Treatment of Malignant Hematological Diseases Using Antibodies that Inhibit Galectin-9

Abstract

The utility of galectin-9 as a potential new drug target has been shown. 【Solutions】In this specification, a method for treating malignant blood diseases (e.g., acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or acute lymphoblastic leukemia (ALL)) using an anti-galectin-9 antibody (e.g., G9.2-17 (IgG4)) is disclosed. In some embodiments, the anti-galectin-9 antibody is used as a monotherapy.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of the filing dates of U.S. Provisional Application No. 63 / 356,840, filed on June 29, 2022; U.S. Provisional Application No. 63 / 394,506, filed on August 2, 2022; U.S. Provisional Application No. 63 / 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.

[0002] Reference to a Sequence Listing This application includes a sequence listing submitted electronically in ASCII format, the entire contents of which are incorporated herein by reference. The above XML copy, created on June 21, 2023, is named 112174 - 0259 - NP013WO00 and has a size of 40,880 bytes.

Background Art

[0003] Galectin-9 is a tandem repeat lectin consisting of two carbohydrate recognition domains (CRDs). It was first discovered and described in 1997 in patients suffering from Hodgkin lymphoma (HL) (Tureci et al., J. Biol. Chem. 1997, 272, 6416-6422). There are three isoforms, which can be located intracellularly or extracellularly. An increase in galectin-9 levels has been observed in a wide range of cancers, including melanoma, Hodgkin lymphoma, hepatocellular carcinoma, pancreatic cancer, gastric cancer, colon cancer, and renal clear cell carcinoma (Wdowiak et al. Int. J. Mol. Sci. 2018, 19, 210). In renal cancer, patients with high galectin-9 expression had larger tumor sizes and showed a more advanced disease progression (Kawashima et al.; BJU Int. 2014; 113:320-332). In melanoma, galectin-9 was expressed in 57% of tumors and was significantly increased in the plasma of progressive melanoma patients compared to healthy controls (Enninga et al., Melanoma Res. 2016 Oct; 26(5):429-441). Many studies have shown the usefulness 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).

[0004] Galectin-9 has been described to play important roles in many cellular processes such as adhesion, cancer cell aggregation, apoptosis, and chemotaxis. Recent studies have shown the role of galectin-9 in immune regulation that supports tumors, for example, through the negative regulation of Th1-type responses, Th2 polarization, and polarization of macrophages to the M2 phenotype. This research also includes studies showing that galectin-9 is involved in the direct inactivation of T cells through its 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).

[0005] Galectin-9 also plays a role in polarizing T cell differentiation towards a tumor-suppressive 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), blocking the checkpoint interaction between galectin-9 and the receptor dectin-1 found on innate immune cells in the tumor microenvironment (TME) has been shown to increase the anti-tumor immune response in the pancreas of the TME and delay tumor progression (Daley et al., Nat Med., 2017, 23, 556-567). Galectin-9 has also been found to bind to CD206, a surface marker of M2 macrophages, resulting in reduced secretion of the macrophage-derived chemokine CVL22 (MDC), which is associated with an extended survival period and reduced risk of recurrence in lung cancer (Enninga et al, J Pathol. 2018 Aug;245(4):468-477). SUMMARY OF THE INVENTION

[0006] This disclosure is based, at least in part, on the development of treatment regimens for malignant blood disorders such as acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), or acute lymphoblastic leukemia (ALL).

[0007] Thus, provided herein is a method for treating a malignant blood disease, 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 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). 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 the subject by intravenous infusion.

[0008] In some embodiments, the anti-Gal9 antibody comprises (a) a light chain variable region (V L ) comprising a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 1, a light chain complementarity determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 2, and a light chain complementarity determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO: 3, and (b) a heavy chain variable region (V H ) comprising a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 4, a heavy chain complementarity determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 5, and a heavy chain complementarity determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO: 6. In some examples, the anti-Gal9 antibody comprises a V L having the amino acid sequence of SEQ ID NO: 8 and a V H having the amino acid sequence of SEQ ID NO: 7.

[0009] Any of the anti-Gal9 antibodies for use in the methods disclosed herein can be an IgG1 molecule. Alternatively, the antibody may be an IgG4 molecule. In some embodiments, the anti-Gal9 antibody can be an IgG4 molecule (e.g., a human IgG4 molecule) that includes an Fc region modified compared to the wild-type IgG4 counterpart (e.g., the wild-type human IgG4 counterpart). In some examples, the modified Fc region includes the amino acid sequence of SEQ ID NO: 14. In a specific example, the anti-Gal9 antibody includes a heavy chain that includes the amino acid sequence of SEQ ID NO: 19 and a light chain that includes the amino acid sequence of SEQ ID NO: 15. Such an anti-Gal9 antibody can be G9.2-17 (IgG4).

[0010] Any of the anti-Gal9 antibodies 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 the subject once a week to once every 6 weeks (e.g., once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 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 6 weeks (e.g., once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks). In some embodiments, the anti-Gal9 antibody is administered once a week. In some embodiments, the anti-Gal9 antibody is administered once every 2 weeks to once every 4 weeks. In some examples, the anti-galectin-9 antibody can be administered to the subject once every 2 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 2 weeks to once every 4 weeks (e.g., once every 2 weeks) at a dose of 6.3 mg / kg, 7.5 mg / kg, 10 mg / kg, 12 mg / kg or 16 mg / kg.

[0011] Alternatively, an anti-Gal-9 antibody such as G9.2-17 (IgG4) can be administered to a subject once every 2 to 6 weeks (e.g., every 2 to 4 weeks), such as once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, at a dose of about 410 mg to about 2056 mg (e.g., about 410 to about 650 mg, about 410 to about 1040 mg, or about 410 to about 1280 mg). In some examples, the anti-Gal-9 antibody is administered to the subject once every 2 to 6 weeks (e.g., every 2 to 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, at a dose of about 410 mg to about 505 mg (e.g., about 410 to about 450 mg or about 410 to about 480 mg). In other examples, the anti-Gal-9 antibody is administered to the subject once every 2 to 6 weeks (e.g., every 2 to 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, at a dose of about 650 mg to about 800 mg (e.g., about 650 to about 700 mg). In still other examples, the anti-Gal-9 antibody is administered to the subject once every 2 to 6 weeks (e.g., every 2 to 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, at a dose of about 1040 mg to about 1280 mg (e.g., about 1040 to about 1120 mg). In yet other examples, the anti-Gal-9 antibody is administered to the subject once every 2 to 6 weeks (e.g., every 2 to 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, at a dose of about 2080 mg to about 2560 mg (e.g., about 2080 to about 2400 mg).

[0012] In other embodiments, any of the anti-Gal9 antibodies 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 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 4 mg / kg, 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 once a week at a dose of about 410 mg to about 2560 (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 the subject once a week at a dose of about 410 mg to about 505 (e.g., about 410 mg to about 450 mg). In some examples, the anti-Gal-9 antibody is administered to the subject once a week at a dose of about 650 mg to about 800 mg (e.g., about 650 to about 700 mg). In yet other examples, the anti-Gal-9 antibody is administered to the subject once a week at a dose of about 1040 mg to about 1280 (e.g., about 1040 mg to about 1120 mg). In yet other examples, the anti-Gal-9 antibody is administered to the subject once a week at a dose of about 2080 mg to about 2560 mg (e.g., about 2080 to about 2400 mg).

[0014] In one example, an anti-Gal9 antibody (e.g., G9.2-17 (IgG4)) disclosed herein can be administered to a subject once a week at a dose of 2 mg / kg. In another example, an anti-Gal9 antibody (e.g., G9.2-17 (IgG4)) disclosed herein can be administered to a subject once a week at a dose of 4 mg / kg. In yet another example, an anti-Gal9 antibody (e.g., G9.2-17 (IgG4)) disclosed herein 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 (e.g., G9.2-17 (IgG4)) disclosed herein 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 (e.g., G9.2-17 (IgG4)) disclosed herein can be administered to a subject once a week at a dose of 12.0 mg / kg. In another example, an anti-Gal9 antibody (e.g., G9.2-17 (IgG4)) disclosed herein can be administered to a subject once a week at a dose of 16.0 mg / kg.

[0015] In some embodiments, the subject of treatment by any of the methods disclosed herein is a human patient having leukemia or lymphoma. In specific examples, the human patient has acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or acute lymphoblastic leukemia (ALL, such as T-cell ALL or B-cell ALL). Alternatively or additionally, the hematologic malignancy is refractory and / or relapsed. In some cases, the human patient has relapsed and / or refractory AML after at least one prior therapy. In other cases, the human patient has relapsed and / or refractory MDS after at least one prior therapy. Alternatively or additionally, the human patient does not have acute promyelocytic leukemia (APL).

[0016] In some embodiments, the human patient has received or is receiving chemotherapy for treating a hematologic malignancy. In other embodiments, the human patient has not received or is not receiving chemotherapy for treating a hematologic malignancy.

[0017] Any of the methods disclosed herein can effectively reduce or eliminate Gal9+ blood cancer cells, such as leukemia or lymphoma cells. Alternatively or additionally, the methods disclosed herein can effectively modulate an immune response targeting cancer cells.

[0018] In some cases, any of the methods disclosed herein includes at least one cycle of treatment consisting of 28 days, during which an anti-Gal9 antibody is administered to the subject once a week. For example, the treatment may include 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, or more.

[0019] In some cases, the methods disclosed herein include an anti-Gal9 antibody as the only active agent for treating a hematologic malignancy (monotherapy).

[0020] Any of the methods disclosed herein may further include monitoring for the occurrence of adverse events during the course of treatment. In some cases, if an adverse event occurs, the dosage of the anti-Gal9 antibody may be decreased.

[0021] 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 for manufacturing a pharmaceutical for use in the treatment methods disclosed herein.

[0022] 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, as well as from the appended claims.

Brief Description of the Drawings

[0023]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0024] Galectin-9, a tandem repeat lectin, is a β-galactoside-binding protein, which has been shown to play a role in regulating cell-cell and cell-matrix interactions. It has been found to be strongly overexpressed in Hodgkin's disease tissue and other pathological conditions. It may also be found circulating within the tumor microenvironment (TME).

[0025] Galectin-9 has been found to interact with TIM-3, a type I cell surface glycoprotein expressed on the surface of leukemia stem cells in all types of acute myeloid leukemia (except M3 (acute promyelocytic leukemia)), but not on normal human hematopoietic stem cells (HSCs). TIM-3 signaling resulting from ligation of galectin-9 has pleiotropic effects on immune cells, inducing apoptosis of Th1 cells (Zhu et al., Nat Immunol., 2005, 6:1245 - 1252) and stimulating the secretion of tumor necrosis factor a (TNF-a), which causes inflammation by innate immunity and leads to the maturation of monocytes into dendritic cells (Kuchroo et al., Nat Rev Immunol., 2008, 8:577 - 580). Furthermore, galectin-9 / TIM-3 signaling has been found to co-activate NF-κB and Wnt signaling, two pathways that promote self-renewal of LSCs (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 with regard to leukemia and other malignant blood disorders.

[0026] Furthermore, galectin-9 is a multifaceted immunomodulator that affects numerous cell types of innate and adaptive immunity. Gal-9 promotes inflammation, induces M2 macrophages and monocyte-derived dendritic cells (mDCs), promotes the differentiation of regulatory T cells, and suppresses NK cell killing ability. There are multiple transcriptional and functional changes induced by galectin-9 independently of Tim-3. The expression of Gal9 has also been found to be elevated in AML patients who have failed chemotherapy. See, for example, OncoImmunology, 5(7):00-00 DOI:10.1080 / 2162402X.2016.1195535. Anti-Gal9 antibodies have been found to significantly inhibit the reconstitution of AML and LSCs. Kikushige et al., Cell Stem Cell 17(3):341-352 (2015).

[0027] Anti-Gal9 antibodies such as G9.2-17 (IgG4) disclosed herein can directly kill Gal9+ hematologic cancer cells through the induction of cytotoxicity. Furthermore, galectin-9 has also been found to affect multiple immunosuppressive pathways. Due to the immunological effects, anti-Gal9 antibodies such as G9.2-17 (IgG4) are enabled as potential monotherapy efficacies.

[0028] Accordingly, provided herein is a method of using an anti-galectin-9 antibody, such as G9.2-17 (IgG4), at a dose of about 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, to treat a malignant blood disease (e.g., acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or acute lymphoblastic leukemia (ALL, T-cell ALL or B-cell ALL, etc.)).

[0029] I. Antibodies that Bind Galectin-9 The present disclosure provides anti-galectin-9 antibodies such as G9.2-17 and functional variants thereof for use in the treatment methods disclosed herein.

[0030] An antibody (used interchangeably in the plural) is an immunoglobulin molecule that can specifically bind to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., via 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, includes not only complete (e.g., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab’, F(ab’)2, Fv), single-chain (scFv), variants thereof, antibody portions, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), as well as glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies, and fusion proteins comprising other immunoglobulin molecules in any modified conformation that contain the antigen recognition site of the required specificity. 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 antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to various classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the various classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structure and three-dimensional configuration of the various classes of immunoglobulins are well known.

[0031] A typical antibody molecule comprises a heavy-chain variable region (V H ) and a light-chain variable region (V L ), which are usually involved in antigen binding. V H and V LThe region can further be subdivided into a more conserved region known as the "framework region" ("FR") and a hypervariable region known as the "complementary determining region" ("CDR") in which more conserved regions are interspersed. Each V H and V L usually consists of three CDRs and four FRs and is arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The ranges of the framework region and the CDR can be accurately identified using methodologies known in the art, for example, the Kabat definition, the Chothia definition, the AbM definition, the EU definition, the "Contact" numbering scheme, the "IMGT" numbering, the "AHo" numbering scheme, and / or the contact definition, all of which are well known in the art. See, for example: E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; Edelman et al., Proc Natl Acad Sci USA. 1969 May;63(1):78-85; and Almagro, J. Mol. Recognit. 17:132-143(2004); MacCallum et al., J. Mol. Biol. 262:732-745(1996), Lefranc M P et al., Dev Comp Immunol, 2003 January;27(1):55-77; and Honegger A and Pluckthun A, J Mol Biol, 2001 Jun. 8;309(3):657-70. See also: hgmp.mrc.ac.uk and bioinf.org.uk / abs).

[0032] In some embodiments, the anti-galectin-9 antibodies described herein are full-length antibodies containing two heavy chains and two light chains, each of which includes a variable domain and a constant domain. Alternatively, the anti-galectin-9 antibody can be an antigen-binding fragment of a full-length antibody. Examples of binding fragments included within the term "antigen-binding fragment" of a full-length antibody include the following: (i) a Fab fragment (a monovalent fragment consisting of V L , V H , C L , and C H 1 domains); (ii) an F(ab’)2 fragment (a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region); (iii) an Fd fragment consisting of V H and C H 1 domains; (iv) an Fv fragment consisting of the V L and V H domains of a single arm of an antibody; (v) a dAb fragment consisting of V H domains (Ward et al., (1989) Nature 341:544-546); and, (iv) isolated complementarity-determining regions (CDRs) that retain function. Further, the two domains of an Fv fragment, V L and V H , are encoded by separate genes, but they can be joined using recombinant methods with a synthetic linker that allows them to be made as a single protein chain, and the V L region and the V H region pair to form a monovalent molecule known as a single-chain Fv (scFv). See, for example: Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883.

[0033] Any of the antibodies described herein, e.g., an anti-galectin-9 antibody, can be either monoclonal or polyclonal. A "monoclonal antibody" refers to a homogeneous population of antibodies, and a "polyclonal antibody" refers to a heterogeneous population of antibodies. These two terms do not limit the source of the antibody or the method of antibody production.

[0034] The reference antibody G9.2-17 refers to an antibody that can bind to human galectin-9 and contains the heavy chain variable region of SEQ ID NO: 7 and the light chain variable domain 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 the G9.2-17 antibody. In some embodiments, the anti-galectin-9 antibody used in the methods disclosed herein is an antibody having the same heavy chain complementarity-determining region (CDR) as the reference antibody G9.2-17 and / or the same light chain complementarity-determining region as the reference antibody G9.2-17. Two antibodies having the same V H and / or V L CDRs, when determined by the same approach, means that their CDRs are identical (e.g., the Kabat approach, Chothia approach, AbM approach, Contact approach, or IMGT approach known herein, see, e.g., bioinf.org.uk / abs / ).

[0035] The heavy and light chain CDRs of the reference antibody G9.2-17 are presented in Table 1 below (determined using the Kabat method).

[0036]

Table 1

[0037] In some examples, the anti-galectin-9 antibodies used in the methods disclosed herein may include a heavy chain complementarity determining region 1 (CDR1) set forth in SEQ ID NO: 4, a heavy chain complementarity determining region 2 (CDR2) set forth in SEQ ID NO: 5, and a heavy chain complementarity determining region 3 (CDR3) set forth in SEQ ID NO: 6 (according to the Kabat scheme), and / or a light chain complementarity determining region 1 (CDR1) set forth in SEQ ID NO: 1, a light chain complementarity determining region 2 (CDR2) set forth in SEQ ID NO: 2, and a light chain complementarity determining region 3 (CDR3) set forth in SEQ ID NO: 3. Anti-galectin-9 antibodies comprising the reference antibody G9.2-17 can be in any format disclosed herein, such as a full-length antibody or a Fab. As used herein, the term "G9.2-17 (IgG4)" refers to the G9.2-17 antibody, which is an IgG4 molecule. Similarly, the term "G9.2-17 (Fab)" refers to the G9.2-17 antibody, which is a Fab molecule.

[0038] In some embodiments, the anti-galectin-9 antibody or a binding portion thereof comprises a heavy chain variable region and a light chain variable region, and the CDR1, CDR2, and CDR3 amino acid sequences of the light chain variable region have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and any increment therein) sequence identity with the amino acid sequences of light chain variable region CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, the anti-galectin-9 antibody or a binding portion thereof comprises a heavy chain variable region and a light chain variable region, and the CDR1, CDR2, and CDR3 amino acid sequences of the heavy chain variable region have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and any increment therein) sequence identity with the amino acid sequences of heavy chain variable region CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 4, 5, and 6, respectively.

[0039] For example, additional galectin-9 antibodies that bind to the CRD1 and / or CRD2 regions of galectin-9 are described in U.S. Patent No. 10,344,091, as well as co-owned and co-pending U.S. Patent Application No. 16 / 173,970 and WO2020 / 198390, the respective relevant disclosures of which are incorporated by reference for the purposes of the subject matter and objects recited herein.

[0040] In some embodiments, the anti-galectin-9 antibodies disclosed herein, individually or collectively, have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increment therein) sequence identity with the corresponding V H CDR of the reference antibody G9.2-17, when compared to the corresponding V H CDR of the reference antibody G9.2-17, when compared to the corresponding V

[0041] 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, modified as 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. The BLAST protein search can be performed using the XBLAST program, score = 50, word length = 3, to obtain an amino acid sequence homologous to the protein molecule of the present invention. If gaps exist between two sequences, gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using the BLAST and gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used.

[0042] In other embodiments, the anti-galectin-9 antibodies described herein comprise a V H comprising HC CDR1, HC CDR2, and HC CDR3, which collectively contain up to 8 amino acid residue diversities (8, 7, 6, 5, 4, 3, 2, or 1 diversity (s)), including additions, deletions, and / or substitutions, compared 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 a V H comprising LC CDR1, LC CDR2, and LC CDR3, which collectively contain up to 8 amino acid residue diversities (8, 7, 6, 5, 4, 3, 2, or 1 diversity (s)), including additions, deletions, and / or substitutions, compared to the LC CDR1, LC CDR2, and LC CDR3 of the reference antibody G9.2-17.

[0043] In one example, the amino acid residue diversity 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 in which the amino acid substitution occurs. The diversity can be adjusted according to methods for altering polypeptide sequences known to those of skill in the art, such as those compiled in references such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0044] In some embodiments, the anti-galectin-9 antibodies disclosed herein having the heavy chain CDRs disclosed herein comprise framework regions derived from subclasses of germline V H fragments. Such germline V HThe field is 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).

[0045] Alternatively or additionally, in some embodiments, an anti-galectin-9 antibody having the light chain CDRs disclosed herein contains a framework region derived from a germline Vκ fragment. Examples include the IGKV1 framework (e.g., IGKV1-05, IGKV1-12, IGKV1-27, IGKV1-33, or IGKV1-39), the IGKV2 framework (e.g., IGKV2-28), the IGKV3 framework (e.g., IGKV3-11, IGKV3-15, or IGKV3-20), and the IGKV4 framework (e.g., IGKV4-1). In other cases, the anti-galectin-9 antibody includes a light chain variable region that contains 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), the IGλ4 framework (e.g., IGλV4-3, IGλV4-60, IGλV4-69), the IGλ5 framework (e.g., IGλV5-39, IGλV5-45), the IGλ6 framework (e.g., IGλV6-57), the IGλ7 framework (e.g., IGλV7-43, IGλV7-46), the IGλ8 framework (e.g., IGλV8-61), the IGλ9 framework (e.g., IGλV9-49), or the IGλ10 framework (e.g., IGλV10-54).

[0046] In some embodiments, the anti-galectin-9 antibody used in the methods disclosed herein can be an antibody having the same heavy chain variable region (V H ) and / or the same light chain variable region (V L ) as the reference antibody G9.2-17, and the amino acid sequences of the V H and V L regions are presented below: VH :

[0047] [Chem.] V L :

[0048] [Chem.]

[0049] In some embodiments, the anti-galectin-9 antibody has a heavy chain variable region of SEQ ID NO: 7 and at least 80% sequence identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity). Alternatively or additionally, the anti-galectin-9 antibody has a light chain variable region of SEQ ID NO: 8 and at least 80% sequence identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity).

[0050] In some cases, the anti-galectin-9 antibodies disclosed herein are functional variants of the reference antibody G9.2-17. Functional variants can have substantially the same binding affinity for human galectin-9 (e.g., having KD values in the same order) and can be structurally similar to the reference antibody, including (e.g., having a limited number of amino acid residue diversities in one or more of the heavy chain and / or light chain CDRs as G9.2-17 disclosed herein, or sequence identity to the heavy chain and / or light chain CDRs of G9.2-17 disclosed herein, or the heavy chain and / or light chain CDRs of G9.2-17).

[0051] In some embodiments, the anti-galectin-9 antibodies described herein bind to galectin-9 and can inhibit its activity by at least 20% (e.g., 31%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein). The apparent inhibition constant (Ki) provides a measure of the potency of the inhibitor appor K i,app ) is related to the concentration of the inhibitor required to reduce enzyme activity and is independent of the enzyme concentration. The inhibitory activity of the anti-galectin-9 antibodies described herein can be measured by a predetermined method known in the art.

[0052] The K of the antibody i, app value can be determined by measuring the inhibitory effect of different concentrations of the antibody on the degree of 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) yields an estimated value of the apparent Ki value. In the case of a competitive inhibitor, Ki app is obtained from the y-intercept extracted from the linear regression analysis of the plot of K i, app against substrate concentration.

[0053]

Number

[0054] A corresponds to v o / E (the initial rate of the enzyme reaction (v o )) in the absence of inhibitor (I) divided by the total enzyme concentration (E)). In some embodiments, the anti-galectin-9 antibodies described herein have a Ki app value of 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 pM or less against the target antigen or antigen epitope. In some embodiments, the anti-galectin-9 antibody has a lower Ki app against the first target (e.g., CRD2 of galectin-9) compared to the second target (e.g., CRD1 of galectin-9). The difference in Ki app (e.g., in the case of specificity or other comparisons) is at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000, or 10 5In some examples, the anti-galectin-9 antibody is greater than or equal to the second antigen (e.g., the first protein or mimetic thereof in a second conformation; or the second protein) and inhibits the first antigen (e.g., the first protein or mimetic thereof in a first conformation). In some embodiments, any of the anti-galectin-9 antibodies further comprises a K app The antibody has been affinity matured to reduce

[0055] In some embodiments, the anti-galectin-9 antibodies described herein have suitable binding affinity for a target antigen (e.g., galectin-9) or an antigenic epitope thereof. As used herein, "binding affinity" refers to the apparent binding constant or K A Refers to. A is the dissociation constant (K D The anti-galectin-9 antibodies described herein have a binding affinity of at least 10 to a target antigen or antigen epitope. -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 Binding affinity (K D The increase in binding affinity can be expressed as K D This corresponds to a decrease in the binding affinity (or binding specificity). Binding affinity (or binding specificity) can be determined in a variety of ways, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for assessing binding affinity are in HBS-P buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 0.005% (v / v) surfactant P20).

[0056] These techniques can be used to measure the concentration of bound binding protein as a function of the concentration of target protein. 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 formula: [Combination] / [Total] = [Target] / (Kd + [Target])

[0057] It is not necessary to accurately determine K A although it is sufficient in some cases to obtain a quantitative measure of affinity (e.g., determined using methods such as ELISA or FACS analysis), since K A is proportional to it, and thus can be used for comparison, e.g., to obtain a qualitative measure of affinity, or to obtain an estimate of affinity based on activity in a functional assay, e.g., an in vitro or in vivo assay, by comparing, e.g., whether the affinity is high, e.g., twice as high. In some cases, in vitro binding assays indicate in vivo activity. In other cases, in vitro binding assays do not necessarily indicate in vivo activity. In some cases, strong binding is beneficial, while in other cases, strong binding is not desirable in vivo and an antibody with a lower binding affinity is more desirable.

[0058] In some embodiments, any heavy chain 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, e.g., human, mouse, rat, or rabbit. In one particular example, the heavy chain constant region is derived from a human IgG (gamma heavy chain) of any of the IgG subfamilies described herein.

[0059] 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 any combination of single domains. In some embodiments, the light chain constant region of the antibodies described herein comprises a single domain of the constant region (e.g., CL). Exemplary light and heavy chain sequences are described below. Exemplary light and heavy chain sequences are described below. The hIgG1 LALA sequence contains two mutations, L234A and L235A (EU numbering), that suppress FcgR binding, and a P329G mutation (EU numbering) that abrogates complement C1q binding, thereby abolishing all immune effector functions. The hIgG4 Fab arm substitution mutant sequence contains a mutation (S228P; EU numbering) that suppresses Fab arm substitution. The IL2 signal sequence (MYRMQLLSCIALSLALVTNS; SEQ ID NO: 9) can be located at the N-terminus of the variable region. This is used in expression vectors and is cleaved during secretion, such that it is not cleaved in the mature antibody molecule. The mature protein (after secretion) begins with "EVQ" for the heavy chain and "DIM" for the light chain. Exemplary amino acid sequences of the heavy chain constant region are provided below: hIgG1 heavy chain constant region (SEQ ID NO: 10) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* hIgG1 LALA heavy chain constant region (SEQ ID NO: 12)

[0060] [Chemistry] hIgG4 heavy chain constant region (SEQ ID NO: 13) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK* hIgG4 heavy chain constant region (SEQ ID NO: 20) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* hIgG4 mutant heavy chain constant region (SEQ ID NO: 14)

[0061] [Chemistry] hIgG4 mutant heavy chain constant region (SEQ ID NO: 21)

[0062] [Chemical]

[0063] In some cases, the heavy chain constant region of 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 without the terminal K residue are presented below: hIgG1 heavy chain constant region without C-terminal lysine (SEQ ID NO: 24) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG* hIgG1 LALA heavy chain constant region without C-terminal lysine (SEQ ID NO: 25)

[0064] [Chemical] hIgG4 heavy chain constant region without C-terminal lysine (SEQ ID NO: 26) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG* hIgG4 heavy chain constant region without a C-terminal lysine (SEQ ID NO: 27) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG* hIgG4 mutant heavy chain constant region without a C-terminal lysine (SEQ ID NO: 28)

[0065]

Chem.

[0066]

Chem.

[0067] In some embodiments, an anti-galectin-9 antibody having any of the above light chain constant regions is paired with a light chain having the following light chain constant region: Light chain constant region (SEQ ID NO: 11) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0068] Exemplary full-length anti-galectin-9 antibodies are presented below: G9.2-17 hIgG1 heavy chain (SEQ ID NO: 16)

[0069] [Chemical formula] G9.2-17 hIgG1 heavy chain without a C-terminal lysine residue (SEQ ID NO: 30)

[0070] [Chemical formula] G9.2-17 hIgG1 LALA heavy chain (SEQ ID NO: 17)

[0071] [Chemical formula] G9.2-17 hIgG1 LALA heavy chain without a C-terminal lysine residue (SEQ ID NO: 31)

[0072] [Chemical formula] G9.2-17 hIgG4 heavy chain (SEQ ID NO: 18)

[0073] [Chemical formula] G9.2-17 hIgG4 heavy chain without C-terminal lysine residue (SEQ ID NO: 32)

[0074]

Chem.

[0075]

Chem.

[0076]

Chem.

[0077]

Chem.

[0078]

Chem.

[0079]

Chem.

[0080]

Chem.

[0081] Any of the above heavy chains can be paired with a light chain of the following (SEQ ID NO: 15) that can be paired.

[0082]

Chemical formula

[0083] 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 increment therein) sequence identity with 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.

[0084] 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 increment therein) sequence identity with 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.

[0085] In some embodiments, the constant region is derived 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 increment therein) sequence identity with 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.

[0086] In some embodiments, the constant region is derived 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 increment therein) sequence identity with 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.

[0087] 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 increment therein) sequence identity with 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.

[0088] In some embodiments, the IgG is a variant having minimal Fc receptor engagement. In one example, the constant region is derived 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 increment therein) sequence identity with 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.

[0089] In some embodiments, the anti-galectin-9 antibody comprises a modified constant region. In some embodiments, the anti-galectin-9 antibody comprises a modified constant region that is immunologically inert, e.g., does not induce complement-mediated lysis or stimulate antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC activity can be evaluated using the method disclosed in U.S. Patent No. 5,500,362. In other embodiments, the constant region is modified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCT Application No. PCT / GB99 / 01441; and / or UK Patent Application No. 9809951.8. In some embodiments, the IgG4 constant region is a variant with reduced heavy chain substitution. In some embodiments, the constant region is derived from the human IgG4 Fab arm substitution mutation S228P.

[0090] 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 increment therein) sequence identity with 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.

[0091] 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 increment therein) sequence identity with 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.

[0092] In some embodiments, the anti-galectin-9 antibody has a light chain having a sequence corresponding to SEQ ID NO: 15; exemplary heavy chain amino acid sequences correspond to SEQ ID NO: 10 (hIgG1); 12 (hIgG1 LALA); 13 (hIgG4); 20 (hIgG4); 14 (hIgG4 variant); and 21 (hIgG4 variant).

[0093] 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 of SEQ ID NO: 15 and a heavy chain consisting 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 essentially SEQ ID NO: 20.

[0094] 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 increment therein) sequence identity with 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.

[0095] 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 increment therein) sequence identity with 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.

[0096] 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 increment therein) sequence identity with 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.

[0097] 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 increment therein) sequence identity with 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.

[0098] 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 increment therein) sequence identity with 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.

[0099] 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 increment therein) sequence identity with 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.

[0100] 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 increment therein) sequence identity with 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.

[0101] In a specific example, the anti-galectin-9 antibody used in the treatment method 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 method disclosed herein is G9.2-17 IgG4. In some examples, such anti-galectin-9 antibodies do not have a C-terminal lysine residue in the heavy chain.

[0102] II. Preparation of Anti-Galectin-9 Antibody Antibodies capable of binding to galectin-9 described herein can be made by any method known in the art, including but not limited to recombinant techniques. An example is presented below.

[0103] Nucleic acids encoding the heavy and light chains of the anti-galectin-9 antibodies described herein can be cloned into one 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 separate promoter. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operably linked to a single promoter such that both the heavy and light chains are expressed from the same promoter. When necessary, an internal ribosome entry site (IRES) can be inserted between the heavy chain coding sequence and the light chain coding sequence.

[0104] 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 such a chain, and the isolated heavy and light chains can be mixed and incubated under suitable conditions that allow for the formation of the antibody.

[0105] Generally, nucleic acid sequences encoding one or all of the chains of an antibody can be cloned into a suitable expression vector operably linked to a suitable promoter using methods known in the art. For example, the nucleotide sequence and the vector can be contacted with a restriction enzyme under suitable conditions to generate complementary ends on each molecule that can pair with each other and be ligated with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the ends of the gene. These synthetic linkers contain nucleic acid sequences corresponding to specific restriction sites within the vector. The choice of expression vector / promoter will depend on the type of host cell used for antibody production.

[0106] A variety of promoters can be used for the expression of the antibodies described herein, including, but not limited to, the cytomegalovirus (CMV) immediate early promoter, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and herpes simplex tk virus promoter.

[0107] Inducible promoters can also be used. Such inducible promoters include those that use the lac repressor from E. coli as a transcriptional modulator to regulate transcription from a mammalian cell promoter having a lac operator [Brown, M. et al., Cell, 49:603-612 (1987)], those that use the tetracycline repressor (tetR) (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 those that use FK506 dimer, VP16, or p65 with estradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech, and Ariad.

[0108] An inducible promoter containing a repressor with an operator can be used. In one embodiment, the lac repressor from E. coli can function as a transcriptional regulator that regulates transcription from a mammalian cell promoter having a lac operator (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)). The tetracycline repressor (tetR) is combined with a transcriptional activator (VP16) to create tTa (tetR-VP16), a tetR mammalian cell transcriptional activator fusion protein, which is combined with a minimal promoter having tetO derived from the human cytomegalovirus (hCMV) major immediate-early promoter to create a tetR-tet operator system that controls gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. Instead of a tetR mammalian cell transcription factor fusion derivative, the tetracycline repressor (tetR) can function as a potent transmodulator to regulate gene expression in mammalian cells when the tetracycline operator is appropriately placed downstream of the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy, 10(16):1392-1399 (2003)). One particular advantage of this tetracycline-inducible switch is that it does not require the use of a tetracycline repressor-mammalian cell transactivator or repressor fusion protein, which in some cases can be toxic to cells in order to achieve its inducible effect (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)).

[0109] Furthermore, the vector may contain, for example, some or all of the following: a selectable marker gene such as the neomycin gene for the selection of stable or transient transfectants in mammalian cells; an enhancer / promoter sequence from the immediate early gene of human CMV for high-level transcription; a transcription termination and RNA processing signal from SV40 for mRNA stability; the SV40 polyomavirus origin of replication and ColE1 for appropriate episomal replication; an internal ribosome entry site (IRES), a multiple cloning site for multiple uses; and T7 and SP6 RNA promoters for in vitro transcription of sense RNA and antisense RNA. Suitable vectors and methods for constructing vectors containing the transgene are well known and available in the art.

[0110] Examples of polyadenylation signals useful for practicing 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.

[0111] One or more vectors (e.g., expression vectors) containing nucleic acids encoding any of the antibodies can be introduced into a host cell suitable for producing the antibody. The host cell can be cultured under conditions suitable for the expression of the antibody or any of its polypeptide chains. Such an antibody or its polypeptide chain can be recovered from the cultured cells (e.g., from the cells or the culture supernatant) by conventional methods, such as affinity purification. Optionally, the polypeptide chains of the antibody can be incubated under suitable conditions for a suitable period to enable the production of the antibody.

[0112] In some embodiments, the method of preparing the antibodies described herein includes a recombinant expression vector encoding both the heavy and light chains of the anti-galectin-9 antibody, as also described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Positive transformed host cells can be selected and cultured under suitable conditions that allow for the expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or the medium. Optionally, the two chains recovered from the host cells can be incubated under suitable conditions that allow for the formation of the antibody.

[0113] In one example, two recombinant expression vectors are provided, one encoding the heavy chain of the anti-galectin-9 antibody and the other encoding the light chain of the anti-galectin-9 antibody. Both of the two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Alternatively, each of the expression vectors can be introduced into a suitable host cell. Positive transformants can be selected and cultured under suitable conditions that allow for the expression of the polypeptide chains of the antibody. When the two expression vectors are introduced into the same host cell, the antibody produced therein can be recovered from the host cell or the medium. Optionally, the polypeptide chains can be recovered from the host cell or the medium and then incubated under suitable conditions that allow for the formation of the antibody. When the two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cell or the corresponding medium. Next, the two polypeptide chains can be incubated under conditions suitable for the formation of the antibody.

[0114] 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 isolated by affinity chromatography using a protein A or protein G binding matrix.

[0115] Any nucleic acid encoding the heavy chain, light chain, or both of the anti-galectin-9 antibody described herein, a vector (e.g., an expression vector) containing the same, and a host cell containing the vector are within the scope of the present disclosure.

[0116] The anti-galectin-9 antibody thus prepared can be characterized using methods known in the art, whereby a decrease, improvement, or neutralization of the biological activity of galectin-9 is detected and / or measured. For example, in some embodiments, ELISA-type assays are suitable for qualitative or quantitative measurement of galectin-9 inhibition of dectin-1 or TIM-3 signaling.

[0117] The biological activity of the anti-galectin-9 antibody can be verified by incubating a candidate antibody with dectin-1 and galectin-9 and monitoring any one or more of the following properties: (a) inhibiting binding between dectin-1 and galectin-9 and signal transduction mediated by the binding; (b) preventing, improving, or treating any aspect of malignant blood diseases; (c) blocking or reducing dectin-1 activation; (d) inhibiting (decreasing) 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.

[0118] In some embodiments, biological activity or efficacy is evaluated in a subject, for example, by measuring peripheral and intratumoral T cell ratios, T cell activation, or by macrophage phenotype analysis.

[0119] Additional assays for determining the biological activity of an anti-galectin-9 antibody include measurement of CD8+ and CD4+ (conventional) T cell activation (e.g., inflammatory cytokine levels such as IFN gamma, TNF alpha, CD44, ICOS granzyme B, perforin, IL2 (upregulated), CD26L, and IL-10 (downregulated)); measurement of macrophage reprogramming (in vitro or in vivo), e.g., from an M2 phenotype to an M1 phenotype (e.g., increase in MHCII, decrease in CD206, increase in TNF-alpha and iNOS), alternatively, for example, in the in vitro assays described herein, the level of ADCC can be evaluated.

[0120] III. Treatment Methods The present disclosure provides a method for treating a malignant blood disease, which is a cancer starting in hematopoietic tissues such as bone marrow or cells of the immune system. Malignant blood diseases include acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndromes, and myeloproliferative neoplasms such as essential thrombocythemia, polycythemia vera, and myelofibrosis. A subject having a malignant blood disease can be identified by routine medical tests such as clinical tests, organ function tests, CT scans, or ultrasounds.

[0121] Pharmaceutical Composition Any of the anti-Gal9 antibodies (e.g., G9.2-17 (IgG4)) disclosed in this specification, and the encoding nucleic acid or nucleic acid set, and vectors containing the same, can be mixed with a pharmaceutically acceptable carrier (excipient) to form a pharmaceutical composition for use in treating a target disease. "Acceptable" means that the carrier is compatible with the active ingredient of the composition (and preferably, can stabilize the active ingredient) and is not harmful to the subject being treated. Pharmaceutically acceptable excipients (carriers) including buffers are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K.E. Hoover.

[0122] The pharmaceutical composition used in this method can contain pharmaceutically acceptable carriers, excipients, or stabilizers in the form of a lyophilized preparation or an aqueous solution. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations used, and these include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzetonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); 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 dextrin; 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 non-ionic surfactants (e.g., TWEEN (trademark), PLURONICS (trademark), or polyethylene glycol (PEG)) may be included.

[0123] In some examples, the pharmaceutical compositions described herein are described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985), Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980), and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with improved circulation times are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse-phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and a PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of a predetermined pore size to obtain liposomes having a desired diameter.

[0124] The antibody, or encoding nucleic acid(s), can also be incorporated, for example, by coacervation techniques or by interfacial polymerization, into microcapsules prepared, for example, from hydroxymethylcellulose or gelatin microcapsules and poly-(methylmethacrylate) microcapsules, respectively, within a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or within a macroemulsion. Such techniques are known in the art. See, for example, Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0125] The pharmaceutical compositions used for in vivo administration must be sterile. This can be readily accomplished, for example, by filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed in a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle.

[0126] In some examples, the pharmaceutical composition is an injectable preparation, and the injectable preparation may contain various carriers, such as vegetable oils, dimethylactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.). In the case of intravenous injection, a water-soluble antibody can be administered by the drip method in which a pharmaceutical preparation containing the antibody and a physiologically acceptable excipient is injected. Physiologically acceptable excipients may include, for example, 5% glucose, 0.9% physiological saline, Ringer's solution, or other suitable excipients. A sterile preparation of a suitable soluble salt form of an antibody, such as an intramuscular preparation, can be administered by dissolving it in a pharmaceutical excipient, such as water for injection, 0.9% physiological saline, or 5% glucose solution.

[0127] (B) Treatment of malignant blood diseases In some embodiments, the present disclosure provides a method for treating a malignant blood disease, such as a malignant blood disease associated with Gal9+ cancer cells, using an anti-galectin-9 antibody described herein, including but not limited to an effective amount of G9.2-17 IgG4 (having the heavy chain of SEQ ID NO: 19 and the light chain of SEQ ID NO: 15).

[0128] Exemplary target malignant blood diseases In some examples, the methods disclosed herein are applied to human patients having leukemia, such as acute myeloid leukemia (AML), or acute lymphoblastic leukemia (ALL) including T-cell ALL and B-cell ALL. In other examples, the methods disclosed herein are applied to human patients having lymphoma, such as those disclosed herein. In yet another example, the methods disclosed herein are applied to human patients having myelodysplastic syndrome (MDS).

[0129] Acute myeloid leukemia (AML) is a blood cancer that begins in the blood and bone marrow and can progress rapidly. AML is associated with abnormal white blood cells produced by the bone marrow, which fill the blood and bone marrow and reduce the space for healthy cells. Symptoms associated with AML include bleeding and anemia. AML can be life-threatening if left untreated.

[0130] Acute lymphoblastic leukemia (acute lymphocytic leukemia or ALL) is a cancer of the bone marrow that can spread rapidly to blood cells. ALL causes the bone marrow to produce an overabundance of immature white blood cells (lymphoblasts). These abnormal cells crowd out healthy red and white blood cells, as well as platelets, in the blood and bone marrow, making it difficult for the body to fight infections and diseases. As the most common childhood leukemia and cancer, ALL typically develops before the age of 15. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of the bone marrow and accounts for approximately 20% of all ALL cases. B-cell acute lymphoblastic leukemia (B-ALL) is the most common cancer type in children and usually affects the patient's B cells.

[0131] Myelodysplastic syndromes (MDS) are conditions that can occur when the hematopoietic cells of the bone marrow become abnormal. This leads to a decrease in the number of one or more types of blood cells. In MDS, some of the bone marrow cells are abnormal (dysplastic) and have problems producing new blood cells. Many of the blood cells formed by 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 with an insufficient number of normal blood cells. Although various cell types can be affected, the most common finding in MDS is a deficiency of red blood cells (anemia).

[0132] In some cases, the subject can be a human patient having a refractory disease, such as refractory AML, refractory ALL, or refractory MDS. As used herein, "refractory" refers to a tumor that does not respond to treatment or becomes resistant thereto. In some cases, the subject can be a human patient having a recurrent disease, such as recurrent AML, recurrent ALL, recurrent MDS, or recurrent cholangiocarcinoma. As used herein, "recurrent" or "recurrence" refers to a tumor that recurs or progresses after a period of improvement (e.g., partial or complete response) by treatment.

[0133] In some cases, the subject is a human patient in whom the level of galectin-9 is elevated compared to a control level. The level of galectin-9 can 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 in cancer cells within a tumor. In other examples, the level of galectin-9 is the level of galectin-9 in immune cells within a tumor. In other examples, the level of galectin-9 can be the level of cell surface galectin-9, e.g., the level of galectin-9 on cancer cells. In one example, the level of galectin-9 can be, for example, the level of galectin-9-expressing cancer cells 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 can be the baseline level before treatment.

[0134] To identify such a subject, a suitable biological sample can be obtained from a subject suspected of having a malignant blood disorder, and the biological sample can be analyzed using conventional methods, such as ELISA or FACS, to determine the level of galectin-9 contained therein (e.g., free, cell surface expressed, or total). In some embodiments, the organoid culture is prepared, for example, as described herein and used to evaluate the subject's galectin-9 level. Single cells derived from a particular fraction obtained as part of the organoid preparation process are also suitable for evaluating the subject's galectin-9 level. In some cases, an assay for measuring the level of galectin-9 in its free form or expressed on the cell surface includes the use of an antibody that specifically binds to galectin-9 (e.g., specifically binds to human galectin-9). Any of the anti-galectin-9 antibodies known in the art can be tested for suitability in any of the above assays and then used in such assays in a predetermined manner. In some embodiments, the antibodies described herein (e.g., the G9.2-17 antibody) can be used in such assays. In some embodiments, the antibodies are described in U.S. Patent No. 10,344,091 and International Publication No. 2019 / 084553, and the respective disclosures of these are incorporated by reference for the purposes and subject matter referenced herein. In some examples, the anti-galectin-9 antibody is a Fab molecule. The assay methods for determining the galectin-9 level disclosed herein are also within the scope of the present disclosure.

[0135] In some embodiments, a human patient to be treated by any of the methods disclosed herein meets one or more of the inclusion and exclusion criteria listed in Example 1 below. For example, such a human patient may meet all of the inclusion and exclusion criteria listed in Example 1 below.

[0136] Exemplary treatment conditions In some embodiments, a pharmaceutical composition comprising an effective amount of an anti-Gal9 antibody (e.g., G9.2-17 IgG4) disclosed herein can be administered to a subject in need of treatment (e.g., a human patient who can be an adult or a pediatric) at, for example, a suitable dosage and a suitable dosing frequency. For example, the antibody can be administered to the subject 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, via a suitable route (e.g., intravenous infusion). See also the disclosure herein.

[0137] The term “about” or “approximately” means within an acceptable error range of 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 measuring system. For example, “about” can mean within acceptable standard deviations in accordance with the conventions of the art. Alternatively, “about” can mean within a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, even more preferably up to ±1% of a given value. Alternatively, especially with respect to biological systems or processes, the term can mean within an order of magnitude of the value, preferably within a factor of two. When a particular value is recited in the application and claims, the term “about” is implicit, in this context, to mean within an acceptable error range of the particular value, unless otherwise stated.

[0138] The effective amount of the pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of treatment via a suitable systemic or local route. In some embodiments, the anti-galectin-9 antibody is administered intravenously, e.g., as a bolus or by continuous infusion over a period of time, intramuscularly, intraperitoneally, intrathecally, subcutaneously, intraarterially, intraarticularly, intrasynovially, intramedullary, intratumorally, suburothelially, orally, by inhalation, or by a topical route. In one embodiment, the anti-galectin-9 antibody is administered to the subject by intravenous infusion. In one embodiment, the anti-galectin-9 antibody is administered to the subject intraperitoneally.

[0139] As used herein, "effective amount" refers to the amount of each active agent necessary to provide a therapeutic effect to a subject, alone or in combination with one or more other active agents. In some embodiments, the therapeutic effect is a decrease in galectin-9 activity and / or amount / expression, a decrease in dectin-1 signaling, a decrease in TIM-3 signaling, a decrease in CD206 signaling, and / or an increase in anti-tumor immune response in the tumor microenvironment. Non-limiting examples of an increase in anti-tumor response include an increase in the activation level of effector T cells, or a switch from the M2 phenotype of TAM to the M1 phenotype. In some cases, the anti-tumor response includes an increase in the ADCC reaction. Determination of whether the amount of the antibody has achieved a therapeutic effect will be apparent to those skilled in the art. As will be appreciated by those skilled in the art, the effective amount varies depending on individual patient parameters including the particular condition being treated, the severity of the condition, age, physical condition, size, gender, and weight, the treatment period, the nature of any combination therapy (if any), the particular route of administration, and similar factors within the knowledge and expertise of the medical practitioner.

[0140] Generally, empirical considerations such as half-life contribute to the determination of the dosage. For example, antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, are used in some cases to extend the half-life of the antibody and prevent the antibody from being attacked by the host immune system. The dosing frequency can be determined and adjusted over the course of the treatment and, although not necessarily, is generally based on the treatment and / or suppression and / or improvement and / or delay of the target disease / disorder. Alternatively, a sustained release formulation of the antibody may be appropriate. A variety of formulations and devices for achieving sustained release are known in the art.

[0141] As used herein, the term "treating" refers to the application or administration of a composition comprising one or more active agents to a subject having a target disease or disorder, a symptom of a disease / disorder, or a predisposition to a disease / disorder, and is intended to treat, cure, alleviate, mitigate, alter, remedy, improve, ameliorate, or affect the disorder, disease or symptom of the disease or disorder, or the predisposition to the disease or disorder. Non-limiting examples of treatment include reduction of tumor size, delay of tumor growth, alleviation of lesions and / or delay of lesion onset, and / or prolongation of survival in a human patient having a target malignant blood disease such as AML, MDS, or ALL (T-cell or B-cell ALL) which may be refractory and / or recurrent.

[0142] Alleviation of a target disease / disorder includes delaying the onset or progression of the disease, or reducing the severity of the disease, or prolonging survival. Alleviation of the disease or prolongation of survival does not necessarily require a therapeutic effect. As used herein, "delaying" the onset of a target disease or disorder means deferring, impeding, decelerating, suppressing, stabilizing, and / or postponing the progression of the disease. This delay can vary in time depending on the disease being treated and / or the medical history of the individual. A method of "delaying" or reducing the onset of a disease, or a method of delaying the onset of a disease, is a method of reducing the probability of developing one or more symptoms of the disease within a given time frame and / or reducing the degree of the symptoms within a given time frame as compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to obtain statistically significant results.

[0143] "Onset" or "progression" of a disease means the initial symptoms of the disease and / or subsequent progression. The onset of a disease is detectable and evaluable using standard clinical techniques well known in the art. However, development can also refer to progression that may not be detectable. For the purposes of the present disclosure, onset or progression refers to the biological course of the symptoms. "Onset" includes occurrence, recurrence, and onset. As used herein, "onset" or "occurrence" of a target disease or disorder includes initial onset and / or recurrence.

[0144] In one example, the dosage of the antibody described herein is determined empirically in an individual to whom one or more administrations (plural possible) of the antibody have been given. The individual is given escalating doses of the antagonist. To assess the effectiveness of the antibody antagonist, one can track an indicator of the disease / disorder, such as any of the disclosed indicators.

[0145] Monotherapy In some embodiments, any of the anti-Gal9 antibodies disclosed herein, such as G9.2-17 (IgG4), can be used as a monotherapy agent (the only active agent) for treating target malignant blood diseases such as AML, MDS, or ALL. Given the direct cell killing effect of the antibody on Gal9+ cancer cells, it is expected that the anti-Gal9 antibodies disclosed herein will be effective as a single therapeutic agent in the treatment of blood cancers involving Gal9+ cancer cells.

[0146] In some embodiments, the anti-Gal9 antibody can be administered to a patient once a week to once every six weeks, for example, by intravenous infusion. In some examples, the antibody can be administered once every one to four weeks, for example, every two to four weeks. In one example, the antibody can be administered once a week. In another example, the antibody can be administered once every two weeks.

[0147] In some embodiments, the anti-galectin-9 antibody disclosed herein (e.g., G9.2-17 IgG4) is administered intravenously over an infusion period of 30 minutes to 6 hours. In some examples, the intravenous infusion of the anti-galectin-9 antibody can be carried out for 30 minutes to 2 hours. In other examples, the anti-galectin-9 antibody can be administered over a long infusion period, for example, about 2 - 6 hours, for example, about 2 - 4 hours or about 4 - 6 hours. In a specific example, the anti-galectin-9 antibody can be intravenously infused for a period of about 3 hours, about 4 hours, about 5 hours, or about 6 hours.

[0148] In some embodiments, the anti-galectin-9 antibodies (e.g., G9.2-17 (IgG4)) disclosed herein for use in the treatment of malignant blood diseases (such as those disclosed herein, e.g., AML, MDS, or ALL) can be administered to a subject at a dose of 0.2 mg / kg to about 32 mg / kg. For example, the dose can be selected from dose levels of 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. In some embodiments, the anti-galectin-9 antibody 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 dose levels of 2 mg / kg, 4 mg / kg, 8 mg / kg, 12 mg / kg, 16 mg / kg, 32 mg / kg, or higher. 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 dose levels of 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.

[0149] In one example, the anti-galectin-9 antibodies disclosed herein (e.g., G9.2-17 (IgG4)) are administered to a subject weekly at a dose of 2 mg / kg. In another example, the anti-galectin-9 antibodies disclosed herein (e.g., G9.2-17 (IgG4)) are administered to a subject weekly at a dose of 4 mg / kg. In yet another example, the anti-galectin-9 antibodies disclosed herein (e.g., G9.2-17 (IgG4)) are administered to a subject weekly at a dose of 6.3 mg / kg. In yet another example, the anti-galectin-9 antibodies disclosed herein (e.g., G9.2-17 (IgG4)) are administered to a subject weekly at a dose of 7.5 mg / kg. In yet another example, the anti-galectin-9 antibodies disclosed herein (e.g., G9.2-17 (IgG4)) are administered to a subject weekly at a dose of 10 mg / kg. In yet another example, the anti-galectin-9 antibodies disclosed herein (e.g., G9.2-17 (IgG4)) are administered to a subject weekly at a dose of 12 mg / kg. In another example, the anti-galectin-9 antibodies disclosed herein (e.g., G9.2-17 (IgG4)) are administered to a subject weekly at a dose of 16 mg / kg.

[0150] In one example, the anti-Gal9 antibodies disclosed herein (e.g., G9.2-17 (IgG4)) can be given to human patients having a target malignant blood disease such as AML, MDS, or ALL at 4 mg / kg (e.g., by i.v.) once a week or once every two weeks. In another example, the anti-Gal9 antibody can be given to a human patient at 7.5 mg / kg (e.g., by i.v.) once a week or once every two weeks. In yet another example, the anti-Gal9 antibody can be given to a human patient at 12 mg / kg (e.g., by i.v.) once a week or once every two weeks. In yet another example, the anti-Gal9 antibody can be given to a human patient at 16 mg / kg (e.g., by i.v.) once a week or once every two weeks.

[0151] In some cases, a flat dosing schedule may be employed in any of the treatment methods disclosed herein. For example, anti-Gal-9 antibodies such as G9.2-17(IgG4) disclosed herein can be administered to a subject once a week to once every four weeks (e.g., once a week or once every two weeks) at a fixed dose, e.g., 410 mg to about 1120 mg. In some examples, the anti-Gal-9 antibody is administered to the subject once a week at about 410 mg to about 505 mg (e.g., about 410 mg to about 450 mg). In some examples, the anti-Gal-9 antibody is administered to the subject once every two weeks at about 410 mg to about 505 mg (e.g., about 410 to about 450 mg). In some examples, the anti-Gal-9 antibody is administered to the subject once a week at about 650 mg to about 800 mg (e.g., about 650 mg to about 700 mg). In some examples, the anti-Gal-9 antibody is administered to the subject once every two weeks at about 650 mg to about 800 mg (e.g., about 650 mg to about 700 mg). In some examples, the anti-Gal-9 antibody is administered to the subject once a week at about 1040 mg to about 1120 mg. In some examples, the anti-Gal-9 antibody is administered to the subject once every two weeks at about 1040 mg to about 1280 mg (e.g., about 1040 mg to about 1120 mg). In some examples, the anti-Gal-9 antibody is administered to the subject once a week at about 2080 mg to about 2560 mg (e.g., about 2080 to about 2400 mg). In some examples, the anti-Gal-9 antibody is administered to the subject once every two weeks at about 2080 mg to about 2560 mg (e.g., about 2080 to about 2400 mg).

[0152] In other cases, a flat dosing schedule may include from 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, or once every six weeks) and about 130 mg to about 320 mg. For example, a flat dosing schedule may include once a week and about 130 mg to about 160 mg. Alternatively, a flat dosing schedule may include once every two weeks and about 130 mg to about 160 mg. In another example, a flat dosing schedule may include once a week and about 260 mg to about 320 mg. Alternatively, a flat dosing schedule may include once every two weeks and about 260 mg to about 320 mg.

[0153] In some cases, the treatment period may be from 6 months to 12 months. In other cases, the treatment period may be from 12 months to 24 months. In other cases, the treatment period may be more than 24 months.

[0154] In some embodiments, the dosage(s) is / are adjusted according to the patient's response to the treatment. In some embodiments, the dosage is changed during the treatment intervals. In some embodiments, the treatment may be temporarily discontinued. In some embodiments, the treatment may be temporarily discontinued. In some embodiments, the anti-galectin-9 therapy is temporarily discontinued. In some embodiments, the checkpoint inhibitor therapy used in combination with the anti-galectin-9 antibody is temporarily discontinued. In some embodiments, both are temporarily discontinued.

[0155] In some cases, a human patient may start with a low dose of an anti-galectin-9 antibody such as G9.2-17 (IgG4) disclosed herein, e.g., 4 mg / kg, 6.3 mg / kg, or 7.5 mg / kg. The dosage can be increased, where applicable, to, e.g., 10 mg / kg, 12 mg / kg, 16 mg / kg, or 32 mg / kg, which can be determined by a physician. Similarly, a human patient can start with a long dosing frequency (e.g., every two weeks) and adjust to a short dosing frequency (e.g., weekly), where applicable, or vice versa.

[0156] Combination therapy In some embodiments, any of the anti-Gal9 antibodies described herein (e.g., G9.2-17 antibody such as G9.2-17 (IgG4) disclosed herein) can be used in combination with a second therapeutic agent, e.g., a chemotherapeutic agent, for treating a target malignant blood disease (e.g., AML, MDS, or ALL) disclosed herein. The selection of a suitable chemotherapeutic agent or other combination agent(s) and treatment modality can depend on various factors including the target malignant blood disease, disease severity, age, gender, treatment history, resistance to previous treatments, etc., which are within the scope of a physician's knowledge.

[0157] For example, anti-Gal9 antibodies such as G9.2-17 (IgG4) can be used in combination with a suitable chemotherapeutic agent for treating AML. Exemplary chemotherapeutic agents include azacitidine, cerubidine (daunorubicin hydrochloride), cyclophosphamide, cytarabine, daunorubicin hydrochloride and cytarabine liposomes, daurismo (glasdegib maleate), dexamethasone, doxorubicin hydrochloride, enasidenib mesylate, gemtuzumab ozogamicin, gilteritinib fumarate, glasdegib maleate, idamycin PFS (idarubicin hydrochloride), idarubicin hydrochloride, idera (enasidenib mesylate), ibosidenib, midostaurin, mitoxantrone hydrochloride, milotarg (gemtuzumab ozogamicin), onureg (azacitidine), prednisone, rubidomycin (daunorubicin hydrochloride), ridapt (midostaurin), tabloid (thioguanine), thioguanine, tibsovo (ibosidenib), trisenox (arsenic trioxide), venclux (venetoclax), venetoclax, vincristine sulfate, vikios (daunorubicin hydrochloride and cytarabine liposomes), zospata (gilteritinib fumarate), sabatolimab, or a combination thereof, but is not limited thereto. In some cases, the second therapeutic agent for use in combination with the anti-Gal9 antibody for treating AML can be a PD-1 inhibitor. Examples include anti-PD-1 antibodies (e.g., pembrolizumab, nivolumab, tislelizumab, dostarlimab, and semaprimab) or anti-PD-L1 antibodies (e.g., durvalumab, avelumab, and atezolizumab), but is not limited thereto.

[0158] In other examples, anti-Gal9 antibodies, such as G9.2-17 (IgG4), can be used in combination with suitable chemotherapeutic agents for treating MDS. Exemplary chemotherapeutic agents include azacitidine (e.g., for both low-risk and high-risk patients of all subtypes of MDS), decitabine (e.g., for both low-risk and high-risk patients of all subtypes of MDS), 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 myelodysplasia / myeloproliferative neoplasms with ring sideroblasts and thrombocytosis (MDS / MPN-RS-T) in whom in some cases erythropoiesis-stimulating agents (ESAs) have been ineffective or are likely to be non-responsive and who require more than 2 units of red blood cell (RBC) transfusions over 8 weeks), ASTX727 (e.g., for adult patients with MDS or CMML in intermediate-1, intermediate-2, and high-risk IPSS groups, including treated and untreated, newly diagnosed and secondary MDS (including patients with refractory anemia, refractory anemia with ring sideroblasts, refractory anemia with excess blasts, and CMML)), luspatercept, decitabine / cedazuridine, pebonexostat, magrolimab, or combinations thereof, but are not limited thereto.

[0159] In some embodiments, the anti-Gal9 antibody can be administered simultaneously with any of the second therapeutic agents disclosed herein. In some embodiments, the anti-Gal9 antibody can be administered prior to the second therapeutic agent disclosed herein. Alternatively, the anti-Gal9 antibody can be administered after the second therapeutic agent. In some cases, the second therapeutic agent is administered systemically. In some cases, the second therapeutic agent is administered locally. In some examples, the second therapeutic agent is administered intravenously, e.g., as a bolus or by continuous infusion over a period of time, intramuscularly, intraperitoneally, intrathecally, subcutaneously, intraarterially, intraarticularly, intravesically, intrasynovially, intramedullarily, intratumorally, or by the urothelial-submucosal route. In one embodiment, the second therapeutic agent is administered to the subject by intravenous infusion.

[0160] Monitoring of treatment response The therapeutic effect on a target malignant blood disease as disclosed herein can be evaluated by methods well known in the art.

[0161] In some embodiments, the anti-tumor activity of any of the treatment methods disclosed herein can be monitored by conventional approaches. For example, before, during, and after administration of an antibody and optionally a second therapeutic agent, the cancer cells and / or biomarkers of a subject can be measured in a biological sample such as blood, serum, plasma, urine, ascites, and / or biopsy derived from a tissue or organ. The results thus obtained can be analyzed to evaluate the therapeutic effect. For example, the levels of Gal9+ cancer cells can be measured before, during, and after treatment to evaluate the direct killing effect of an anti-Gal9 antibody such as G9.2-17 (IgG4).

[0162] Alternatively or additionally, the response to treatment can also be characterized by one or more of the immune phenotypes of blood and tumors (e.g., to evaluate the immunomodulatory effect), cytokine profile (serum), soluble galectin-9 levels in blood (serum or plasma), immunohistochemistry (tumor, stroma, immune cells), tumor mutational burden (TMB), mismatch repair status, or the expression level and expression pattern of galectin-9 in tumor tissue by a disease-related tumor marker (e.g., measured at about 3 months, 6 months, or 12 months, or later, or at any other clinically indicated time point).

[0163] In some examples, changes in the levels of immune cells and immune cell markers in the blood or tumor, such as immune activation, can be determined before and after treatment. Such changes can be measured in a patient's blood and tissue samples using methods known in the art such as multiplex flow cytometry and multiplex immunohistochemistry. For example, a panel of phenotypic and functional PBMC immune markers can be evaluated at baseline before treatment initiation and at various time points during treatment. Table 2 lists non-limiting examples of markers useful for these assessment methods. Flow cytometry (FC) is an optimal technique that provides fast and useful information for analyzing the phenotype and function of cells and has attracted attention in immune phenotype monitoring. It enables the characterization of many subsets of cells, including rare subsets, in complex mixtures such as blood and represents a way to rapidly acquire large amounts of data. The advantages of FC are speed, sensitivity, and specificity. Standardized antibody panels and procedures can be used to analyze and classify immune cell subtypes. Multiplex IHC is a powerful investigative tool that provides objective quantitative data explaining the immune status of tumors in both the number and location of immune subsets and allows the evaluation of multiple markers on a single tissue section. Computer algorithms can be used to combine chromogenic IHC methods and staining with a digital pathology approach to quantify IHC-based biomarker content from whole-slide images of patient biopsies.

[0164]

Table 2

[0165] A subject being treated with any of the anti-Gal9 antibodies (e.g., G9.2-17) disclosed herein, alone or in combination with a second therapeutic agent (e.g., a chemotherapeutic agent) disclosed herein, can be monitored for the occurrence of adverse effects (e.g., severe side effects). Exemplary adverse effects to be monitored are shown in Example 1 below. If the occurrence of an adverse effect is observed, the treatment conditions can be changed for that subject. For example, the dose of the anti-galectin-9 antibody can be reduced and / or the dosing interval can be extended. The appropriateness and extent of the reduction can be evaluated by a qualified clinician. In some embodiments, one or more dose reductions of from about 10% to about 80% of the previous dose level can be implemented. In some embodiments, one or more dose reductions of from about 10% to about 20%, from about 20% to about 30%, from about 30% to about 40%, from about 40% to about 50%, from about 50% to about 60%, or from about 70% to about 80% of the previous dose level can be implemented. In some embodiments, one or more dose reductions of from 10% to 20%, from 20% to 30%, from 30% to 40%, from 40% to 50%, from 50% to 60%, or from 70% to 80% of the previous dose can be implemented. In some embodiments, one or more dose reductions of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% of the previous dose can be implemented. In some embodiments, one or more dose reductions of 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the previous dose level are implemented. Alternatively or additionally, the dose of the second therapeutic agent can be reduced and / or the dosing interval of the checkpoint inhibitor can be extended. In some cases (e.g., the occurrence of life-threatening adverse effects), treatment can be discontinued.

[0166] Any of the methods disclosed herein can include reducing the dose of the anti-Gal9 antibody if one or more adverse events (e.g., those disclosed herein) are observed during treatment.

[0167] IV. Kits for Use in Treating Diseases Associated with Galectin-9 The present disclosure also provides kits for use in treating or alleviating malignant blood diseases (such as, for example, AML, MDS, or ALL) such as those disclosed herein. Such kits can include an anti-Gal9 antibody, such as any of those described herein (e.g., G9.2-17 (IgG4)), and optionally, one or more containers containing a second therapeutic agent (e.g., a second therapeutic agent disclosed herein) (also described herein) to be used in combination with the anti-Gal9 antibody.

[0168] In some embodiments, the kit can include instructions for use to be used according to any of the methods described herein. The instructions included can include instructions for administration of the anti-Gal9 antibody, and optionally, a second therapeutic agent, for treating the target disease described herein, delaying its onset, or alleviating it. In some embodiments, the kit further includes instructions, for example, for selecting a suitable individual for treatment based on identification, using, for example, diagnostic methods described herein, of whether an individual is suffering from the target disease. In still other embodiments, the instructions for use include instructions for administration of the antibody to individuals at risk of the target disease.

[0169] Instructions for use regarding the use of the anti-Gal9 antibody typically include information regarding dosage, dosing schedule, and route of administration for the intended treatment. The container can be a unit dose, a bulk package (e.g., a multi-dose package), or a sub-unit dose. The instructions for use provided in the kits of the present invention are typically instructions written on a label or package insert (e.g., a paper sheet included in the kit), although instructions for use that are machine-readable (e.g., instructions recorded on a magnetic or optical storage disk) are also acceptable.

[0170] The label or package insert indicates that the composition is for use in treating, delaying the onset of, and / or alleviating the target malignant blood disease. In some embodiments, instructions for use for performing any of the methods described herein are provided.

[0171] The kit of the present invention is present in a suitable packaging. Suitable packagings include, but are not limited to, vials, bottles, flasks, flexible packagings (e.g., sealed Mylar or plastic bags). Also contemplated are packages for use in combination with a particular device, e.g., an inhaler, a nasal administration device (e.g., an atomizer), or an infusion device, e.g., a minipump. In some embodiments, the kit has a sterile access port (e.g., the container can be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). In some embodiments, the container also has a sterile access port (e.g., the container is an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an anti-Gal9 antibody as described herein.

[0172] The kit can optionally provide additional components such as buffers and interpretive information. Typically, the kit includes a container and a label or package insert(s) on or associated with the container. In some embodiments, the present invention provides a product comprising the contents of the above kit.

[0173] General procedures The practice of the present invention, unless otherwise indicated, uses conventional techniques in the fields of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology that are within the scope of the relevant art. Such techniques are fully described in the following documents: for example, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8) J.Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988 - 1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

[0174] Without further elaboration, it is believed that one of ordinary skill in the art can, based on the above description, make maximum use of the present invention. Accordingly, the following specific embodiments should be construed as merely illustrative and not limiting in any way the remainder of the disclosure herein. All publications cited herein are hereby incorporated by reference for the purposes or subject matter referred to herein.

Example

[0175] 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) G9.2-17(IgG4) is a fully human immunoglobulin gamma (IgG)4 monoclonal antibody (mAb) that targets carbohydrate recognition domain 2 (CRD2) of the galectin-9 (gal-9) protein and is being developed for the treatment of patients with recurrent and refractory solid tumors and hematologic malignancies. Gal-9 plays an important regulatory role in the anti-tumor immune response. Gal-9, which is overexpressed and / or secreted in many cancer types (solid tumors and hematologic malignancies), functions as an immunosuppressive factor, controlling macrophages, T cells, natural killer (NK) cells, and myeloid-derived suppressor cells (MDSC), and conferring immune privilege to tumor cells and abrogating immune-mediated cancer attack by interfering with the sensitivity of cancer cells to cytotoxic T cell-induced cell death. G9.2-17(IgG4) is designed to induce gal-9 blockade and interfere with the immunosuppressive function of gal-9, which may result in immune reactivation and inhibition / control of tumor growth.

[0176] G9.2-17(IgG4) is expected to benefit the treatment of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), such as recurrent and / or refractory AML or MDS, at least with respect to the following: · A favorable safety profile. · Potential immunomodulation in the periphery and bone marrow monitored by PD and efficacy assessments. · Clinical improvement in patients.

[0177] A schematic diagram of the study is shown in Figure 1. An overview of the study objectives and endpoints is shown in Table 3 below.

[0178]

Table 3-1

Table 3-2

Table 3-3

[0179] I. Objectives of the Study Primary Objectives · Establish the safety and tolerability of G9.2-17 (IgG4) as a single agent · Confirm the dose(s) of G9.2-17 (IgG4) in the second phase (R2PD) · Determine the incidence of DLT across dose levels during Cycle 1 (28 days)

[0180] Secondary Objectives · Determine the preliminary efficacy of G9.2-17 (IgG4) as a single treatment · Characterize the pharmacokinetic (PK) profile of G9.2-17 (IgG4)

[0181] Exploratory Objectives · Test pharmacodynamic markers, including immunological and molecular changes in peripheral blood, bone marrow, and serum Gal-9 · Evaluate the immunogenicity of G9.2-17 (IgG4)

[0182] II. Study Endpoints Primary Endpoints · Evaluate safety parameters including adverse events, vital sign measurements, clinical safety laboratory tests, 12-lead electrocardiogram (ECG), echocardiogram / cardiac ultrasound (ECHO), and physical examination · Determine the biologically effective dose of G9.2-17 (IgG4) · Determine the maximum tolerated dose (MTD) of G9.2-17 (IgG4) · Determine the RP2D dose(s) of G9.2-17 (IgG4) · Evaluate DLT during Cycle 1

[0183] Secondary Endpoints The secondary endpoints for determining preliminary efficacy (disease response) are as defined in Tables 4 and 5 below:

[0184]

Table 4-1

Table 4-2

[0185]

Table 5-1

Table 5-2

[0186] PK endpoints Secondary PK endpoints include the evaluation of the PK parameters of G9.2-17 (IgG4) (including, but not limited to, the area under the curve [AUC 0-168h from time 0 to 168 hours, the observed maximum serum concentration [C max , and the time to reach C max [T max , and the trough serum concentration [C トラフ .

[0187] Exploratory endpoints The exploratory endpoints are shown in Tables 6 and 7 below.

[0188]

Table 6

[0189]

Table 7

[0190] Exploratory immunological and molecular changes at endpoints in peripheral blood and bone marrow · Serum / plasma cytokine multiplexing · Mass cytometry whole blood immune profiling · Whole blood gene array · Gal-9 in serum by ELISA

[0191] Exploratory Immunogenicity Endpoints · Evaluation of ADA

[0192] III. Study Design This is a non-blind, non-randomized, multi-center, phase 1 dose-escalation study in patients with AML who are refractory / relapsed to at least one prior therapy, with or without allogeneic hematopoietic stem cell transplantation, or who have a documented diagnosis of high-risk MDS that is refractory / relapsed after at least one prior therapy and for whom standard therapies that may provide clinical benefit are not available. A 4+2 algorithm-based dose-escalation design (Lin et al., Biostatistics, 2001; Wheeler et al., PLoS One, 2016) is used to establish the DLT and facilitate the identification of the RP2D. This study will be conducted at approximately 15 sites in the United States. The study duration is estimated to be 24 months. Survival follow-up will continue for up to 2 years.

[0193] The outline of the study design is shown in Figure 1, and the visit schedule and planned evaluations at each visit are detailed in Table 8.

[0194] Dose Escalation and De-escalation Treatment Periods This study consists of the following periods: Screening Period: Up to 2 weeks prior to the first dose (-14 days to -1 day) Treatment Period: A 28-day treatment cycle (SoA) as presented in the evaluation schedule (Table 8) Post-treatment Period: 30 days after the last treatment (end of treatment visit / early termination visit) Follow-up Period: Long-term follow-up for up to 2 years (every 3 months).

[0195] Dose Escalation This dose - setting trial was conducted using a 4 + 2 algorithm - based dose - escalation design ((Lin et al., Biostatistics, 2001; Wheeler et al., PLoS One, 2016)) to determine the MTD or biologically active dose. This helps to identify the dose(s) of RP2D. The definition of a biologically effective dose is the dose at which 0 - 100% of the 6 patients in a cohort experience the following: · In the case of AML: either molecular remission of minimal residual disease (MRD) or a decrease to 5% or less of blasts (absence of circulating blasts with Auer rods). · In the case of MDS: either a transfusion - independent period of at least 8 weeks or a decrease to 5% or less of blasts.

[0196] Up to 6 patients per treatment cohort 1 - 5 start at a dose of 2.0 mg / kg and receive a 60 - minute intravenous (IV) infusion of G9.2 - 17 (IgG4) on days 1, 8, 15, and 22 of each 28 - day cycle, once weekly (QW).

[0197] Patients assigned to a specific dose - escalation cohort are administered the study dose corresponding to that cohort. They are administered the investigational drug until disease progression, unacceptable toxicity, or withdrawal from the trial for other reasons. Only patients who withdraw for reasons other than toxicity or tolerance issues during the first treatment cycle are replaced.

[0198]

Table 8 - 1

Table 8 - 2

Table 8 - 3

Table 8 - 4

Table 8 - 5

Table 8-6

[0199] A total of five dose levels were evaluated within the 4+2 design, with the starting dose being 2.0 mg / kg QW: · Dose escalation cohort 1 = 2.0 mg / kg QW (maximum n = 6) · Dose escalation cohort 2 = 4.0 mg / kg QW (maximum n = 6) · Dose escalation cohort 3 = 7.5 mg / kg QW (maximum n = 6) · Dose escalation cohort 4 = 12.0 mg / kg QW (maximum n = 6) · Dose escalation cohort 5 = 16.0 mg / kg QW (maximum n = 6).

[0200] One cycle (28 days) consists of four doses of G9.2-17 (IgG4).

[0201] The start of the next higher dose cohort is based on an analysis of patient safety data focused on the occurrence of DLT, other relevant safety, and available PK data from the previous cohort. Skipping of dose levels is not permitted.

[0202] Patients treated before the RP2D is identified can escalate to the highest dose level if they clear the 28-day DLT period of the originally enrolled cohort. Dose escalation may not occur during the course of a cycle. Patients can continue to escalate to the approved highest dose level until they are discontinued due to toxicity, disease progression, or other reasons (e.g., the patient chooses to discontinue the study). They may escalate only after completing the full cycle at the current dose level, not during the course of the cycle.

[0203] If no clinical benefit has been demonstrated, the duration of the investigational drug is limited to 6 cycles, i.e., 6 months. If a patient has not achieved a minimal PR as a clinical benefit, the patient discontinues the investigational drug even in the absence of unacceptable toxicity. If a patient has shown at least a PR as a clinical benefit within 6 cycles (6 months) from the start of the investigational drug, that patient can continue the administration beyond 6 months until disease progression, patient refusal, or unacceptable toxicity occurs.

[0204] If DLT occurs in any patient during the first 28 days of treatment, that patient permanently discontinues the administration of the investigational drug.

[0205] For patients who experience toxicity (including IMAR) outside the DLT window, dose reduction is permitted only if the principal investigator assesses that a clinical benefit has been obtained and can continue to be obtained even with a lower dose of G9.2-17 (IgG4). The dose of G9.2-17 (IgG4) is first reduced by 50% according to the definition of dose change guidance presented in the protocol, and may be further reduced by 50%. Further dose reduction is not permitted. If a patient discontinues for reasons other than toxicity or tolerability issues only during the first treatment cycle, replace until the MTD is identified.

[0206] Dose escalation (cohort-1 only) Only during cycle 1 of cohort 1 (2.0 mg / kg G9.2-17 (IgG4)), a new cohort 1 can be started with a lower dose of G9.2-17 (IgG4) as follows: · If two or more patients reach DLT, a lower-dose cohort 1 may be started at a lower dose. · If signs of clinical benefit are observed in cohort 1 based on secondary, exploratory endpoints, or reduction of other markers of disease activity such as myeloblasts that have decreased by more than 50% compared to pre-treatment, a lower-dose cohort 1 may be added to explore the dose-response relationship as an element of dose selection for further development. The lower-dose selection is based on available PK, PD data, and markers of disease activity.

[0207] Completion of dose escalation based on the 4 + 2 algorithm Dose escalation based on the 4 + 2 algorithm is completed when one or more of the evaluated G9.2-17 (IgG4) doses are identified as the RP2D dose(s).

[0208] Dose-limiting toxicity criteria The DLTs evaluated in this study are defined as clinically significant hematological and / or non-hematological AEs, or abnormal test values that clearly do not result from underlying leukemia or external causes during the first cycle (28 days) of the study. Patients who experience DLTs during the first 28 days of treatment have their administration of the investigational agent permanently discontinued.

[0209] DLT is a toxicity that meets any of the following criteria: · Any death clearly not due to underlying leukemia or external causes · Signs of potential drug-induced liver injury, as follows (in the case of Hy's Law): o ALT or AST exceeds 3 times the upper limit of normal (ULN) and is confirmed by repeat testing after 24 hours, and o Serum total bilirubin (TBL) exceeds 2 × ULN (confirmed by repeat testing after 24 hours) o For elevations in TBL and / or aminotransferase (AT), such as viral hepatitis (A, B, or C), alcoholic hepatitis, or autoimmune hepatitis, existing or acute liver disease, biliary obstruction or duct disease, Gilbert's syndrome, disease progression, or another pharmaceutical that may cause the observed effects, no other explanation can be found. · All grade ≥ 4 hematotoxicities lasting more than 7 days and clearly not due to underlying leukemia or external causes. Events beyond the 28-day DLT monitoring period are also counted. All grade 3 - 5 hematotoxicities clearly not due to underlying leukemia or external causes, with the following exceptions: · Grade 3 fatigue, asthenia, fever, anorexia (Grade 3 anorexia can only be excluded if it does not result in hospitalization, use of tube feeding, or total parenteral nutrition), or constipation. · Grade 3 nausea, vomiting, or diarrhea that does not require tube feeding, total parenteral nutrition, or hospitalization or extension of hospitalization. · Infections, bleeding, or other expected direct complications due to underlying active leukemia cytopenia. · Grade 3 infusion reactions (including cytokine release syndrome (CRS)) if managed successfully and recovered within 72 hours. · Grade 3 or 4 TLS if clinically managed successfully and recovered within 7 days without peripheral organ damage. · Grade 3 or 4 single electrolyte abnormalities lasting less than 72 hours are not considered DLTs.

[0210] End of the trial The end of the study is defined as the point at which all patients have been treated with G9.2-17 (IgG4) until the RP2D or dose is identified, disease progression is confirmed, or treatment is discontinued for other reasons, and all patients have completed the OS follow-up.

[0211] Follow up the patients for OS for up to 2 years after the last administration of G9.2-17 (IgG4).

[0212] The end date of the trial is defined as the last visit date of the last patient.

[0213] Trial stopping rules The dose escalation phase of the 4+2 design is completed when one of the doses or intermittent doses in cohorts 1-5 is identified as the RP2D. The RP2D is determined based on the MTD, DLT, PK, PD, and additional safety data observed at each dose level, and any other factors that may be considered by the trial sponsor. If the lower limit of the Agresti and Coull CI for the lowest study dose level exceeds the target DLT rate, the trial is stopped for safety (Agresti et al., The American Statistician, 1998).

[0214] IV. Test Population Inclusion Criteria 1. Patients aged 18 years or older at the time of obtaining informed consent. 2. Patients with morphologically documented primary or secondary AML according to the World Health Organization (WHO) criteria (Arber et al., Blood, 2016), who are refractory / relapsed to at least one prior therapy, regardless of the presence or absence of allogeneic stem cell transplantation, and for whom standard treatment that can provide clinical benefit is not available or has been refused. 3. Patients with a documented diagnosis of high-risk myelodysplastic syndrome (MDS), who are refractory / relapsed after at least one prior therapy based on the Revised International Prognostic Scoring System (IPSS-R) (Greenberg et al., Blood, 2012), and for whom standard treatment that can provide clinical benefit is not available. 4. The patient can understand, sign, and date a written informed consent form at the time of screening visit before any protocol-specific procedures. 5. The patient can comply with and is willing to comply with the test procedures according to the protocol, including bone marrow biopsy. 6. The patient's ECOG performance status is 2 or less. 7. The interval between the patient's pre-treatment and the time of test drug administration is at least 2 weeks for cytotoxic drugs (excluding hydroxyurea administered for cytoreduction), or at least 5 half-lives for previous investigational or non-cytotoxic drugs. If the patient has recovered from clinically relevant safety issues and has recovered from grade 1 or less toxicity from prior therapy, a shorter washout period than the described washout period may be considered after consultation with the medical monitor. 8. The patient must meet the following criteria shown by clinical examinations: a. The WBC count at the first administration is less than 25,000 / uL. b. Aspartate aminotransferase or alanine aminotransferase is less than or equal to 3 × ULN (less than or equal to 5.0 × ULN if considered due to leukemia involvement). c. Total bilirubin is less than or equal to 2 × ULN (less than or equal to 3 × ULN if considered due to leukemia involvement or Dubin-Johnson syndrome). d. Creatinine clearance is 60 mL / min or more. 9. The patient's minimum remaining life expectancy is 3 months or more. 10. Female patients must meet one of the following: i) Postmenopausal before screening (defined as no menstruation for at least 1 year), or the state after surgical sterility or hysterectomy is documented (at least 1 month before screening), ii) If pregnancy is possible, a negative pregnancy test in serum or urine must be obtained at screening and, starting at screening, throughout the study period, and for 90 days after the last dose of the study drug, two forms of contraception (at least one of which must be a barrier method) must be used. 11. Female patients must not breastfeed at the time of screening, during the study period, and for 90 days after the last dose of the study drug. 12. Female patients must not donate eggs starting at screening, throughout the study period, and for 90 days after the last dose of the study drug. 13. Male patients and the spouses / partners of their female partners who are capable of pregnancy must use a highly effective contraceptive method consisting of two forms of contraception (one of which must be a barrier method) starting at screening.

[0215] Exclusion Criteria Subjects who meet any of the following exclusion criteria are not eligible to be included in this study: 1. Patients diagnosed with acute promyelocytic leukemia (APL). 2. The patient has an active malignancy other than AML. Participants with a history of a malignancy that has been appropriately treated and for which anti-cancer systemic therapy (i.e., chemotherapy, immunotherapy, small molecule inhibitors, radiation therapy, or surgery) has not been continued or is not required during the course of the trial. Participants receiving hormonal therapy or adjuvant therapy such as zoledronic acid or denosumab are eligible. 3. The patient has persistent non-hematological toxicity of grade 2 or higher (CTCAE v5.0) and has symptoms and objective findings due to previous AML treatment (including chemotherapy, kinase inhibitors, immunotherapy, investigational drugs, radiation, hematopoietic stem cell transplantation [HSCT], or surgery). 4. The patient has received HSCT and meets any of the following: received HSCT within 6 months prior to the first study dose; has persistent non-hematological toxicity of grade 2 or higher related to donor lymphocyte infusion of the transplant. 5. The patient has active graft-versus-host disease (GVHD) and is receiving immunosuppressive treatment for GVHD. Calcineurin inhibitors require at least 4 weeks of washout prior to screening. 6. The patient has symptomatic central nervous system (CNS) involvement due to other CNS diseases related to the underlying and secondary effects of leukemia or malignancy. 7. The patient has disseminated intravascular coagulation disorder (DIC). 8. The patient has undergone major surgery within 4 weeks prior to the first study dose. 9. The patient has received radiation therapy within 4 weeks prior to the first study dose. 10. The patient has congestive heart failure of New York Heart Association (NYHA) class 3 or 4 or has a history of congestive heart failure of NYHA class 3 or 4 in the past (except when the left ventricular ejection fraction (LVEF) is 45% or higher as determined by a screening echocardiogram or multigated (MUGA) scan performed within 3 months prior to participation in the trial). 11. The patient has any factor that increases the risk of QTc prolongation or the risk of arrhythmia events, such as i) rhythm, ii) conduction, iii) the morphology of the resting ECG (e.g., complete left bundle branch block, second-degree heart block, second-degree heart block, PR interval exceeding 250 milliseconds); and / or iv) congenital long QT syndrome or a family history of long QT syndrome that is not medically managed, based on the judgment of the responsible investigator of the clinical trial. 12. The patient is known to have a symptomatic active infection, including any identified active COVID-19 infection. 13. The patient is known to have a known human immunodeficiency virus infection. 14. The patient has a known active hepatitis B or C, or other active liver impairment. 15. The patient has any condition that, in the opinion of the responsible investigator of the clinical trial, makes the patient unsuitable for participation in the trial. 16. Any other medical, mental, or social condition that, in the opinion of the responsible investigator of the clinical trial, may interfere with trial participation or compliance, or endanger the safety of the patient. 17. A patient who has no intention or is unable to comply with the protocol. 18. Live vaccines administered within 30 days before the start of treatment. 19. A history of severe hypersensitivity reactions to any monoclonal antibody (mAb) and / or their excipients. 20. Known hereditary or acquired hemorrhagic disorders. 21. Active bleeding. 22. A patient who has a new thrombosis, embolism, cerebral hemorrhage, or other disease, or a medical history within 1 year before enrollment. 23. Substance abuse or long-term alcohol abuse that affects the evaluation results. 24. Progressive autoimmune diseases requiring systemic therapy (except for autoimmune hypothyroidism treatable with synthetic thyroid hormone).

[0216] V. Description of Trial Evaluation Demographics and Other Screening Evaluations During screening, collect patient background. These include age, gender, race, and ethnicity. The safety assessment, which is also part of the screening evaluation, is described below.

[0217] Medical history The medical history includes oncology history, surgery / transplant history, radiotherapy history, as well as COVID-19 history and tests. · Personal history including prior treatments / surgeries (record of any in situ implants or past implants, prior and / or current use of medical devices, concomitant medications (name, indication, dose, route, dose changes if any starting and ending dates and reasons), existing symptoms, and AEs), family history based on the patient's best knowledge and hereditary diseases at risk based on the complete family history). · Records of dental treatments performed in the past 12 months · In the case of patients with previously resected pancreatic adenocarcinoma, record whether the primary tumor was located in the head, body, or tail of the pancreas. · Bowel habits / typical frequency and consistency · Record any dietary requirements or preferences (e.g., practice of specific diets: intermittent fasting, keto diet, etc.). · Records of past and current allergies (allergen, severity)

[0218] ECHO / MUGA ECHO and / or MUGA are obtained at the time points shown in the SoA (Tables 5 - 6). If clinically necessary, the evaluation is repeated once every 3 months.

[0219] ECOG The ECOG performance status is evaluated using the following grading at the time points shown in the SoA (Table 8): · Grade 0: Completely active and able to continue all pre-disease performance without limitation. · Grade 1: Strenuous physical activity is limited, but able to walk and perform light work or sedentary jobs, e.g., light housework, office work. · Grade 2: Able to walk, can perform all self-care, but unable to perform any work activities. More than about 50% of waking hours are able to move vigorously · Grade 3: Can only perform limited self-care, restricted to bed or chair for more than 50% of waking hours · Grade 4: Completely physically disabled. Unable to continue any self-care. Completely restricted to bed or chair · Grade 5: Death.

[0220] Efficacy assessment Efficacy assessment (secondary) The efficacy assessment is derived from the recommendations of the latest International Working Group (IWG) and Key Opinion Leaders regarding AML and MDS. The efficacy endpoints and definitions for AML and MDS are provided herein. All endpoints for both AML and MDS are derived from four types of assessments collected according to the SoA (Table 8): · Hematology (usual components of clinical laboratory evaluation; see below) · MRD MRD includes central multiparameter flow cytometry (MFC) in blood with respect to immunophenotypic markers and is performed according to the SoA (Table 8) · Bone marrow biopsy / aspiration Bone marrow aspirates are used for MRD analysis by RT-qPCR / NGS molecular genetic analysis and, according to the SoA, also include MFC for lao (Table 8). Bone marrow biopsies are locally evaluated to assess the degree and nature of bone marrow cellularity. After cycle 3, if not medically feasible, bone marrow biopsy / aspiration may not need to be completed. · Hematology blood smear Hematology blood smear specimens (stained for myeloperoxidase [MPO], Sudan black, alpha-naphthyl acetate esterase [NAE], and periodic acid Schiff [PAS]) are used to determine the following: · % of blasts · Morphology of blasts with particular focus on whether Auer rods are present (typical of AML) · Dyshematopoiesis Hematological blood smear specimens are performed locally according to the SoA (Table 8).

[0221] Safety evaluation Adverse events See the following disclosure.

[0222] Clinical laboratory evaluation Patients have blood samples collected for specified clinical tests according to the SoA (approx. 5 mL at each time point) (Table 8); additional tests may be performed at any time during the study if determined necessary by the principal investigator of the clinical trial or if required by local regulations. Protocol-specific requirements for patient inclusion or exclusion are detailed above.

[0223] Clinical laboratory parameters are analyzed at the local testing facility of the site. Completed laboratory evaluations include hematology and serum chemistry, defined as follows: · Serum chemistry: glucose, total protein, albumin, electrolytes [sodium, potassium, chloride, magnesium, phosphorus], calcium, bilirubin (total, direct), SGPT (ALT) or SGOT (AST), alkaline phosphatase, gamma-glutamyl transferase (gamma GT), lactate dehydrogenase (LDH), creatinine, hemoglobin A1c (HgbA1c) (only if there is a history of type 1 or type 2 diabetes), blood urea nitrogen, creatine phosphokinase (CPK). o Thyroid-stimulating hormone (TSH), fT4, lipase, amylase, PTH, FSH, luteinizing hormone (LH), free cortisol additionally specified at designated visits o Fasting blood glucose is evaluated only if clinically necessary. · Hematology: complete blood count, differential, platelets, hemoglobin. · Coagulation: PT and PTT, activated partial thromboplastin time (APTT) and INR (for acceptable anticoagulants), C-reactive protein (CRP), and troponin. · Urine test: The patient collects a urine sample for a predetermined urine test. The urine test includes color, appearance, and specific gravity gauge, protein, leukocyte esterase, glucose, ketone, urobilinogen, nitrite, WBC, RBC, as well as pH, and urine culture (if the patient is clinically symptomatic).

[0224] Abnormal test findings related to underlying diseases are considered clinically insignificant unless they are judged to be more severe than expected for the participant's condition.

[0225] All clinical tests with values considered clinically significantly abnormal during the study or within 30 days after the last dose of the study drug need to be repeated until the values return to normal or baseline or are considered clinically insignificant.

[0226] If clinically significant values do not return to normal / baseline or Grade 1 within the period, the cause must be identified.

[0227] All clinical tests required by the protocol must be performed according to the test manual and SoA (Table 8).

[0228] If test values of clinical tests not specified in the protocol, performed by the local testing institution of the facility, require a change in the management of the participant or are considered clinically significant (e.g., SAE or AE or dose change), the results must be recorded.

[0229] 12-lead electrocardiogram The 12-lead ECG is obtained using an ECG machine that automatically calculates the heart rate and measures the heart rate, PR interval, QRS duration, the time distance in the ECG from the start of the QRS complex to the end of the T wave (QT) interval, and the QTcF interval, as outlined in the SoA (Table 8).

[0230] Vital signs Vital signs are measured after 5 minutes of rest in the supine position according to SoA (Table 8) and include body temperature, blood pressure (systolic and diastolic), heart rate, and respiratory rate.

[0231] To monitor the G9.2-17 (IgG4) infusion reaction on Day 1 of Cycle 1, vital signs must be recorded every 15 minutes during the 1-hour infusion of the antibody and then every 30 minutes for 1 hour after the infusion, and then hourly if applicable, thereafter.

[0232] Physical examination Medical and physical examinations must be performed by a qualified physician, nurse, or physician assistant and must include a thorough examination of all body systems. Additionally, height is measured only at screening for the determination of body surface area. Weight is included at all scheduled test times. Neurological examinations are performed only on patients with stable and / or pre-treatment brain metastases.

[0233] Monitoring and management of tumor lysis syndrome To monitor and manage potential TLS, the following precautions are implemented: · In addition to the blood sampling scheduled on Day 1 of Cycle D1, a second blood sampling is performed on Day 2 of Cycle 1 to appropriately monitor clinical laboratory parameters related to TLS during the first week of G9.2-17 (IgG4) treatment. · Patients can be evaluated for the risk of TLS prior to each G9.2-17 (IgG4) infusion and, if clinically necessary (e.g., if there has been an experience of TLS in a previous treatment plan), can be treated according to the facility's standard treatment, such as appropriate hydration and uric acid-lowering drugs, prior to the start of G9.2-17 (IgG4) infusion. · If the disease burden of the subject is high and the risk of TLS is high, it may be appropriate to maintain / contain them for additional monitoring after G9.2-17 (IgG4) administration.

[0234] Pharmacokinetics If possible, calculate the following serum PK parameters for G9.2-17 (IgG4): ·AUC 0~168時間 ·C max ·T max ·Serum concentration vs. time profile

[0235] Collect approximately 5 mL of blood samples and process them into serum at each time point specified by the SoA (Table 8). A complete list of PK parameters will be provided in the statistical analysis plan.

[0236] Pharmacodynamic biomarker The planned time points for biomarker evaluation are provided by the SoA (Table 8), and sampling may decrease until the third cycle 6 months after treatment. The following samples are required for the biomarker study and will be collected from all participants in the trial as specified by the SoA · Blood samples to be collected prior to administration of the test article (approximately 15 mL pre-dose) · Samples will be tested for PD biomarkers (by flow cytometry, ELISA, IHC, or multiplex phenotyping) to evaluate their association with the observed clinical response to G9.2-17 (IgG4) using a validated assay.

[0237] In this trial, the following biomarkers are planned to be evaluated (note that the following list includes expected biomarkers but is not limited to these): · Multiplexing of serum / plasma cytokines · Mass cytometry whole blood immune profiling · Whole blood gene array · Gal-9 in serum by ELISA

[0238] Immunogenicity assessment Blood samples (approximately 3 mL) will be collected from all participants according to the SoA (Table 8) and processed into serum. Additionally, serum samples should also be collected from patients who discontinued the test article or withdrew from the trial at the end / early termination of treatment visit.

[0239] VI. Treatment Preparation, Handling, and Storage G9.2-17(IgG4) is supplied in single-use vials. The G9.2-17(IgG4) formulation is diluted to the target dose prior to administration. All dilutions should be performed in a controlled aseptic environment (patient dose is prepared and administered by IV infusion over approximately 60 minutes).

[0240] Dosage and Administration Administer G9.2-17(IgG4) to all patients. Administer G9.2-17(IgG4) by IV infusion QW over approximately 60 minutes. Record the start and duration of the infusion.

[0241] In accordance with the 4+2 design, start at 2.0 mg / kg and sequentially increase the dose to administer G9.2-17(IgG4) as a single agent to patients.

[0242] Patients who experience DLT in Part 1 do not resume treatment.

[0243] Dosage Administration If a reaction related to the infusion occurs, interrupt the infusion and administer the relevant pharmaceutical(s) (e.g., antihistamine, antiemetic, steroid, antipyretic, beta blocker(s), etc.) if clinically necessary. If it is determined appropriate to resume the infusion, resume at a slower infusion rate. Record the new rate, as well as the time of infusion cessation and resumption when resuming the infusion.

[0244] In subsequent cycles for the same patient, consider applying appropriate premedication (e.g., antihistamine, antiemetic, steroid, antipyretic, beta blocker(s), etc. if clinically necessary) and using a slower infusion rate.

[0245] Dose Modification The decision to proceed to the next dose level of G9.2-17 IgG4 is based on safety, tolerability, and preliminary PK data obtained in at least four patients at the previous dose level. The dosing schedule can also be adjusted based on the PK data obtained.

[0246] The detailed dose change procedures described in Tables 9 and 10 are available.

[0247]

Table 9-1

Table 9-2

Table 9-3

Table 9-4

Table 9-5

[0248]

Table 10

[0249] Administration Delay If there may be a relationship to one or more investigational agents or a clinically meaningful grade 3 or higher AE related to the investigational agent occurs, consult with the medical monitor before continuing administration. In the case of grade 3 or higher AE, an administration delay may be necessary.

[0250] Dose Reduction For patients being evaluated for DLT (within the 28-day DLT window), dose reduction is not permitted.

[0251] Dose reduction is only permitted if there is a clinical benefit and it can be continuously obtained under dose reduction conditions. See Table 9 (for IMAR) or Table 9 (for other AEs).

[0252] Dose change of IMAR If IMAR occurs, see Table 9 (IMAR) for guidance on dose management of G9.2-17 (IgG4).

[0253] All relevant medical examinations / tests should be performed to confirm that the adverse event is IMAR.

[0254] Discontinuation of investigational drug Rarely, the patient may need to permanently discontinue the investigational drug. If the investigational drug is permanently discontinued for reasons other than disease progression and the patient is not being treated with other anti-cancer therapy(ies), continue to evaluate the patient for disease progression for up to 2 years. See Table 8 (SoA) for data to be collected at the time of discontinuation of the investigational drug and follow-up, and for any additional optional evaluations that need to be completed.

[0255] The investigator must make every effort to continue the patient's study treatment until one of the reasons for discontinuation of study treatment (disease progression, toxicity related to the investigational drug, withdrawal of consent) is met. If the patient has progression on imaging but no clear clinical progression and no alternative treatment is initiated, the patient may continue study treatment at the discretion of the study responsible physician. However, if the patient has no progression on imaging but has clear clinical progression, study treatment should be stopped and the patient should be advised of the available treatment options.

[0256] The patient may be discontinued prior to disease progression for any of the following reasons: · DLT · AE occurs / recurs outside the DLT window where discontinuation of study treatment(s) is required · IMAR occurs / recurs where discontinuation of study treatment(s) is required ·Termination of the trial by PureTech Health, LLC ·Concurrent diseases or conditions that may endanger the patient's safety if they prevent further administration of the treatment or if the trial treatment is continued ·At the discretion of the patient or the principal investigator of the clinical trial ·Pregnancy ·Use of non-protocol anti-cancer therapy ·Serious deviation from the protocol by the patient (including lack of compliance) ·Serious protocol violation by the principal investigator of the clinical trial

[0257] The reason for the patient to discontinue the trial treatment should be documented in the CRF. If the patient discontinues the trial treatment due to toxicity, record "dose-limiting toxicity" or "adverse event" as the main reason for withdrawal. If the patient discontinues the trial prematurely at any time due to an AE or SAE, the patient should be followed until recovery to grade 2 or lower, unless the underlying disease is unlikely to improve

[0258] Combination therapy Pre-dose and co-administration, including vaccines and complementary treatments / supplements, should be documented for each patient at each scheduled visit (Table 8).

[0259] Any pharmaceutical or vaccine (including over-the-counter or prescription drugs, recreational drugs, vitamins, and / or herbal supplements) that the participant is taking at the time of registration or during the trial should be recorded along with the following information: ·Reason for use ·Dates of administration, including start and end dates ·Dosing information, including dose and frequency

[0260] Permitted pharmaceuticals / both The following co-administrations are permitted: ·In patients in the trial, the WBC count exceeds 25,000 / uL. The use of hydroxyurea is permitted in combination with G9.2-17 (IgG4) according to standard institutional practice for the first 2 cycles ·The principal investigator of the clinical trial must, in accordance with standard institutional practices, manage any of the following: ·Infusion reactions ·Tumor lysis syndrome ·Cytokine release syndrome. ·Bisphosphonate treatment (e.g., zoledronic acid) or denosumab for bone metastases has been stable for at least 6 months prior to treatment (C1D1). ·Use of inhaled corticosteroids and mineralocorticoids (e.g., fludrocortisone), topical steroids, intranasal steroids, intra-articular steroids, and ophthalmic steroids. ·Prophylactic or therapeutic use of anticoagulants ·Vaccination against COVID-19, seasonal influenza, and / or other common clinically necessary indications (e.g., tetanus, pneumococcus, HBV, etc.) is permitted before or during the study period. The timing and type of vaccine must be recorded. ·Carefully monitor highly sensitive narrow therapeutic index CYP3A4 substrates such as, but not limited to, alfentanil, dihydroergotamine, ergotamine, fentanyl, pimozide, and quinidine, and adjust the dose as necessary, taking into account that G9.2-17 (IgG4) can potently inhibit TGF-β and IL-10 in vitro. Reducing cytokine levels from the patient's baseline level may change CYP expression / activity and exposure to sensitive CYP3A4 substrates. Note: Immunosuppressive CYP3A4 substrates such as tacrolimus, cyclosporine, everolimus, and sirolimus are prohibited.

[0261] Prohibited medications / both During this trial, the following medications are not permitted. ·Concomitant administration of investigational drugs other than G9.2-17 (IgG4) for any indication. ·Although not limited thereto, systemic immunosuppressive treatments including cyclophosphamide, azathioprine, methotrexate, thalidomide, tacrolimus, cyclosporine, everolimus, sirolimus, and anti-TNF agents. However, patients are permitted to take acute low-dose systemic immunosuppressive drugs (e.g., prednisone or equivalent at 10 mg / day or less). ·Replacement therapies (e.g., thyroxine for adrenal or pituitary insufficiency, insulin, physiological corticosteroid replacement therapy [e.g., prednisone equivalent at 10 mg / day or less]) are not considered a form of systemic treatment.

[0262] VII. Adverse Events Definitions Adverse Event AE refers to any event, side effect, or other unfavorable medical occurrence that occurs in association with the use of a pharmaceutical product in humans, regardless of whether it is considered to have a causal relationship with this treatment. Thus, an AE can be any unfavorable, unintended sign (which may include clinically significant laboratory finding abnormalities), symptom, or disease that is temporally related to the use of a pharmaceutical product, regardless of whether it is considered to be related to the pharmaceutical product.

[0263] Events that meet the definition of AE include the following: ·Worsening of chronic or intermittent pre-existing conditions, including an increase in either the frequency and / or intensity of the condition ·New conditions detected or diagnosed after administration of the investigational drug that occur during the reporting period, even if they may have been present prior to the start of the trial ·Signs, symptoms, or clinical sequelae of suspected interactions ·Signs, symptoms, or clinical sequelae suspected of overdose of either the investigational drug or concomitant medication (overdose of the substance itself is not reported as an AE / SAE).

[0264] Events that do not meet the definition of AE include the following: ·Medical or surgical procedures (e.g., endoscopy, appendectomy); if the condition leading to the procedure meets the criteria for AE, it should be reported as an AE · A situation where no unfavorable medical event occurred (e.g., social and / or convenience hospitalization at the hospital) · The expected within-day variation (without deterioration) of pre-existing disease(s) or condition(s) present or detected at the start of the trial

[0265] If there is evidence of an AE by report or observation, the principal investigator or designee shall further evaluate and record the following information: · Time to onset and recovery · Severity · Causality / relationship to the test procedure · Actions taken regarding the test article · Outcome

[0266] Serious adverse event An SAE is an event that meets any of the following criteria: · Results in death · Threatens life · Requires hospitalization or prolongation of an existing hospitalization · Results in persistent or significant disability / incapacity · Is a congenital anomaly / congenital defect · Is a serious medical event that does not result in death, threaten life, or require hospitalization. Based on appropriate medical and scientific judgment, if the event places the subject at risk and medical or surgical intervention may be required to prevent one of the above outcomes, the event is considered an SAE. Examples of such events include emergency room or home intensive treatment for allergic bronchospasm, blood disorders or convulsions that do not result in hospitalization of an inpatient, or the occurrence of drug dependence or drug abuse

[0267] Suspicion of unexpected serious side effects Suspicion of unexpected serious side effects (USAR) refers to an adverse event that is unexpected (i.e., not described in the reference safety information of the investigational medicinal product) and meets the definition of a serious drug adverse reaction, and whose specificity or severity does not match that described in the reference safety information (i.e., the IB of the investigational medicinal product)

[0268] Evaluation of Clinical Laboratory Abnormalities and Other Abnormalities Abnormal test findings (e.g., clinical chemistry, hematology, and urinalysis) or other abnormal evaluations (e.g., ECG or vital signs) that are judged to be clinically significant are recorded as AEs and SAEs if they meet the definitions of AE and SAE. Clinically significant abnormal test findings or other abnormal evaluations that are detected during the trial or present at screening and significantly worsen after the start of the trial are reported as AEs or SAEs. However, clinically significant abnormal test findings or other abnormal evaluations that are related to the disease of the trial subjects or present or detected at the start of the trial and do not worsen are not reported as AEs or SAEs unless the treating investigator determines that the patient's condition is more severe than expected.

[0269] Clinical laboratory values that deviate significantly from pre - determined values (determined by the treating investigator) may be repeated. If warranted, additional or more frequent tests than those specified in the protocol should be performed to provide adequate documentation of the AE and recovery from the AE.

[0270] The treating investigator exercises his or her medical and scientific judgment in determining whether an abnormal test finding or other abnormal evaluation is clinically significant.

[0271] Adverse Events of Special Interest An AESI is an AE that is a scientific and medical concern specific to the sponsor's product or program and requires additional monitoring. If such an event occurs, further investigation may be necessary to better characterize and understand it.

[0272] In this trial, when the severity of the event is grade 3 or higher, IMAR and IRR are considered AESIs: · IMAR · IRR · CRS · TLS.

[0273] AESI must be reported to the PureTech Health Pharmacovigilance department or its designee within the same time frame applicable to SAE.

[0274] Evaluation of Adverse Events Severity All AEs will be graded by severity using the CTCAE version 5.0. If an AE is not described in the CTCAE criteria, the corresponding grading will be done based on the best medical judgment of the Investigator as follows: · Mild (Grade 1): Asymptomatic or mild symptoms; only clinical or diagnostic observations; no intervention required · Moderate (Grade 2): Minimal, local, or non-invasive intervention required; age-appropriate activities of daily living (ADL) are limited · Severe (Grade 3): Medically significant but not immediately life-threatening; hospitalization or prolongation of hospitalization required; physically disabling; or limitation of self-care ADL · Life-threatening (Grade 4): Life-threatening outcome; urgent intervention required · Death (Grade 5): Death related to the AE.

[0275] The terms "severe" and "serious" are not synonymous. Severity is a measure of intensity (characterized above), while seriousness as defined herein defines the reporting requirements.

[0276] VIII. Statistics Sample Size In this dose escalation study, to determine the RP2D dose(s) of G9.2-17 (IgG4), either the MTD and / or the minimum safe and effective biological dose will be established using a 4+2 algorithm-based dose escalation design. Due to the potential inclusion of patient backfill, a maximum of 50 patients are expected to be enrolled in this study as the total sample size.

[0277] Analysis Population The intention-to-treat (ITT) population is defined as patients who received at least one administration of G9.2-17 (IgG4). The efficacy analysis is performed for ITT. In ITT, the treatment of patients is carried out. The efficacy population is defined as all patients within ITT who showed at least one measurable overall response at the 1-month time point. This population is used for sensitivity analysis.

[0278] The per-protocol (PP) population includes patients who received at least one cycle of G9.2-17 (IgG4) and had no major protocol deviations.

[0279] The safety population (SAF) is defined as all patients who received at least one administration of G9.2-17 (IgG4). The safety analysis is performed for SAF.

[0280] The PK population includes any patient who received at least one dose of G9.2-17 (IgG4) and had at least one post-dose concentration that was evaluable.

[0281] The pharmacodynamics (PD) population is defined as patients who received at least one cycle of G9.2-17 (IgG4) and had at least one post-treatment PD evaluation in addition to the baseline PD evaluation.

[0282] General Statistical Plan Database lock and primary analysis are performed after the last patient has had a primary endpoint event. The final study analysis is performed after the study is completed. All analyses are descriptive.

[0283] Unless otherwise specified, all continuous endpoints are summarized using descriptive statistics including the number of patients (n), mean, standard deviation, median, minimum, and maximum. All categorical endpoints are summarized using frequencies and percentages and are 95% CIs. Screening measurements are the first study treatment date or the last value prior to that. Kaplan-Meier survival curves for PFS and OS are created from the date treatment was initiated using the Kaplan-Meier method, but there is no comparative analysis between treatment arms in any part of the trial. Waterfall plots and Swimmer plots are used to graphically present the ORR and DoR for all patients in each treatment arm within each tumor type evaluated in Part 2. All statistical analyses are performed using SAS® version 9.2. (SAS, Cary, NC).

[0284] Safety Analysis Unless otherwise specified, all safety analyses are performed in the SAF and analyzed using descriptive statistics.

[0285] Efficacy Analysis Disease response is descriptively summarized for ITT and PP. Sensitivity analyses are performed using the efficacy population.

[0286] Pharmacokinetics, Pharmacodynamics, and Immunogenicity PK, PD, and immunogenicity are descriptively summarized for the PK / PD population.

[0287] G9.2-17 (IgG4) exhibits a rapid clearance rate in human subjects compared to conventional antibody therapeutics. After the safety of the anti-Gal-9 antibody was established, a treatment plan including a once-weekly dosing schedule was developed to maintain a consistent systemic exposure level of the anti-galectin 9 antibody.

[0288] The treatment methods provided herein, which include administering an anti-galectin-9 antibody such as G9.2-17 (IgG4) at a specific dosage and dosing schedule provided herein (e.g., as used in the clinical trials provided herein), are expected to result in one or more favorable clinical outcomes, e.g., tumor size reduction, tumor growth delay, lesion remission and / or delay in lesion onset, and / or extension of survival in human patients having a target malignant blood disease such as AML, MDS, or ALL (T-cell or B-cell ALL), which can be refractory and / or recurrent.

[0289] Example 2: Analysis of AML Patients Treated with Anti-Galectin 9 Antibody Biological samples such as blood samples are collected from AML patients participating in the clinical trial described in Example 1 above. The pharmacokinetics (PK), immunogenicity, and pharmacodynamic (PD) profiles of the anti-galectin 9 antibody (G9.2-17 (IgG4)) are investigated to examine immunological and molecular changes in peripheral blood and bone marrow. The evaluation schedule of the clinical trial is shown in Figure 2.

[0290] The following assays are used when evaluating the PD profile of the anti-galectin 9 antibody.

[0291] Analytical Assays: 1. Luminex 2. Immunophenotyping PBMC Flow Cytometry 3. CyTOF (Cytometry by Time-of-Flight) 4. MRD Evaluation by PCR 5. Gene Array: Advanta Immuno-Oncology Gene Expression Assay 6. Total G9.2-17 (IgG4) (PK) LC / MS / MS Analysis - Already in use in G9.2-17 (IgG4) Solid Tumor Studies 7. Anti-G9.2-17 (IgG4) MSD-ECL - Already in use in G9.2-17 (IgG4) Solid Tumor Studies 8. Galectin-9 serum ELISA - G9.2-17 (IgG4) solid tumor test is already in use. Total G9.2-17 (IgG4) (PK) LC / MS / MS analysis, anti-G9.2-17 (IgG4) MSD-ECL, and galectin-9 serum ELISA are already being conducted at the PPD central testing facility using developed and validated methods in the G9.2-17 (IgG4) solid tumor trial.

[0292] Luminex: Luminex enables the evaluation of cytokine patterns, thereby providing a more comprehensive description of complex cell signaling networks than previously possible, making it an important tool in the investigation of cytokine environments in physiological and pathological processes. It offers higher throughput and density than traditional methods, provides data delivery with accuracy equivalent to ELISA but more robust. Luminex requires significantly less sample volume and shorter assay times than ELISA, revolutionizing our ability to rapidly assess the relative concentrations of various soluble factors. Luminex is unique in that it simultaneously offers both high density (multiplexing 100 assays per sample) and high throughput (capable of testing up to 1,000 samples per day). It offers the advantages of higher throughput, less sample volume, and lower cost, facilitating the simultaneous evaluation of multiple immune mediators. Markers of interest are: IFN-γ, IL-10, IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-6, IL-8, TNF-α, MIP 1b, MCP-1, MIP 1a, IL-17a, IL-5, TGF-β.

[0293] Immunophenotyping PBMC Flow Cytometry: Multiparameter flow cytometry technology is used to comprehensively monitor the immune response to immunotherapy from a single blood tube, often maximizing information from limited patient materials. The immune markers included in PBMC immunophenotyping do not overlap with those in the CyTOF panel and include standard immune cell class markers (e.g., CD3, CD4, CD8, CD11b / c CD14, CD15, CD16, CD19, CD29, CD25, CD27, CD28, CD33, CD38, CD45, CD45RO, CD45RA, CD56, CD57, CD66b, CD95, CD127, CD161, CD163, CD294, FOXP3, CCR4, CCR6, CXCR3, CXCR5, CXCR7, TCRγδ, HLA-DR, IgD, Ki67, TIGIT, Lag-3, and PD-1), in addition to molecules such as gal-9, TIM-3, TIGIT, PD-1, PD-L1, CTLA-4, CD47, CD73, CD123, KI67, etc. These enable the stratification of the expression of immune checkpoint molecules and other immune targets in AML across these immune cell populations, and as a result, their potential changes / regulation can be dynamically tracked upon exposure to anti-galectin 9 antibody. Granulocytes are excluded as PBMC rather than whole blood is analyzed. Analysis of these markers by flow cytometry may indicate potential combination therapies that are clinically exploitable in combination with G9.2-17 (IgG4), and some, e.g., anti-CD47, anti-CD123, anti-TIGIT, anti-CD73, anti-TIM-3, are already in clinical trials.

[0294] CyTOF: Time-of-flight cytometry is an application of mass cytometry used to quantify surface and intracellular labeling targets in single cells. CyTOF utilizes antibodies conjugated to rare heavy metal isotopes that are present in low cell numbers and not normally present in biological samples. CyTOF uses an ICP-MS detector to enable simultaneous quantification of multiple cellular components. Clinical trial samples are processed using the most comprehensive Maxpar® Human Immune Monitoring Panel, which includes 30 intracellular and extracellular parameters / markers, enabling highly multiplexed analysis of patient samples and reliable quantification of 37 immune cell populations in human whole blood or PBMCs. Clinical trial samples are valuable, and this method is selected because it requires only 270 μl of whole blood. Flow cytometry is limited by the number of parameters that can be analyzed simultaneously, significantly restricting its usefulness, so this method is used. The markers to be investigated are provided in Figure 1. Additional markers to be investigated are provided above.

[0295] MRD evaluation by RT-qPCR: Monitoring minimal residual disease (MRD) is a routine clinical practice in hematological malignancies. MRD techniques need to be highly sensitive, widely applicable, accurate, reliable, fast, and inexpensive. MRD serves several important functions. Clinicians use this prognostic marker to evaluate response to treatment, predict the likelihood of relapse, and identify whether a patient is in remission or has relapsed. The sensitivity of the test determines its effectiveness in detecting MRD, and the main outcome is the MRD cell level. The cutoff level is 0.01% MRD cells, or 1 MRD cell per 10,000 cells. Measurements above this indicate a higher risk of relapse compared to results below 0.01%, and as the number increases (e.g., MRD above 1%), the risk of relapse also rises, and the likelihood of survival decreases. Currently, flow cytometry and polymerase chain reaction (PCR) analysis are the preferred methods. In clinical trials, MRD evaluation by flow cytometry is performed by local testing facilities, and samples are sent for PCR analysis. Peripheral blood can be used for both methods, but the MRD level is 10-fold lower compared to bone marrow. Therefore, whenever possible, bone marrow samples are also collected. PCR has a 1-log higher sensitivity compared to flow cytometry, which is the reason for implementing this modality to measure MRD in addition to flow cytometry.

[0296] Gene Array: The Immuno-Oncology Gene Expression Assay for Translation and Clinical Research consists of 170 informative genes involved in immune cell identification, immune and cancer cell functions, immune regulation and cell fate, and response to checkpoint inhibitors (and thus immunotherapeutic drugs). It is designed for use on the Biomark™ HD System, and the Advanta Immuno-Oncology Gene Expression Assay detects these gene expression markers with high sensitivity across defined cell subsets. This assay can enable and accelerate the development of immunotherapies by identifying predictive biomarker signatures for treatment response. Tumor gene expression profiling has been shown to be effective in measuring the immune response and treatment response during cancer progression. However, previous approaches using large pre-constructed panels or transcriptome analysis containing hundreds of genes are costly and time-consuming. Also, large pre-constructed panels can be difficult to customize to the specific experimental needs. The Advanta Immuno-Oncology Gene Expression Assay was developed in collaboration with excellent researchers from academia and biopharmas to provide an appropriate balance of biomarker breadth, assay flexibility, and workflow efficiency. The Advanta Immuno-Oncology Gene Expression Assay is available as a 2-panel set. The first panel contains 91 major markers of tumor immune response previously shown in multi-site international clinical trials to convey tumor progression and immunotherapy response. The second panel contains 74 additional informative immuno-oncology markers and 17 open assay inlets for additional customization. Both panels contain the same 5 reference genes. When combined with Fluidigm microfluidics technology, this Advanta assay uniquely offers significant workflow efficiency compared to conventional gene expression profiling methods.Each reaction is miniaturized to nanoliter volumes, which means that clinical trial blood samples are used in the most efficient way and controlled using accurate automation to achieve accurate and cost-effective qPCR testing over a wide dynamic range.

[0297] Galectin-9 ELISA: Collect the patient's blood according to the normal procedure and isolate the serum for processing with the Boster Picokine™ Human Galectin-9 Precoated Enzyme-Linked Immunosorbent Assay (ELISA) kit at the designated central laboratory (Flagcip Biosciences). This product is a solid-phase immunoassay specially designed to measure human galectin-9 using 96-well strip plates. The detection antibody is a biotinylated antibody specific for gal-9. The capture antibody is a mouse-derived monoclonal antibody and the detection antibody is a goat-derived polyclonal antibody. Measure the optical density of the wells with a FLUOstar Optima microplate reader (BMG Labtech). Calculate the serum galectin-9 concentration with BMG Optima 2.10R2 software.

[0298] The anti-galectin 9 antibody (G9.2-17 (IgG4)) can reduce the amount of leukemia cells in the bone marrow and periphery, reactivate at least effector T cells and macrophages, significantly regulate the ratio and phenotype of immune cell populations and / or cytokines in the blood, and may reflect the reversal of immunosuppression present in AML / MDS. The anti-galectin 9 antibody can also reduce serum levels of galectin-9 when elevated at baseline. The research steps outlined herein are designed to efficiently advance Part 1 of the clinical trial, inform and potentially accelerate further clinical trials in Part 2. In accordance with this vision, this plan focuses on the timely acquisition, analysis, and reporting of samples while collecting safety and any efficacy measurements in treated patients. Clinical trials can be long-term and costly, and any unnecessary exposure of non-responders to ineffective immunomodulatory drugs should be avoided. Practically, one goal of this trial is to identify and develop assays as complementary diagnostics to determine the probability that an individual patient will respond to treatment with the anti-galectin 9 antibody and reflect its anti-leukemic and immunomodulatory effects in vivo. This would enable the effective selection of likely responders and potentially accelerate further clinical development of the antibody.

[0299] Since the anti-galectin 9 antibody may rapidly affect peripheral immunodynamics and circulating target levels (within the first week after dosing), sampling during the trial covering early and late time points is applied. Similarly, many blood draw time points coincide with the trial PK sampling, which may enable derivation of correlations between PK parameters and any immunomodulatory signals detected through phenotypic assays and galectin-9 serum levels.

[0300] In this study, while ensuring the following, the immunological pathways and treatment responses will be elucidated: i) the planned assays measure the desired endpoints and include both phenotypic and functional immune markers; ii) assays addressing relevant stimulatory and regulatory pathways are considered; iii) both host and tumor-related markers that can affect the outcome are measured; iv) the assays are reproducible and the variability is within an acceptable range; v) the methods for quality assurance and quality control are appropriate; vi) the central testing agency is validated; vii) the compartment (peripheral blood / bone marrow) in which the assays need to be measured is carefully considered; viii) the time points for sample collection during the treatment plan for performing the assays are selected to best determine the kinetics of the effect; ix) an analysis using appropriate statistical and bioinformatics methods is planned; and x) whenever possible, specimens are banked (cells, serum, plasma, bone marrow) for future assays (functional, phenotypic, DNA, and mRNA).

[0301] NanoString assay: Collect a patient's blood sample and isolate PBMCs following normal procedures. Extract RNA from the PBMC sample using the RNeasy Mini Kit (WI-84) and use an elution volume of 30 μL. Then, measure the RNA concentration using fluorometric analysis with either the Quant-iT RiboGreen RNA Reagent Kit (WI-82) or the Qubit RNA Assay Kit (WI-258). The optimal concentration of the sample is 60 ng / μL in a volume of 5 μL. In this study, the following nCounter gene expression panel is used: the human nCounter® PanCancer IO360 panel, including RLF:NS_IO_360_v1.0. The nCounter gene expression analysis is performed in batches of up to 11 clinical samples. Additionally, all runs also include panel standard samples for each nCounter assay.

[0302] For data analysis, the raw data is normalized using background correction, normalization using spike-in positive controls, and normalization using housekeeping genes.

[0303] Equivalents From the above description, those skilled in the art can easily identify the essential features of the present invention and, without departing from its spirit and scope, can adapt the present invention to various modifications and adaptations for various usage methods and conditions. Therefore, other embodiments are also within the scope of the claims.

[0304] Although several embodiments of the invention have been described and demonstrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein, and each such variation and modification is believed to be within the scope of the embodiments of the invention described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are exemplary, and that actual parameters, dimensions, materials, and / or configurations will depend on the particular application(s) for which the teachings of the invention are used. Those skilled in the art can understand or confirm many equivalents of the specific embodiments of the invention described herein using only conventional experimentation. Therefore, it is understood that the above-described embodiments are presented by way of example only and that embodiments of the invention other than those specifically described and recited within the scope of the appended claims and their equivalents may be practiced. Embodiments of the invention disclosed herein are directed to the individual features, systems, articles, materials, kits, and / or methods described herein. Further, any combination of two or more of such features, systems, articles, materials, kits, and / or methods is included within the scope of the invention disclosed herein if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0305] All definitions defined and used herein are to be understood as controlling dictionary definitions, definitions in incorporated by reference documents, and / or ordinary meanings of defined terms.

[0306] All references, patents, and patent applications disclosed in this specification are hereby incorporated by reference in their entirety for the subject matter for which each is cited, and in some cases, this may include the entire document.

[0307] As used in this specification and the claims, the indefinite articles "a" and "an" are to be understood to mean "at least one" unless explicitly indicated to the contrary.

[0308] As used in this specification and the claims, the phrase "and / or" as used herein means "either or both" of the elements so conjoined, i.e., in some cases, elements that exist conjunctively and in other cases, separately existing elements. A plurality of elements listed with "and / or" are to be construed in the same way, i.e., as "one or more" of the elements so conjoined. Other elements may optionally be present whether or not they are related to or unrelated to the specifically identified elements, which are specifically identified by the "and / or" clause. Thus, by way of non-limiting example, reference to "A and / or B" when used in combination with language without limitations such as "comprising" may, in one embodiment, refer to only A (optionally including elements other than B); in another embodiment, only B (optionally including elements other than A); and in yet another embodiment, both A and B (optionally including other elements).

[0309] In this specification and the claims, as used herein, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as including the number of elements or the list and, optionally, items not listed in the additional list, i.e., including at least one of them, but also including a plurality of them. Terms that clearly indicate the contrary, such as "only one of" or "exactly one of", or when used in the claims, "consisting of" refers to including exactly the number of elements or one element of the list. Generally, the term "or" as used herein should be interpreted as indicating an exclusive alternative (i.e., "either one but not both") only when an exclusive condition precedes, such as "either", "one of", "only one of", or "exactly one of". When used in the claims, "consisting essentially of" shall have the ordinary meaning as used in the field of patent law.

[0310] As used in this specification and the claims, the phrase "at least one" with respect to a list of one or more elements means at least one selected from any one or more of the elements in the list of elements, but necessarily does not include at least one or more of every element specifically recited in the list of elements, and does not exclude any combination of elements in the list of elements. This definition is allowed to optionally allow for the presence of elements other than those specifically recited in the list of elements referred to by the expression "at least one", whether or not they are related to the specifically identified ones of those elements. Thus, by way of non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B", or, equivalently, "at least one of A and / or B") can, in one embodiment, optionally refer to at least one that includes a plurality of A's in which B is absent (and, optionally, includes elements other than B); in another embodiment, optionally refer to at least one that includes a plurality of B's in which A is absent (and, optionally, includes elements other than A); in yet another embodiment, optionally refer to at least one that includes a plurality of A's; and, optionally, refer to at least one that includes a plurality of B's (and, optionally, includes other elements), etc.

[0311] Further, it should also be understood that, unless expressly indicated to the contrary, in any method claimed in this specification that includes a plurality of steps or acts, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

Claims

**Claim 1** A method for treating a malignant blood disorder, 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 once a week to once every four weeks at a dose of about 2 mg / kg to about 32 mg / kg. **Claim 2** The method of claim 1, wherein the anti-Gal9 antibody comprises: (a) A light chain containing a variable region of the light chain (V Ｌ ), the light chain (LC) complementarity determining region 1 (CDR1) containing the amino acid sequence of SEQ ID NO: 1, the LC complementarity determining region 2 (CDR2) containing the amino acid sequence of SEQ ID NO: 2, and the LC complementarity determining region 3 (CDR3) containing the amino acid sequence of SEQ ID NO: 3, said light chain, and (b) a heavy chain comprising a heavy chain variable region (V Ｈ ) that includes a heavy chain (HC) complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 4, an HC complementarity determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 5, and an HC complementarity determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO:

6. **Claim 3** The anti-Gal9 antibody includes V containing the amino acid sequence of SEQ ID NO: 8 Ｌ and V containing the amino acid sequence of SEQ ID NO: 7 Ｈ The method according to claim 2, comprising the same **Claim 4** The method according to any one of claims 1 to 3, wherein the anti-Gal9 antibody is an IgG1 molecule or an IgG4 molecule. **Claim 5** The method of claim 4, wherein the anti-Gal9 antibody is an IgG4 molecule comprising a modified Fc region compared to the wild-type human IgG4 counterpart. **Claim 6** The method of claim 5, wherein the modified Fc region comprises the amino acid sequence of SEQ ID NO:

14. **Claim 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. **Claim 8** The method according to 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. **Claim 9** The method according to 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. **Claim 10** The method of claim 8 or claim 9, wherein the anti-Gal9 antibody is administered once every two weeks. **Claim 11** The method of claim 8 or claim 9, wherein the anti-Gal9 antibody is administered once a week. **Claim 12** The method according to 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. **Claim 13** The method according to any one of claims 1 to 8, wherein the anti-Gal9 antibody is administered at a dose of 4.0 mg / kg per week, 7.5 mg / kg per week, 12 mg / kg per week, or 16.0 mg / kg per week. **Claim 14** The method according to any one of claims 1 to 13, wherein the subject is a human patient having leukemia or lymphoma.

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

16. The method according to any one of claims 1 to 15, wherein the malignant blood disease is refractory and / or relapsed.

17. The method according to claim 16, wherein the malignant blood disease is refractory and / or relapsed high-risk MDS.

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

19. The method according to claim 15, wherein the human patient has relapsed and / or refractory MDS after at least one prior therapy.

20. The method according to any one of claims 1 to 19, wherein the human patient has received or is receiving chemotherapy for treating the malignant blood disease.

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

22. The method according to any one of claims 1 to 21, wherein the method comprises the anti-Gal9 antibody as the only active agent for treating the malignant blood disease.

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

24. The method according to claim 21, further comprising reducing the dose of the anti-Gal9 antibody in the subject in whom the adverse event has occurred.

Images