected and serum was separated according to routine practice at a designated central laboratory (Flagship Biosciences) for use at Boster Picokine TM The samples were processed on a human galectin-9 pre-coated enzyme-linked immunosorbent assay (ELISA) kit. This is a solid phase immunoassay specifically designed for measuring human galectin-9 using a 96-well strip plate. The detection antibody is a biotinylated antibody specific for gal-9. The capture antibody is a monoclonal antibody from mouse, and the detection antibody is a polyclonal antibody from goat. The optical density of the wells was measured using a FLUOstar Optima microplate reader (BMG Labtech). Serum galectin-9 concentrations were calculated using BMG Optima 2.10R2 software.

[0626] Anti-galectin-9 antibodies (G9.2-17 (IgG4)) can reduce leukemic cell load in the bone marrow and periphery and reactivate at least effector T cells and macrophages, and broadly modulate the proportions and phenotypes of immune cell populations and / or cytokines in the blood to reflect the reversal of immunosuppression present in AML / MDS. Anti-galectin-9 antibodies can also reduce galectin-9 serum levels, where it is elevated at baseline. The study steps outlined here are designed to move efficiently during part 1 of the clinical trial and can inform and potentially accelerate further clinical trials in part 2. In line with this view, the plan focuses on timely sample collection, analysis, and reporting, while collecting safety and any preliminary efficacy readouts for treated patients. Clinical trials can be lengthy and expensive, and any unnecessary exposure of non-responders to ineffective immunomodulatory drugs should be avoided. Indeed, one goal of this study is to identify and develop assays as complementary diagnostics to determine the likelihood that an individual patient will respond to treatment with anti-galectin-9 antibodies and reflect their anti-leukemic and immunomodulatory effects in vivo. This could effectively select possible responders and accelerate further clinical development of the antibodies.

[0627] Anti-Galectin-9 antibodies are likely to have rapid effects on peripheral immune dynamics and circulating target levels (within the first week after administration), which is why study sampling was applied to cover both early and late time points. Likewise, many blood collection time points coincided with study PK sampling, which may allow for the derivation of correlations between PK parameters and any immunomodulatory signals detected by phenotype and Galectin-9 serum levels.

[0628] This study elucidates immune pathways and therapeutic responses while ensuring that: i) planned assays measure the desired endpoints and include phenotypic and functional immune markers; ii) assays that address relevant stimulatory and regulatory pathways are considered; iii) host-related and tumor-related markers that may influence the results are measured; iv) assays are reproducible with acceptable variation; v) quality assurance and quality control methods are in place; vi) central laboratory validation is performed; vii) careful consideration is given to the compartment in which the assay needs to be measured (peripheral blood / bone marrow); viiii) time points during the treatment regimen in which the assay is performed are selected to best determine the kinetics of the effect; ix) appropriate statistical and bioinformatic methods are planned for analysis; and x) samples (cells, serum, plasma, bone marrow) are pooled for future assays (functional, phenotypic, DNA, and mRNA) if possible.

[0629] NanoString Assay: Patient blood samples were collected and PBMCs were isolated according to routine procedures. RNA was extracted from PBMC samples using the RNeasy Mini Kit (WI-84) with an elution volume of 30 μL. Subsequently, RNA concentration was measured using a fluorescent analysis method using the Quant-iT RiboGreen RNA Reagent Kit (WI-82) or the Qubit RNA Assay Kit (WI-258). The optimal concentration of the sample was 60 ng / μL in a 5 μL volume. The following nCounter gene expression panels were used in this study: Human with RLF PanCancer IO360 Set: NS_IO_360_v1.0. nCounter gene expression analysis is performed in batches of up to 11 clinical samples. In addition, each run also contains a set of standard samples for the corresponding nCounter assay.

[0630] For data analysis, raw data were normalized using background correction, normalized using spiked positive controls, and normalized using housekeeping genes.

[0631] Equivalent solutions

[0632] From the above description, those skilled in the art can easily determine the basic characteristics of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention to adapt it to various uses and conditions. Therefore, other embodiments are also within the scope of the claims.

[0633] Although several inventive embodiments have been described and illustrated herein, it is easy for a person of ordinary skill in the art to think of various other means and / or structures for performing the functions described herein and / or obtaining results and / or one or more advantages, and each such change and / or modification is considered to be within the scope of the inventive embodiments described herein. More generally, it is easy for a person skilled in the art to understand that all parameters, dimensions, materials and configurations described herein are intended to be exemplary, and the actual parameters, dimensions, materials and / or configurations depend on one or more specific applications for which the present invention is taught. A person skilled in the art recognizes or is able to determine many equivalents of the specific inventive embodiments described herein using only routine experiments. Therefore, it should be understood that the foregoing embodiments are presented only by way of example, and within the scope of the appended claims and their equivalents, the inventive embodiments may be implemented in a manner different from that specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit and / or method described herein. In addition, if such features, systems, articles, materials, kits and / or methods are not mutually contradictory, any combination of two or more such features, systems, articles, materials, kits and / or methods is included within the scope of the invention disclosed herein.

[0634] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0635] All references, patents, and patent applications disclosed herein are incorporated by reference for each subject matter cited, and in some cases, in their entirety.

[0636] Unless explicitly indicated to the contrary, as used herein in the specification and claims, the indefinite articles "a" and "an" should be understood to mean "at least one".

[0637] As used herein in the specification and claims, the phrase "and / or" should be understood to mean "either or both" of the connected elements, i.e., the elements are present in combination in some cases and separately in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so connected. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with an open-ended phrase such as "comprising", may refer to A only (optionally including elements other than B) in one embodiment; to B only (optionally including elements other than A) in another embodiment; to A and B (optionally including other elements) in another embodiment; and so on.

[0638] 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 understood to be inclusive, i.e., including at least one, but also including multiple elements or more than one in a list of elements, and optionally, including items that are not listed in addition. Only terms that clearly indicate the opposite, such as "only one of..." or "exactly one of..." or "consisting of..." when used in a claim will refer to the inclusion of multiple elements or exactly one element in a list of elements. Generally, the term "or" as used herein should be interpreted as indicating an exclusive selection (i.e., "one or the other but not two") only when it is crowned with an exclusive term such as "any", "one of...", "only one of..." or "exactly one of...". "Substantially consisting of...", when used in a claim, should have its ordinary meaning used in the field of patent law.

[0639] As used herein in the specification and claims, the phrase "at least one" with respect to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related 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 refer in one embodiment to at least one (optionally including more than one) A without B present (and optionally including elements other than B); in another embodiment to at least one (optionally including more than one) B without A present (and optionally including elements other than A); in another embodiment to at least one (optionally including more than one) A and at least one (optionally including more than one) B (and optionally including other elements); and so on.

[0640] It should also be understood that the order of steps or acts in any method claimed herein that includes more than one step or act is not necessarily limited to the order in which the steps or acts of the method are described unless explicitly indicated to the contrary.

Claims

1. A method for treating a hematological malignancy, comprising administering to a subject in need thereof a pharmaceutical composition comprising an antibody that binds to human galectin-9 (anti-Gal9 antibody), wherein the anti-Gal9 antibody is administered to the subject at a dose of about 2 mg / kg to about 32 mg / kg once a week to once every 4 weeks.

2. The method of claim 1, wherein the anti-Gal9 antibody comprises: (a) a light chain, the light chain comprising a light chain variable region (V L ), which comprises a light chain (LC) complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 1, a LC complementarity determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO: 2, and a LC complementarity determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO: 3, and (b) a heavy chain, said heavy chain comprising a heavy chain variable region (V H ), which comprises a heavy chain (HC) complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:4, a HC complementarity determining region 2 (CDR2) comprising the amino acid sequence of SEQ ID NO:5, and a HC complementarity determining region 3 (CDR3) comprising the amino acid sequence of SEQ ID NO:

6.

3. The method of claim 2, wherein the anti-Gal9 antibody comprises: V L , which comprises the amino acid sequence of SEQ ID NO: 8; and V H , which comprises the amino acid sequence of SEQ ID NO:

7. The method according to any one of claims 1 to 3, wherein the anti-Gal9 antibody is an IgG1 molecule or an IgG4 molecule.

5. The method of claim 4, wherein the anti-Gal9 antibody is an IgG4 molecule comprising a modified Fc region relative to a wild-type human IgG4 counterpart. The method of claim 5 , wherein the modified Fc region comprises the amino acid sequence of SEQ ID NO:

14. 7 . The method of claim 6 , wherein the anti-Gal9 antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 19; and a light chain comprising the amino acid sequence of SEQ ID NO:

15.

8. The method of any one of claims 1 to 7, wherein the anti-Gal9 antibody is administered to the subject at a dose of about 2 mg / kg, about 4 mg / kg, about 6.3 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 12 mg / kg, or about 16 mg / kg.

9. The method of any one of claims 1 to 8, wherein the anti-Gal9 antibody is administered to the subject at a dose of about 2 mg / kg, about 4 mg / kg, about 7.5 mg / kg, about 12 mg / kg, or about 16 mg / kg.

10. The method of claim 8 or claim 9, wherein the anti-Gal9 antibody is administered once every two weeks.

11. The method of claim 8 or claim 9, wherein the anti-Gal9 antibody is administered once a week.

12. The method of any one of claims 1 to 8, wherein the anti-Gal9 antibody is administered at a dose of 4.0 mg / kg per week, 6.3 mg / kg per week, 7.5 mg / kg per week, 10.0 mg / kg per week, 12 mg / kg per week, or 16.0 mg / kg per week.

13. The method of any one of claims 1 to 8, wherein the anti-Gal9 antibody is administered at a dose of 4.0 mg / kg per week, a dose of 7.5 mg / kg per week, a dose of 12 mg / kg per week, or a dose of 16.0 mg / kg per week.

14. The method of any one of claims 1 to 13, wherein the subject is a human patient suffering from leukemia or lymphoma.

15. The method of claim 14, wherein the human patient has acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or acute lymphoblastic leukemia (ALL).

16. The method of any one of claims 1 to 15, wherein the hematological malignancy is refractory and / or relapsed.

17. The method of claim 16, wherein the hematological malignancy is refractory and / or relapsed and high-risk MDS.

18. The method of claim 15, wherein the human patient has relapsed and / or refractory AML after at least one prior line of treatment.

19. The method of claim 15, wherein the human patient has relapsed and / or refractory MDS after at least one prior line of treatment.

20. The method of any one of claims 1 to 19, wherein the human patient has undergone or is undergoing chemotherapy for treatment of the hematological malignancy.

21. The method of any one of claims 1 to 20, comprising at least one treatment cycle consisting of 28 days, during which the anti-Gal9 antibody is administered to the subject once a week.

22. The method of any one of claims 1 to 21, wherein the method comprises the anti-Gal9 antibody as the only active agent used to treat the hematological malignancy.

23. The method of any one of claims 1 to 22, further comprising monitoring the occurrence of adverse events during treatment.

24. The method of claim 21, further comprising reducing the dose of the anti-Gal9 antibody in the subject experiencing the adverse event.

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