Antibody against galectin-3 and method of use thereof

Abstract

The present invention provides methods and uses for compositions containing antibodies for managing, treating, or preventing diseases (e.g., cancer). [Solution] Provided herein are compositions, methods, and uses comprising an antibody that immunospecifically binds to the galectin-3 (LGALS3) carbohydrate-binding domain (CBD).

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority over U.S. Provisional Patent Application 62 / 625,166 (filed February 1, 2018) (which is incorporated herein by reference in its entirety). (Technical field) The present invention relates to compositions, methods, and uses comprising an antibody that immunospecifically binds to the galectin-3 (LGALS3) carbohydrate-binding domain (CBD). [Background technology]

[0002] Galectins are a family of small, highly conserved eukaryotic proteins that recognize specific complex sugars of glycosylated cell surface proteins. They are essential for linking cancer cells to the stromal microenvironment and regulating development, adhesion, signaling, invasion, and immune system interactions. Galectins can be found in the nucleus, cytoplasm, and pericellular space. Extracellular galectins are primarily released into exosomes and do not appear to have classical secretion originating from the ER or Golgi. Humans have at least 12 different galectins, which are expressed in diverse tissues and developmental stages. In the last decade, human galectins, particularly galectin-3 (LGALS3), have become clear as important linkers between the microenvironment and tumor cells. In particular, the biological function of glycoproteins and other surface glycans depends primarily on specific sugar chains bound to the Golgi (resulting in unique galectin selectivity). Extracellularly, LGALS3 participates in regulating cell membrane residence time, adhesion, migration, invasion, and vasculogenesis. While LGALS3 binds to other native ligands, its highest affinity ligand is the nearest lactosamine disaccharide of the polylactosamine chain, which adorns many O- and N-glycan species. Through binding and polymerization, LGALS3 forms a lattice that regulates the position and residence time of growth factor receptors (including EGFR, PDGFR, integrins, and CTLA4, among others) (Figure 1). Activation of downstream signaling molecules (e.g., SRC, ERK, AKT, and FAK) drives the production of key molecules required for metastasis and invasion. On the surface of T cells, CTLA4 surface concentration is stabilized by LGALS3, resulting in immunosuppression. Disappearance of the LGALS3 lattice inhibits the behavior of multiple cancer cells and immune cells. In addition to cancer, LGALS3 expression is also associated with renal disease, hepatic fibrosis, pulmonary fibrosis, heart failure, and parasitic diseases (see Figure 3). [Overview of the project] [Problems that the invention aims to solve]

[0003] Provided herein are compositions, methods, and uses comprising an antibody that immunospecifically binds to galectin-3 (LGALS3) and modulates the expression and / or activity of LGALS3 to manipulate or treat LGALS-mediated diseases (e.g., cancer). [Means for solving the problem]

[0004] In certain embodiments, what is provided herein is an antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment immunospecifically binds to the carbohydrate-binding domain (CBD) of galectin-3 (LGALS3). In some embodiments, the LGALS3 CBD includes SEQ ID NO: 27. In some embodiments, the antibody is a monoclonal antibody, a single-chain antibody, or any composition comprising the aforementioned antigen-binding fragment. In some embodiments, an antibody or its antigen-binding fragment inhibits in vitro invasion of tumor cells in a Matrigel invasion assay. In some embodiments, the tumor cells are ovarian tumor cells. In some embodiments, the antibody or its antigen-binding fragment inhibits the binding of LGALS3 to glycosylated cell surface proteins. In some embodiments, the antibody or its antigen-binding fragment inhibits the binding of LGALS3 to glycosylated cell surface receptors. In some embodiments, the antibody or its antigen-binding fragment inhibits the binding of LGALS3 to glycosylated growth factor receptors. In some embodiments, the antibody or its antigen-binding fragment inhibits the binding of LGALS3 to the following: glycosylated mucin-1 (MUC-1), mucin-4 (MUC4), mucin-16 (MUC16), disiaroganglioside, GD2, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), insulin-like growth factor receptor (IGFR), cMET / hepatocyte growth factor receptor (HGFR), integrin, and CTLA4. In some embodiments, glycosylated MUC16 is N-glycosylated with Asn1800 or Asn1806. In some embodiments, an antibody or its antigen-binding fragment inhibits the growth of tumors expressing the glycosylated form of MUC16.

[0005] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) comprising: (a) VH complementarity-determining region (CDR) 1 comprising the amino acid sequence SYGVH (SEQ ID NO: 5); (b) VH CDR2 comprising the amino acid sequence VIWSDGSTTYNSTLKS (SEQ ID NO: 6); and (c) VH CDR3 comprising the amino acid sequence HISNYGTMDY (SEQ ID NO: 7). In some embodiments, the antibody or its antigen-binding fragment comprises a VH comprising: (a) VH complementarity-determining region (CDR) 1 comprising the amino acid sequence GFSLSSY (SEQ ID NO: 11); (b) VH CDR2 comprising the amino acid sequence WSDGS (SEQ ID NO: 12); and (c) VH CDR3 comprising the amino acid sequence HISNYGTMDY (SEQ ID NO: 13). In some embodiments, the antibody or its antigen-binding fragment comprises VH including: (a) VH complementarity-determining region (CDR) 1 comprising the amino acid sequence GFSLSSYG (SEQ ID NO: 17); (b) VH CDR2 comprising the amino acid sequence IWSDGST (SEQ ID NO: 18); and (c) VH CDR3 comprising the amino acid sequence ARHISNYGTMDY (SEQ ID NO: 19). In some embodiments, the antibody or its antigen-binding fragment comprises VH, wherein VH comprises the amino acid sequence QVQLKESGPGLVAPSQSLSITCTISGFSLSSYGVHWVRQPPGKGLEWLVVIWSDGSTTYNSTLKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARHISNYGTMDYWGQGTSVTVS (SEQ ID NO: 24). In some embodiments, the antibody or its antigen-binding fragment comprises a light chain variable region (VL) comprising: (a) VL CDR1 comprising the amino acid sequence RASQDIRNYLN (SEQ ID NO: 8); (b) VL CDR2 comprising the amino acid sequence YTSRLHS (SEQ ID NO: 9); and (c) VL CDR3 comprising the amino acid sequence QHFNTLPPT (SEQ ID NO: 10). In some embodiments, the antibody or its antigen-binding fragment comprises a VL comprising: (a) VL CDR1 comprising the amino acid sequence RASQDIRNYLN (SEQ ID NO: 14); (b) VL CDR2 comprising the amino acid sequence YTSRLHS (SEQ ID NO: 15); and (c) VL CDR3 comprising the amino acid sequence QHFNTLPPT (SEQ ID NO: 16). In some embodiments, the antibody or its antigen-binding fragment comprises a VL containing: (a) VL CDR1 containing the amino acid sequence QDIRNY (SEQ ID NO: 20); (b) VL CDR2 containing the amino acid sequence YTS (SEQ ID NO: 21); and (c) VL CDR3 containing the amino acid sequence QHFNTLPPT (SEQ ID NO: 22). In some embodiments, the antibody or its antigen-binding fragment comprises a VL, the VL containing the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGSIKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIRNLEQEDIATYFCQHFNTLPPTFGGGTKLEIK (SEQ ID NO: 26).

[0006] In some embodiments, the antibody or its antigen-binding fragment comprises VH containing the amino acid sequence of SEQ ID NO: 24 and VL containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region (VH), the VH including VH CDR1, VH CDR2, and VH CDR3 of the antibody provided in any one of Figures 13A, 13B, or 13C. In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (VL), the VL including VL CDR1, VL CDR2, and VL CDR3 of the antibody provided in any one of Figures 13A, 13B, or 13C. In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region (VH) (including VH CDR1, VH CDR2, and VH CDR3 of the antibody provided in any one of Figures 13A, 13B, or 13C) and a light chain variable region (VL) (including VL CDR1, VL CDR2, and VL CDR3 of the antibody provided in any one of Figures 13A, 13B, or 13C). In some embodiments, the antibody or its antigen-binding fragment comprises the heavy and / or light chain of the antibody provided in any one of Figures 12A, 12B, 13D, 13E, 13F, 13G, 13H, 13I, 13J, 13K, 13L, 13M, 13N, or 13O. In some embodiments, the antibody comprises human-derived heavy chain and light chain constant regions. In some embodiments, the heavy chain constant region has an isotype selected from the group consisting of gamma 1, gamma 2, gamma 3, and gamma 4. In some embodiments, the light chain constant region has an isotype selected from the group consisting of kappa and lambda. In some embodiments, the antibody or its antigen-binding fragment is humanized. In some embodiments, the antibody or its antigen-binding fragment is a humanized form of a rodent antibody. In some embodiments, the antibody is an immunoglobulin comprising two identical heavy chains and two identical light chains. In some embodiments, the immunoglobulin is IgG.

[0007] Further provided in certain embodiments are antibody conjugates, which include an antibody or its antigen-binding fragment provided herein that is conjugated with a certain agent. In some embodiments, the agent is an imaging agent or a cytotoxic agent. In some embodiments, the antibody or its antigen-binding fragment is a bispecific antibody. In some embodiments, the bispecific antibody binds immunospecifically to CD3. In some embodiments, the bispecific antibody comprises an immunoglobulin that binds immunospecifically to LGALS3, where the light chain of the immunoglobulin is complexed via a peptide linker with a single-chain variable fragment (scFv) that binds immunospecifically to CD3. Further provided in certain embodiments are bispecific antibody complexes, which include a bispecific antibody provided herein that is complexed with a certain agent. In some embodiments, the agent is an imaging agent or a cytotoxic agent. In some embodiments, the antigen-binding fragment is an scFv. Further provided in certain embodiments is an scFv complex comprising the scFv provided herein, conjugated with a drug. In some embodiments, the drug is an imaging drug or a cytotoxic drug. Further provided in certain embodiments are chimeric antigen receptors (CARs) comprising the antibodies or antigen-binding fragments provided herein or the scFv provided herein. Further provided in certain embodiments are T cells that recombinantly express the CARs provided herein.

[0008] Further provided in certain embodiments are polynucleotides comprising nucleic acid sequences encoding an antibody heavy chain, an antibody light chain, an scFv, and / or a CAR as provided herein. Further provided in certain embodiments is a vector comprising the polynucleotides provided herein, ligated to a promoter so as to act. Further provided in certain embodiments are isolated cells containing the polynucleotides or vectors provided herein. Further provided in certain embodiments are pharmaceutical compositions comprising: a therapeutically effective amount of the antibody or antigen-binding fragment thereof provided herein, an antibody conjugate provided herein, a bispecific antibody provided herein, a bispecific antibody conjugate provided herein, an scFv provided herein, an scFv conjugate provided herein, a CAR provided herein, a polynucleotide provided herein, a vector provided herein, or a cell provided herein; and a pharmaceutically acceptable carrier.

[0009] Further provided in certain embodiments is a method for treating cancer in a patient in need, the method comprising the step of administering to the patient a pharmaceutical composition provided herein. In some embodiments, the cancer is of the ovary, lung, pancreas, breast, uterus, fallopian tube, or primary peritoneum. In some embodiments, the pharmaceutical composition inhibits metastasis in the patient. In some embodiments, the patient is a human patient. In some embodiments, the method further comprises the step of administering to the patient a therapeutically effective amount of an additional therapeutic agent. Further provided in certain embodiments is the immunogenic peptide of SEQ ID NO: 2. In some embodiments, the immunogenic peptide is complexed with an immunogenic carrier protein. Further provided in certain embodiments are (a) the aforementioned fragment comprising the LGALS3 protein or LGALS3 carbohydrate-binding domain and (b) a fusion protein comprising an Fc domain. In some embodiments, the LGALS3 carbohydrate-binding domain comprises domain SEQ ID NO: 32. In some embodiments, the LGALS3 carbohydrate-binding domain comprises amino acids 117-244 of SEQ ID NO: 1. In some embodiments, the Fc domain is the human IgG1 Fc domain. Further provided in certain embodiments are methods for producing antibodies or antigen-binding fragments thereof that specifically bind to LGALS3 CBD, the methods comprising the step of immunizing a target animal with an immunogenic peptide or fusion protein provided herein. In some embodiments, the target animal is a goat, sheep, donkey, chicken, guinea pig, rat, rabbit, or mouse. In some embodiments, the immunogenic peptide is complexed with an immunogenic carrier protein. In some embodiments, the immunogenic carrier protein is keyhole limpet hemocyanin. [Brief explanation of the drawing]

[0010] [Figure 1A] Figure 1A shows the structure and activity of extracellular galectin-3 (LGALS3). LGALS3 forms a pentamer and ligates ovarian cancer cell surface oncocytes (e.g., MUC16) to signaling molecules via contact with signaling receptors (e.g., EGFR and integrins). Activation of downstream signaling molecules (e.g., SRC, ERK, AKT, and FAK) drives the production of molecules crucial for metastasis and invasion. [Figure 1B] Figure 1B shows how antibody inhibitors that bind to the LGALS3 carbohydrate-binding domain block the activation of “outside-to-inside” signaling by disrupting the LGALS3 surface complex and destabilizing the cell surface receptor. [Figure 2A] Figure 2A shows LGALS3 shRNAs that inhibit MUC16 oncogene activation, as assessed by phosphorylation of EGFR, AKT, ERK, and SRC (P-EGFR, P-AKT, P-ERK, and P-SRC, respectively). Reduction of LGALS3 strongly inhibits MUC16 activation (MUC16c344) of various oncogenes in two ovarian models. [Figure 2B]Figure 2B shows that LGALS3 competition inhibits Matrigel invasion by ovarian cancer cell lines (OVCAR3, OVCA-432, OVCA-433, and CAOV3), but not by LGAL1 (galectin-1). The carbohydrate-binding domain of LGALS3 (117-244LGAL3-pFUSE) inhibits ovarian cancer cell invasion, but 11-119LGAL1-pFUSE does not. Both swinesonin and kifunensin inhibit the synthesis of the LGALS3 ligand, polylactosamine. [Figure 2C] Figure 2C shows the suppression of A2780 ovarian cancer by LGALS3 function inhibitors in a tumor xenograft model. The reduction in LGALS3 expression by shLGALS3 inhibition is highly inhibitory. Moderate tumor growth inhibition was also observed with the low-affinity protodrug LGALS3-pFUSE, a fusion protein of the unmodified carbohydrate-binding domain of LGALS3 linked to the Fc backbone. Figure 2D shows that antisense knockdown of MGAT5 and LGALS3 inhibits tumor growth. In SKOV3 cells, MUC16 expression enhanced tumor growth (top line). In contrast, loss of the N-glycosylation site, or shRNA against MGAT5 or LGALS3, reduced growth to control levels. [Figure 2D] Figure 2D shows that antisense knockdown of MGAT5 and LGALS3 inhibits tumor growth. In SKOV3 cells, MUC16 expression enhanced tumor growth (top line). In contrast, loss of N-glycosylation sites or shRNA against MGAT5 or LGALS3 reduced growth to control levels. [Figure 3] This paper demonstrates the in vitro and in vivo experimental functions of galectin 3 and the potential inhibitory effects of the donor antibody on therapy. [Figure 4]The primary structure of the LGALS3 carbohydrate-binding domain (CBD) is shown. LGALS3 CBD is divided into five subdomains (each binding to only one sugar residue), usually labeled A, B, C, D, and E. In the figure, highly conserved amino acids are underlined, while domains C, D, and E are shown within a framed region. This sequence was used in mouse peptide immunization. R186(*) is particularly important for LGALS3 function. The LGALS3 CBD sequence is highly homologous to mouse galectin-3 but differs from GAL1, GAL7, and GAL9. While each of these family members possesses similar sugar-binding domains, LGALS3 is the only lectin with a polymerization domain. [Figure 5] This paper presents the ELISA reactivity and sequences of various galectin family members. Abbreviations: CBD-3 = carbohydrate-binding domain of galectin-3 (human); LGALS3 = whole human galectin-3 protein; GAL1 CBD = galectin-1 carbohydrate-binding domain (human). The selected antibody was positive for both the whole protein (LGALS3) and the LGALS3 carbohydrate-binding domain (CBD-3). Several group D antibodies were cloned to determine co-inhibitory levels of both galectin-1 and LGALS3 and correlated with in vivo toxicity profiles. [Figure 6] The candidate antibody inhibits laminin binding to LGALS3. [Figure 7] This shows the binding of antibody 14D11.2D2 to LGALS3 in an ELISA assay. The assay utilized the following: a nickel-plated 96-well microplate, 500 ng / well of polyhistidine-labeled LGALS3 as the primary protein, a dilution of primary antibody 14D11.2D2 at a starting concentration of 1 mg / mL, and a 1:3000 dilution of goat anti-mouse IgG1-HRP as a secondary antibody. [Figure 8]The binding of the antibody 14D11.2D2 to LGALS3 in a surface plasmon resonance (SPR) assay is shown. The top curve represents 14D11.2D2 at 250.0 nM, the second curve at 14D11.2D2 at 125.0 nM, the third curve at 31.25 nM, and the bottom curve at 7.813 nM. A dissociation constant (KD) of 14.6 nM was obtained. [Figure 9] This ELISA assay demonstrates the inhibition of LGALS3 binding to laminin by 14D11.2D2. At a concentration of 250 nM, 14D11.2D2 reduced LGALS3 binding by 36.6%. [Figure 10] In the Matrigel invasion assay, 14D11.2D2 inhibited invasion in SKOV3-MUC16c344 (SKOV3c344), A2780-MUC16c344 (A2780c344), and OVCAR3 cell lines compared to untreated cells. ** or *** indicates a statistically significant difference between antibody-untreated and treated cells. The Matrigel invasion assay was performed using SKOV3--MUC16c344, A2780--MUC16c344, and wild-type OVCAR3 cell lines. When treated with the antibody, significantly less Matrigel membrane invasion was observed in MUC16-transfected SKOV3 cells (p=0.001), and similar results were observed in antibody-treated OVCAR3 cells (p=0.0009). MUC16-transfected A2780 cells showed reduced cellular invasion in the presence of 14D11.2D2, but this was not statistically significant (p=0.1067). [Figure 11]This study demonstrates the increased survival effect of 14D11.2D2 antibody-treated mice in an ovarian tumor xenograft model. Mice immunized with 14D11.2D2 antibody had a higher survival rate than mice that were not administered the antibody. Ovarian tumor cells (A2780-phrGFP-MUC16c344 (A2780c344)) were transplanted into the flanks of 20 athymic female nude mice. Ten mice were immunized by intravenous (iv) injection of 50 μg / mouse of 14D11.2D2 twice a week for a total of eight doses. Tumor volume was measured twice a week. Mice were sacrificed when the tumor volume reached 1500 mm3. Pilot mouse experiments were conducted using the A2780-MUC16c344 cell line with 14D11.2D2. 14D11.2D2 was administered at a dose of 50 μg / mouse twice a week for a total of eight doses. After 28 days, animals that did not receive antibodies had a statistically significantly lower survival rate than animals that received antibodies (p=0.012). [Figure 12A] Figure 12A shows the heavy chain DNA, amino acid sequence, and CDR sequence of the 14D11.2D2 antibody. The CDR sequence was identified by Kabat, Chothia, and IMGT numbering. Shaded regions are highly conserved regions of the heavy chain. Figure 12B shows the light chain DNA, amino acid sequence, and CDR sequence of the 14D11.2D2 antibody. The CDR sequence was identified by Kabat, Chothia, and IMGT numbering. Shaded regions are highly conserved regions of the light chain. [Figure 12B] Figure 12B shows the light chain DNA, amino acid sequence, and CDR sequence of the 14D11.2D2 antibody. The CDR sequence was identified by Kabat, Chothia, and IMGT numbering. Shaded regions are highly conserved regions of the light chain. [Figure 13A] 13A-C show the CDR sequences of isolated LGALS3 antibodies obtained by peptide immunization. The CDR sequences were identified by Kabat (13A), Chothia (11B), and IMGT (13C) numbering. [Figure 13B]13A-C show the CDR sequences of isolated LGALS3 antibodies obtained by peptide immunization. The CDR sequences were identified by Kabat (13A), Chothia (11B), and IMGT (13C) numbering. [Figure 13C] 13A-C show the CDR sequences of isolated LGALS3 antibodies obtained by peptide immunization. The CDR sequences were identified by Kabat (13A), Chothia (11B), and IMGT (13C) numbering. [Figure 13D] Figure 13D-O shows the VH and / or VL chain sequences of the same LGALS3 antibody. The light chain sequences for 3G10.D11, 11D1.A12, and 21A12.A6 were not obtained. [Figure 13E] Figure 13D-O shows the VH and / or VL chain sequences of the same LGALS3 antibody. The light chain sequences for 3G10.D11, 11D1.A12, and 21A12.A6 were not obtained. [Figure 13F] Figure 13D-O shows the VH and / or VL chain sequences of the same LGALS3 antibody. The light chain sequences for 3G10.D11, 11D1.A12, and 21A12.A6 were not obtained. [Figure 13G] Figure 13D-O shows the VH and / or VL chain sequences of the same LGALS3 antibody. The light chain sequences for 3G10.D11, 11D1.A12, and 21A12.A6 were not obtained. [Figure 13H] Figure 13D-O shows the VH and / or VL chain sequences of the same LGALS3 antibody. The light chain sequences for 3G10.D11, 11D1.A12, and 21A12.A6 were not obtained. [Figure 13I] Figure 13D-O shows the VH and / or VL chain sequences of the same LGALS3 antibody. The light chain sequences for 3G10.D11, 11D1.A12, and 21A12.A6 were not obtained. [Figure 13J] Figure 13D-O shows the VH and / or VL chain sequences of the same LGALS3 antibody. The light chain sequences for 3G10.D11, 11D1.A12, and 21A12.A6 were not obtained. [Figure 13K]Figure 13D-O shows the VH and / or VL chain sequences of the same LGALS3 antibody. The light chain sequences for 3G10.D11, 11D1.A12, and 21A12.A6 were not obtained. [Figure 13L] Figure 13D-O shows the VH and / or VL chain sequences of the same LGALS3 antibody. The light chain sequences for 3G10.D11, 11D1.A12, and 21A12.A6 were not obtained. [Figure 13M] Figure 13D-O shows the VH and / or VL chain sequences of the same LGALS3 antibody. The light chain sequences for 3G10.D11, 11D1.A12, and 21A12.A6 were not obtained. [Figure 13N] Figure 13D-O shows the VH and / or VL chain sequences of the same LGALS3 antibody. The light chain sequences for 3G10.D11, 11D1.A12, and 21A12.A6 were not obtained. [Figure 13O] Figure 13D-O shows the VH and / or VL chain sequences of the same LGALS3 antibody. The light chain sequences for 3G10.D11, 11D1.A12, and 21A12.A6 were not obtained. [Figure 14A] Figure 14A shows reduced metastatic lung tumor development in athymic mice injected with a breast cancer cell line lacking LGALS3 expression. Female athymic nude mice (6-8 weeks old at the start of the experiment) were injected via tail vein 1.5 x 10⁶ cells of either MDA-MB-231-TGL wt or MDA-MB-231-TGL shGAL3 into Group 1 (10 mice) and Group 2 (10 mice), respectively. Dorsal images are shown (week 1* indicates 1 week after injection). The MDA-MB-231 cell line is an epithelial human breast cancer cell line. [Figure 14B] Figure 14B shows the extended survival of LGALS3-deficient mice, as indicated by the Kaplan-Meier survival curve (p=0.0178). [Figure 14C] Figure 14C shows the effect of treatment with the 14D11.2D2 antibody on tumor development in mice injected with the MDA-MB-231-TGL breast cancer cell line. [Figure 15]The exemplary nucleotide (NM_002306.3) and amino acid (NP_002297.2) sequences of human LGALS3 are shown. [Modes for carrying out the invention]

[0011] This disclosure should not be limited to the individual embodiments described in this application, which are intended as illustrative examples of the individual features of this disclosure. Not all diverse embodiments of this disclosure are disclosed herein. As will be obvious to those skilled in the art, many modifications and variations of this disclosure can be made without departing its scope. In addition to the methods and apparatus enumerated herein, functionally equivalents within the scope of this disclosure will be obvious to those skilled in the art from the foregoing description. Such modifications and variations are considered to fall within the scope of the appended claims. This disclosure should be limited only by the appended claims and the equivalents of the full scope that such claims are to enjoy. This disclosure is not limited to specific uses, methods, reagents, compounds, compositions, or biological systems, and it should be understood that such uses, methods, reagents, compounds, compositions, or biological systems are, of course, subject to change. It should also be understood that the terms used herein are solely for the purpose of describing specific embodiments and are not intended to be limiting. In addition, if any feature or characteristic of this disclosure is described as a Markush Group, a person skilled in the art will recognize that this disclosure is describing any individual member or subgroup of the members of that Markush Group. As will be understood by those skilled in the art, for all purposes, and especially with respect to the provision of written descriptions, all scopes disclosed herein also encompass all possible sub-scopes and combinations thereof. Any described scope is readily recognized as sufficiently described, and such same scope can be broken down into at least equal 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, etc. As a non-limiting example, each scope considered herein can readily be broken down into the smaller 1 / 3, the middle 1 / 3, the larger 1 / 3, etc. As will also be understood by those skilled in the art, all words, e.g., “up to,” “at least,” “greater than,” “less than,” etc., include the described number and can subsequently be broken down into sub-scopes as considered above. Finally, as will be understood by those skilled in the art, a scope includes each of the individual numbers. Thus, for example, a group having 1–3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1–5 cells refers to a group having 1, 2, 3, 4, or 5 cells, and so on.

[0012] definition Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this disclosure belongs. The following references provide general definitions of many of the terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., 1994; The Cambridge Dictionary of Science and Technology, Walker ed., 1988; The Glossary of Genetics, 5th Ed., R. Rieger et al., eds., Springer Verlag, 1991; and Hale & Marham, The Harper Collins Dictionary of Biology, 1991. As used herein, unless otherwise specified, the following terms have the meanings attributed to them below. Terms used herein are used solely for the purpose of describing individual embodiments and are not intended to limit this disclosure. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. As used herein, when the term “approximately” is used to modify a number or range of numbers, it indicates that deviations of 5% to 10% above and 5% to 10% below the value or range fall within the intended meaning of the stated value or range. As used herein, the term “administration” of a drug to a target animal includes any route by which the drug is introduced or delivered to the target animal in order to achieve the intended function of the drug. Administration may be achieved by any suitable route, including, but not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, and other suitable routes as described herein. Administration includes self-administration and administration by another person.

[0013] The term “amino acid” refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrrolidine and selenocysteine. Amino acid analogs refer to substances that have the same chemical structure as naturally occurring amino acids; that is, they have a hydrogen atom, a carboxyl group, an amino group, and an α-carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as naturally occurring amino acids. In some embodiments, the amino acids forming the polypeptide are of the D type. In some embodiments, the amino acids forming the polypeptide are L-type. In some embodiments, the first plurality of amino acids forming the polypeptide are D-type, and the second plurality of amino acids are L-type. Amino acids are referred herein by their commonly known three-letter codes or by the one-letter codes recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides are referred by their commonly accepted single-letter codes. The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acid residues. These terms apply to polymers of naturally occurring amino acids, as well as amino acid polymers in which one or more amino acid residues are non-naturally occurring amino acids (e.g., amino acid analogs). These terms encompass amino acid chains of any length (full-length proteins), where amino acid residues are linked by covalent peptide bonds.

[0014] As used herein, “control” is another sample used in an experiment for comparative purposes. Controls can be “positive” or “negative.” For example, if the purpose of the experiment is to determine the correlation of the effectiveness of therapeutic agents for the treatment of a particular type of disease, then typically a positive control (a composition known to exhibit the desired therapeutic effect) and a negative control (a subject animal or sample that is not given the treatment or is given a placebo) are used. As used herein, the terms “effective dose” or “therapeutably effective dose” refer to an amount of drug sufficient to achieve the desired therapeutic effect. In the context of the application of therapeutic drugs, the amount of therapeutic peptide administered to the target animal may depend on the type and severity of the infection, as well as the individual characteristics (e.g., general health status, age, sex, weight, and drug resistance). The same may also depend on the degree, severity, and type of the disease. Those skilled in the art can determine an appropriate dosage according to these and other requirements.

[0015] As used herein, the term “expression” refers to the process by which polynucleotides are transcribed into mRNA, and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotides are derived from genomic DNA, expression may include the splicing of mRNA in eukaryotic cells. The level of gene expression can be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In some features, the level of gene expression in one sample can be directly compared to the level of that gene expression in a control or reference sample. In other features, the level of gene expression in one sample can be directly compared to the level of that gene expression in the same sample after administration of the composition disclosed herein. The term “expression” also refers to one or more of the following events: (1) the generation of an RNA template from a DNA sequence in a cell (e.g., by transcription); (2) the processing of an RNA transcript in a cell (e.g., by splicing, editing, 5' cap formation, and / or 3' end formation); (3) the translation of an RNA sequence into a polypeptide or protein in a cell; (4) post-translational modification of a polypeptide or protein in a cell; (5) the presentation of a polypeptide or protein on the cell surface; and (6) the secretion, presentation, or release of a polypeptide or protein from a cell.

[0016] The term “linker” refers to a synthetic sequence (e.g., an amino acid sequence) that connects or ligates two sequences (e.g., two polypeptide domains). In some embodiments, the linker comprises an amino acid sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. As used herein, the term “antibody” refers not only to complete antibody molecules but also to fragments of antibody molecules that retain immunogenic binding ability. Such fragments are also well known in the art and are frequently used both in vivo and in vitro. Therefore, as used herein, the term “antibody” refers not only to complete immunoglobulin molecules but also to the well-known active fragments F(ab')2 and Fab. Fab fragments lacking F(ab')2 and the Fc fragment of the complete antibody are more rapidly removed from circulation and may result in less nonspecific tissue binding of the complete antibody (Wahl et al., J. Nucl. Med. 24:316-325, 1983). The antibodies of the present invention include whole naive antibodies, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, multispecific antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', single-chain V-region fragments (scFv), single-domain antibodies (e.g., nanobody and single-domain camel antibodies), V NAR The antibodies include fragments, bispecific T cell interlocking (BiTE) antibodies, minibodies, disulfide-linked Fv (dsFv), and anti-idiotype (anti-Id) antibodies, intrabodies, fusion polypeptides, unconventional antibodies, and any of the antigen-binding fragments described above. In particular, the antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules (i.e., molecules containing antigen-binding sites). The immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass.

[0017] In certain embodiments, the antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain has a heavy chain variable region (V in this specification). H (abbreviated as) and heavy chain steady state (C H The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain has a light chain variable region (V in this specification).L (abbreviated as) and the light chain constant (C L ) region. The light chain constant region consists of one domain, C L is composed of. V H and V L regions are further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)), with more conserved regions called framework regions (FRs) interspersed. Each V H and V L is composed of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors (including various cells of the immune system (such as effector cells) and the first component of the classical complement system (Clq)). As used interchangeably herein, the terms "antigen-binding portion," "antigen-binding fragment," or "antigen-binding region" of an antibody refer to the region or portion of the antibody that binds to an antigen and further confers antigen specificity to a fragment of the antibody, antigen-binding protein. For example, an antibody includes one or more fragments of the antibody that retain the ability to specifically bind to an antigen (such as a peptide / HLA complex). It has been shown that the antigen-binding function of an antibody can be exerted by fragments of a full-length antibody. Examples of antigen-binding portions included within the term "antibody fragment" of an antibody include the following: Fab fragment, V L , V H , a monovalent fragment consisting of C L and CH1 domains; F(ab)2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; Fd fragment consisting of V H and CH1 domains; Fv fragment consisting of the V L and V H domains of a single arm of an antibody; dAb fragment consisting of V H domain (Ward et al., Nature 341: 544-546, 1989); and isolated complementarity-determining regions (CDRs).

[0018] Antibodies and antibody fragments can be derived whole or partially from mammals (e.g., humans, non-human primates, goats, guinea pigs, hamsters, horses, mice, rats, rabbits, and sheep) or non-mammalian antibody-producing animals (e.g., chickens, ducks, geese, snakes, and tailed amphibians). Antibodies and antibody fragments can be produced in animals or from non-animal sources, such as yeast or phages (e.g., as single antibodies or antibody fragments or as part of an antibody library). Furthermore, the two domains of the Fv fragment (V L and V H Although these are encoded by separate genes, they can be joined together by a synthetic linker using recombination methods. The linker can make them into a single protein chain, and in that single chain, V L and V H The region pairs form monovalent molecules. These are known as single-chain Fv (scFv) (see, for example, Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. 85: 5879-5883, 1988). These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as whole antibodies. "Isolated antibodies" or "isolated antigen-binding proteins" are identified and isolated and / or recovered from their natural environmental components. "Synthetic antibodies" or "recombinant antibodies" are generally produced using recombinant techniques or peptide synthesis techniques known to those skilled in the art. As used herein, the term “single-chain variable fragment” or “scFv” refers to the heavy chain (V) of an immunoglobulin (e.g., mouse or human). H ) and light chain (V L ) is a fusion protein of the variable region, and the said region is covalently bonded to V H :V L It forms a heterodimer. Heavy chain (V H) and light chain (V L ) are either directly linked or linked by peptide code linkers (e.g., about 10, 15, 20, 25 amino acids). The linker is V H The N-terminus of V L Connect to the C-terminal of or V H The C-terminus of V L It connects to the N-terminus. Typically, the linker is rich in glycine for flexibility and serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain.

[0019] Despite the removal of the constant region and the introduction of a linker, the scFv protein retains its intrinsic immunoglobulin specificity. Single-chain Fv polypeptide antibodies are similar to those described by Huston et al. H - and V L-It can be expressed from nucleic acids containing coding sequences (Huston, et al., Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See also U.S. Patents 5,091,513, 5,132,405 and 4,956,778 and U.S. Patent Publications No. 20050196754 and No. 20050196754. Inhibitory antagonist scFv has been described (see, for example, the following: Zhao et al., Hybridoma (Larchmt) 27(6):455-51, 2008; Peter et al., J Cachexia Sarcopenia Muscle, 2012; Shieh et al., J Imunol 183(4):2277-85, 2009; Giomarelli et al., Thromb Haemost 97(6):955-63, 2007; Fife eta., J Clin Invst 116(8):2252-61, 2006; Brocks et al., Immunotechnology 3(3): 173-84, 1997; Moosmayer et al., Ther Immunol 2(10):31-40, 1995). Antagonists scFv with stimulant activity have been described (see, for example, Peter et al., J Biol Chem 25278(38):36740-7, 2003; Xie et al., Nat Biotech 15(8):768-71, 1997; Ledbetter et al., Crit Rev Immunol 17(5-6):427-55, 1997; Ho et al., Bio Chim Biophys Acta 1638(3):257-66, 2003).

[0020] As used herein, “F(ab)” refers to a fragment of an antibody structure that binds to an antigen but is monovalent and lacks an Fc region. For example, an antibody digested by the enzyme papain produces two F(ab) and Fc fragments (e.g., a heavy (H) chain constant region; and an Fc region that does not bind to an antigen). As used herein, “F(ab')2” refers to an antibody produced by pepsin digestion of a total IgG antibody, where the fragment has two antigen-binding (ab') (bivalent) regions, and each (ab 1 The ) region includes two separate amino acid chains, a portion of the H chain for antigen binding linked by a disulfide bond and a light (L) chain, and the remaining H chain portion is further linked together. The “F(ab')2” fragment can be split into two individual Fab' fragments. As used herein, “CDR” is defined as the complementarity-determining region amino acid sequence of an antibody, which is the hypervariable region of the immunoglobulin heavy and light chains. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th US Department of Health and Human Services, National Institutes of Health, 1987. Generally, an antibody contains three heavy and light chain CDRs or CDR regions in its variable region. The CDR provides the majority of contact residues for the binding of the antibody to the antigen or epitope. In certain embodiments, the CDR region is outlined using the Kabat system (Kabat, EA, et al. Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, 1991).

[0021] As used herein, the terms “constant region” or “constant domain” are interchangeable and have meanings well known in the industry. The constant region is a portion of an antibody, such as the carboxyl-terminal portion of the light and / or heavy chain, which does not participate in antibody-antigen binding but can exhibit diverse effector functions (e.g., interaction with Fc receptors). The constant region of an immunoglobulin molecule has a generally more conserved amino acid sequence compared to the immunoglobulin variable domain. As used herein, “epitope” is a term in the art and can refer to a localization region of an antigen to which an antibody can bind immunospecifically. An epitope may be, for example, a sequence of amino acids in a polypeptide (linear or continuous epitope), or it may be composed of two or more discontinuous regions of one polypeptide or more polypeptides (stereostructural, nonlinear, discontinuous, or discontinuous epitope). As used herein, the term “ligand” refers to a molecule that binds to a receptor. In particular, ligands bind to receptors on other cells to enable cell-to-cell recognition and / or interaction.

[0022] As used herein, the term “affinity” refers to a measure of binding strength. While not theoretically bound, affinity depends on the closeness of stereochemical agreement between the antibody binding site and the antigenic determinant, the size of the contact area between them, and the distribution of charged and hydrophobic groups. Affinity also includes the term “avidity,” which refers to the strength of antigen-antibody binding after the formation of a reversible complex (e.g., monovalent or polyvalent). Methods for calculating the affinity of an antibody to an antigen are known in the art, and such methods include the use of binding experiments for affinity calculations. Antibody activity in functional assays (e.g., flow cytometry assays) also reflects antibody affinity. Antibodies and affinity can be phenotypically characterized and compared using functional assays (e.g., flow cytometry assays). Nucleic acid molecules useful in this disclosure include any nucleic acid molecules encoding polypeptides or fragments thereof. In certain embodiments, nucleic acid molecules useful in this disclosure include nucleic acid molecules encoding antibodies or their antigen-binding moieties. Such nucleic acid molecules do not need to be 100% identical to the endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantially homologous" or "substantially identical" status to an endogenous sequence can typically hybridize with at least one strand of a double-stranded nucleic acid molecule. "Hybridization" means a pair that forms a double-stranded molecule under various stringency conditions with a complementary polynucleotide sequence (e.g., a gene described herein) or a portion thereof (see, for example, Wahl, GM and SL Berger, Methods Enzymol. 152:399, 1987; Kimmel, AR Methods Enzymol. 152:507, 1987).

[0023] As used herein, “immunely binding,” “immunely recognizing,” “specifically binding,” and “specifically recognizing” are synonymous terms with respect to antibodies and refer to antibodies and their antigen-binding fragments that bind to an antigen (e.g., an epitope or immune complex) via an antigen-binding site, as understood by those skilled in the art, without precluding cross-reactivity between the antibody or its antigen-binding fragment and other antigens. The terms “substantially homologous” or “substantially identical” mean a polypeptide or nucleic acid molecule that exhibits at least 50% or higher homology or identity with a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). For example, such a sequence is at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% homology or identity at the amino acid level or nucleic acid level with respect to a sequence used for comparison (e.g., a wild-type or natural sequence). In some embodiments, a substantially homologous or substantially identical polypeptide includes one or more amino acid substitutions, insertions, or deletions compared to a sequence used for comparison. In some embodiments, a substantially homologous or substantially identical polypeptide includes one or more non-natural amino acids or amino acid analogs (including D-amino acids and reversed enantiomers) in the substitution of the homologous sequence. Sequence homology or sequence identity is typically measured using sequence analysis software, such as the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs. Such software matches identical or similar sequences by selecting the degree of homology for various substitutions, deletions, and / or modifications. An exemplary approach to determining the degree of identity involves using the BLAST program to show closely related sequences.-3 and e -100 It can be used for probability scores between these two points. As used herein, the term “analog” refers to a structurally related polypeptide or nucleic acid molecule that has the function of a reference polypeptide or nucleic acid molecule.

[0024] As used herein, “conservative sequence modification” means an amino acid modification that does not significantly affect or alter the binding properties of the anti-LGALS3 antibody or its antigen-binding fragment containing the amino acid sequence. Conservative modifications may include amino acid substitutions, additions, and deletions. Modifications can be introduced into the scFv of the anti-LGALS3 antibody or its antigen-binding fragment by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Amino acids can be classified into groups according to their physicochemical properties (e.g., charge and polarity). A conservative amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid within the same group. For example, amino acids can be classified by charge. Specifically, positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In addition, amino acids can be classified by polarity. Specifically, polar amino acids include arginine (basic polarity), asparagine, aspartic acid (acidic polarity), glutamic acid (acidic polarity), glutamine, histidine (basic polarity), serine, threonine, and tyrosine; and nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Therefore, by replacing one or more amino acid residues within the CDR region with other amino acid residues within the same group, the modified antibody can be tested for the retained function (i.e., the function shown in (c) to (1) above) using the functional assay described herein. In certain embodiments, one, two or more, three, four or five or more residues in the specified sequence or CDR region are not modified.

[0025] As used herein, the term “heterogeneic nucleic acid molecule or polypeptide” means a nucleic acid molecule (e.g., cDNA, DNA, or RNA molecule) or polypeptide that is not normally present in a cell or a sample obtained from a cell. This nucleic acid may be of another organism's origin, or it may be, for example, an mRNA molecule that is not normally expressed in a cell or sample. As used herein, the term “adjust” refers to changing something in a positive or negative direction. Illustrative adjustments include changes of approximately 1%, 2%, 5%, 10%, 25%, 50%, 75%, or 100%. As used herein, the term “increase” means a change of at least about 5% in the positive direction (including, but not limited to, about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or about 100%). As used herein, the term “decrease” means a change in the negative direction of at least about 5% (including, but not limited to, about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or about 100%). As used herein, an “isolated” polynucleotide or nucleic acid molecule is one that has been separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule (e.g., in mice or humans). Furthermore, an “isolated” nucleic acid molecule, such as a DNA molecule, when produced by recombinant technology, is substantially free of other cellular material or culture media, or when chemically synthesized, is substantially free of chemical precursors or other chemical substances. For example, the term “substantially free” includes preparations of polynucleotides or nucleic acid molecules containing less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% of other substances (e.g., cellular material, culture media, other nucleic acid molecules, chemical precursors, and / or other chemical substances). As used herein, the term “isolated cell” refers to a cell that has been separated from the molecular and / or cellular components that naturally accompany it.

[0026] An “effective dose” (or “therapeutably effective dose”) is a sufficient amount to produce a beneficial or desired clinical outcome for treatment. An effective dose may be administered to the animal in question in one or more doses. In terms of treatment, an effective dose is a sufficient amount to alleviate, improve, stabilize, reverse or delay the progression of a disease (e.g., neoplasia), or to mitigate the pathological consequences of the disease (e.g., neoplasia). The effective dose is generally determined on a case-by-case basis by a physician and is within the scope of the skill of a person skilled in the art. When determining an appropriate dosage to reach an effective dose, several requirements are typically considered. These requirements include the age, sex, and weight of the animal in question, the symptoms being treated, the severity of the symptoms, and the morphology and effective concentration of the manipulated immune cells administered. As used herein, the term “neoplasia” refers to a disease characterized by the pathological proliferation of cells or tissues and subsequent migration or invasion into other tissues or organs. Neoplasia is typically unregulated and progressive and occurs under conditions that do not elicit or cause cessation of normal cell proliferation. Neoplasia can affect a variety of cell types, tissues, or organs. These include organs selected from the group consisting of (but not limited to) the bladder, colon, bone, brain, breast, cartilage, gliosa, esophagus, fallopian tubes, gallbladder, heart, intestines, kidneys, liver, lungs, lymph nodes, nerve tissue, ovaries, pleura, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract, ureters, urethra, uterus, and vagina, or the aforementioned tissues or cell types. Neoplasia includes cancer, such as sarcoma, carcinoma, or plasmacytoma (malignant tumor of plasma cells).

[0027] As used herein, the terms “to treat” or “treatment” refer to a clinical intervention in an attempt to alter the course of a disease in an individual or cell being treated, which may be performed for prevention or during the course of a clinical lesion. Therapeutic effects in treatment include, but are not limited to, prevention of the onset or recurrence of the disease, improvement of symptoms, disappearance of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of the rate of disease progression, improvement or alleviation of symptoms, and remission or improved prognosis. By preventing the progression of the disease or abnormality, treatment can not only prevent exacerbation of the abnormality in an affected or diagnosed subject animal or a subject animal suspected of having the disease, but treatment can also prevent the onset or symptoms of the abnormality in a subject animal at risk of or suspected of having the abnormality. As used herein, the term “target animal” refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc. (for example, the animal may be the recipient of the treatment in question, or cells may be collected from the animal).

[0028] General Galectin-3 (LGALS3) is a β-galactoside-binding protein that can be secreted by many cells, but lacks signaling sequences for translocation to the endoplasmic reticulum and Golgi compartment, and entry into the classical secretory pathway. LGALS3 is found in multiple intracellular and extracellular locations and has multifaceted biological functions (e.g., cell proliferation, cell adhesion, and cell-cell interaction). Depending on its localization and post-translational modifications (e.g., cleavage and phosphorylation), LGALS3 can exhibit anti-apoptotic or pro-apoptotic activity. Cleavage of galectin-3 has been reported to be related to vascularization potential and apoptosis resistance. Phosphorylation of galectin-3 regulates its sugar-binding capacity. Our preliminary study in ovarian cancer using artificially synthesized chimeric LGALS3 determined that LGALS3 plays an essential role in the proliferation, spread, and invasive characteristics of ovarian cancer (Rao, et al. (2017) ACS Chem. Biol. 12 (8): 2085-2096 (the entire text is incorporated herein by reference)). This study utilized both LGALS3 knockdown shRNA repression and chimeric antibodies (in the chimeric antibody, the naturally occurring low-affinity LGALS3 carbohydrate-binding domain was replaced with an antibody variable region). Knockdown experiments showed that LGALS3 tumor expression is required for the oncogene-activating effect of the ovarian cancer mucin MUC16 / CA125 (Figure 2A). When LGALS3 knockdown cells were transplanted into nude mice, these cells showed very limited proliferation, confirming the essential role of LGALS3 in in vivo tumor growth (Figure 2D). Furthermore, it was shown that the proliferation / invasiveness enhancement by LGALS3 is extracellular. Invasive studies have shown that Matrigel invasion by ovarian cancer cells is dependent on LGALS3. Low-affinity chimeric antibodies containing a fusion protein of the unmodified carbohydrate-binding domain of LGALS3 linked to the Fc backbone can inhibit LGALS3 function and prevent Matrigel invasion, whereas chimeric inhibitory antibodies against galectin-1 were ineffective (Figure 2B). In addition, low-affinity chimeric anti-LGALS3 inhibitory antibodies reduced in vivo proliferation of xenografts in mice (Figure 2C). These chimeric antibodies did not cause any significant side effects in host mice and were consistent with reported minimal LGALS3 knockout efficacy (Wright et al., (2017) J Leukoc Biol 101(3): 717-726).

[0029] This specification describes an immunological strategy for isolating inhibitory, high-affinity antibodies against LGALS3. The primary sequence of the LGALS3 carbohydrate-binding domain (CBD) is shown in Figure 4. The boxes indicate the C, D, and E domains of the CBD, which are important for lactosamine binding. Highly conserved amino acids are underlined. R186( *The LGALS3 CBD domain is important for the function of GAL3. The GAL3 CBD sequence is highly homologous to mouse galectin-3 but differs from GAL1, GAL7, and GAL9. Each of these family members has a similar sugar-binding domain, while LGALS3 is the only lectin that has a polymerization domain. As described in the examples herein, mice were immunized with LGALS3 CBD or peptides and / or fusion constructs containing a portion thereof to produce antibodies against LGALS3 CBD. Antibodies were screened against whole protein CBD and whole protein for GAL1, LGALS3, GAL7, and GAL9 to select antibodies. Isolated antibodies bind to native human LGALS3 and mouse homolog Lgal3. In addition, the antibodies showed functional inhibition of LGALS3 binding to polylactosamine in laminin. Furthermore, the selected antibodies were shown to inhibit tumor cell invasion when evaluated by the Matrigel invasion assay and to increase survival in a mouse ovarian cancer xenograft model. Various studies have highlighted the role of LGALS3 in tumor biology and inflammation. A partial list of the effects and expected results of LGALS3 targeting is shown in Figure 3. Many of the expected effects are benefits anticipated in cancer treatment. Although the effects of LGALS3 are diverse, even complete elimination of LGALS3 in knockout mouse models is almost consistent with normal development, and some immunosuppressive effects are substantially indistinguishable from those of TNF inhibitors. Based on these studies, the antibodies provided herein are expected to be useful therapeutic agents for the treatment of LGALS3-mediated diseases and conditions (including, but not limited to, cancer and inflammation).

[0030] Provided are antibodies and their antigen-binding fragments, as well as polypeptides (e.g., fusion proteins, complexes, and / or chimeric antigen receptors) containing such antibodies and fragments, and cells expressing the said. The antibodies and fragments specifically bind, among other things, to epitopes of the LGALS3 protein. Such antibodies are referred to herein as “anti-LGALS3 antibodies.” Such epitopes are typically epitopes within the carbohydrate-binding domain (CBD) of the LGALS3 molecule or substantially within the said domain. In some embodiments, the epitopes are located within or include amino acid residues 117-244 of LGALS3 (SEQ ID NO: 1). In some embodiments, the antibody or fragment binds to an epitope within or including amino acid residues 117-244 of LGALS3 (SEQ ID NO: 1). In some embodiments, the epitope is located within or includes amino acid residues 152-195 of LGALS3 (SEQ ID NO: 1). In some embodiments, the antibody or fragment binds to an epitope within amino acid residues 152-195 of LGALS3 (SEQ ID NO: 1) or an epitope containing the aforementioned. In some embodiments, the epitope is located within or contains amino acid residues 170-186 of LGALS3 (SEQ ID NO: 1). In some embodiments, the antibody or fragment binds to an epitope within or contains amino acid residues 170-186 of LGALS3 (SEQ ID NO: 1). In some embodiments, the epitope is a portion of the CBD containing the sequence CNTKLDNNWGREERQSVFPFESG (SEQ ID NO: 2). In some embodiments, the antibody or fragment binds to an epitope within or containing the sequence CNTKLDNNWGREERQSVFPFESG. In some embodiments, the epitope is located within or contains domains C, D, and E of the CBD of GALS3. In some embodiments, the antibody or fragment binds to an epitope within amino acid residues 170-186 of LGALS3 (SEQ ID NO: 1) or an epitope containing the above.

[0031] In some embodiments, the anti-LGALS3 antibodies and their antigen-binding fragments described herein inhibit laminin binding by LGALS3. In some embodiments, the anti-LGALS3 antibodies and their antigen-binding fragments described herein inhibit the binding of LGALS3 to lactosamine. In some embodiments, the anti-LGALS3 antibodies and their antigen-binding fragments described herein suppress or treat cancer. In some embodiments, the anti-LGALS3 antibodies and their antigen-binding fragments described herein inhibit the invasion and proliferation of tumor cells. In some embodiments, the anti-LGALS3 antibodies and their antigen-binding fragments described herein inhibit tumor vascularization. In some embodiments, the anti-LGALS3 antibodies and their antigen-binding fragments described herein inhibit metastasis. In some embodiments, the anti-LGALS3 antibodies and their antigen-binding fragments described herein inhibit the proliferation of metastatic tumors. In some embodiments, the anti-LGALS3 antibodies and their antigen-binding fragments described herein inhibit the activation of cancer-related signaling molecules (e.g., AKT and ERK). In some embodiments, the anti-LGALS3 antibodies and their antigen-binding fragments described herein inhibit LGALS3-induced T cell apoptosis. In some embodiments, the anti-LGALS3 antibodies and their antigen-binding fragments described herein inhibit T cell immunosuppression. In some embodiments, the anti-LGALS3 antibodies and their antigen-binding fragments described herein suppress or treat cardiovascular diseases. In some embodiments, the anti-LGALS3 antibodies and their antigen-binding fragments described herein suppress or treat heart failure, coronary heart disease, or myocardial infarction. In some embodiments, the anti-LGALS3 antibodies and their antigen-binding fragments described herein suppress or treat pulmonary fibrosis. In some embodiments, the anti-LGALS3 antibody and its antigen-binding fragment described herein suppress or treat kidney disease. In some embodiments, the anti-LGALS3 antibody and its antigen-binding fragment described herein suppress or treat glomerulonephritis. In some embodiments, the anti-LGALS3 antibody and its antigen-binding fragment described herein suppress or treat renal cell carcinoma.

[0032] Also provided herein are polynucleotides (e.g., isolated polynucleotides) comprising such antibodies and nucleic acid sequences (e.g., complementary DNA (cDNA)) encoding their antigen-binding fragments, heavy chains, or light chains. Furthermore, vectors (e.g., expression vectors) and cells (e.g., isolated cells or ex vivo cells) comprising such antibodies and polynucleotides (e.g., isolated polynucleotides) comprising nucleic acid sequences (e.g., complementary DNA (cDNA)) encoding their antigen-binding fragments, heavy chains, or light chains. Also provided are methods for producing such antibodies, their antigen-binding fragments, heavy chains, light chains, vectors, and cells. In other features, provided herein are methods and uses for anti-LGALS3 antibodies for treating or manipulating the symptoms or abnormalities described herein, for example, for treating or manipulating cancer. Related compositions (e.g., pharmaceutical compositions), kits, and diagnostic methods are also provided. As used herein, the terms “LGALS3,” “LGALS3 polypeptide,” “LGALS3 peptide,” “Gal-3,” “Gal-3 polypeptide,” and “Gal-3 peptide” are interchangeable herein to refer to galectin-3 and its functional homologues and fragments that bind to β-galactoside. TM Accession number NM_002306.3 (Sequence ID: 3) provides an exemplary human LGALS3 nucleic acid sequence. GenBank TM Accession number NP_002297.2 (SEQ ID NO: 4) provides an exemplary human LGALS3 amino acid sequence.

[0033] Anti-GALS3 antibody Anti-LGALS3 antibodies or their antigen-binding fragments may include, for example, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetramer antibodies containing two heavy chains and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain variable fragments (scFV), camelized antibodies, aphibodies, and disulfide-bonded Fv (dsFv) or their fragments. Such antibodies can be produced by methods known in the art. A multispecific antibody or fragment thereof refers to an antibody or fragment thereof that can simultaneously bind to at least two targets of different structures (e.g., two different antigens, two different epitopes on the same antigen, or one hapten and one antigen or epitope). One specificity may be, for example, against B cell, T cell, myeloid cell, plasma cell, or mast cell antigen or epitope, such as CD3. The other specificity may be against different antigens on the same or different cell types, such as LGALS3. A multispecific, multivalent antibody is a construct having two or more binding sites, where the binding sites have different specificities (e.g., a bispecific diabody), where one binding site reacts with one antigen and the other with another.

[0034] A bispecific antibody is an antibody that can simultaneously bind to two targets of different structures. Bispecific antibodies (bsAb) and bispecific antibody fragments (bsFab) have at least one arm that immunospecifically binds to a first target (e.g., LGALS3) and at least one other arm that immunospecifically binds to a second target (e.g., CD3). A variety of bispecific fusion proteins can be generated using molecular manipulation. In one form, the bispecific fusion protein is bivalent and consists, for example, (i) an scFv with a single binding site for one antigen and (ii) an antibody or Fab for a second antigen. In another form, the bispecific fusion protein is tetravalent and consists, for example, an IgG with two binding sites for one antigen and two identical scFvs for a second antigen. See, for example, International Publication WO 2011 / 1160119 (which is incorporated herein by reference in its entirety). Recent methods for producing bispecific monoclonal antibodies involve the use of engineered recombinant monoclonal antibodies. These monoclonal antibodies possess additional cysteine ​​residues and therefore can crosslink more strongly than more conventional immunoglobulin isotypes. See, for example, FitzGerald et al., Protein Eng. 10(10): 1221-1225, 1997. Another approach involves engineering recombinant fusion proteins by linking fragments of two or more different single-chain antibodies or antibody fragments that possess the required bispecificity. See, for example, Coloma et al., Nature Biotech. 15: 159-163, 1997. A wide variety of bispecific fusion proteins can be produced using molecular manipulation.

[0035] Bispecific fusion proteins can be constructed in a similar manner by linking two or more different single-chain antibodies or antibody fragments. A variety of fusion proteins can be constructed using recombinant methods. In certain features, a flexible linker ligates an scFv (e.g., a CD3-targeting scFv) to the constant region of the light chain of a monoclonal antibody (e.g., the anti-LGALS3 antibody described herein). An appropriate linker sequence necessary for in-frame linkage between the heavy chain Fc and scFv is introduced into the VL and V kappa domains via a PCR reaction. Subsequently, the DNA fragment encoding the scFv is bound to a staging vector containing a DNA sequence encoding the CH1 domain. The resulting construct is excised and bound to a vector containing a DNA sequence encoding the VH region of the antibody (e.g., anti-LGALS3 antibody). The resulting vector is used to transfect a suitable host cell (e.g., a mammalian cell) to express the bispecific fusion protein. In certain embodiments, functional bispecific single-chain antibodies (bscAbs) (also called diabodies) can be prepared using the anti-LGALS3 antibodies and their antigen-binding fragments described herein and generated in mammalian cells by recombinant methods. See, for example, Mack et al., Proc. Natl. Acad. Sci., 92: 7021-7025, 1995 (the above is incorporated herein by reference in its entirety). For example, a bscAb can be generated by linking two single-chain Fv fragments via a glycine-serine linker using a recombinant method. The VL and VH domains of the two antibodies in question are isolated using standard PCR methods known in the art. Bispecific single-chain antibodies and bispecific fusion proteins are within the scope of the present invention.

[0036] In certain embodiments, the anti-GALS3 antibody or its antigen-binding fragment described herein refers to an scFv. scFv is an industry-recognized term. An scFv comprises a fusion protein of the variable regions of the heavy (H) and light (L) chains of an immunoglobulin, where the fusion protein retains the same antigen specificity as the whole immunoglobulin. VH is fused to VL via a peptide linker. In certain embodiments, the peptide linker is an amino acid residue having a length of 5 to 25, 5 to 15, 10 to 20, 10 to 15, or 15 to 25. In certain embodiments, the scFv peptide linker exhibits one or more features suitable for peptide linkers known to those skilled in the art. In certain embodiments, the scFv peptide linker comprises an amino acid that makes the scFv peptide linker soluble, such as serine and threonine. In certain embodiments, the scFv peptide linker comprises an amino acid that makes the scFv peptide linker flexible, such as glycine. In certain embodiments, the scFv peptide linker connects the N-terminus of VH to the C-terminus of VL. In certain embodiments, the scFv peptide linker can connect the C-terminus of VH to the N-terminus of VL. In certain embodiments, the anti-GALS3 antibody or its antigen-binding fragment described herein refers to a chimeric antigen receptor (CAR). CAR is an industry-recognized term. CARs can target tumor-associated antigens (e.g., LGALS3). The CARs provided herein consist of scFv derived from LGALS3 glycosylation.

[0037] In some embodiments, the antibody, the transmembrane main is a transmembrane main derived from a T cell co-stimulatory molecule (e.g., CD28, CD8, CD38, OX-40, or 4-IBB-derived transmembrane main), and the primary signaling domain (e.g., a T cell receptor (TCR) zeta (ζ) chain cytoplasmic signaling domain). In some embodiments, the CAR further also comprises one or more additional regions or domains, e.g., one or more spacers or linkers (including extracellular spacers). These are derived, for example, from an antibody or other cell surface molecule, e.g., a spacer comprising one or more antibody CH2, CH3, and / or hinge regions, or a spacer derived from a CD28 molecule or a CD8 molecule, or other spacers. Also provided herein are cells, e.g., T cells engineered to express such a CAR, e.g., cells that recombinantly express such a CAR. CAR-expressing T cells preferably induce T cell activation, proliferation, and / or lysis of cells of such tumors upon recognition of LGALS3-expressing tumors.

[0038] An anti-LGALS3 antibody may be any type of immunoglobulin (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b). In certain embodiments, the antibody described herein is an IgG antibody or one of the aforementioned classes or subclasses. In certain embodiments, the antibody described herein is an IgG1 antibody. In certain embodiments, the antibody described herein is an IgG2 antibody. In certain embodiments, the antibody described herein is an IgG2a antibody. In certain embodiments, the antibody described herein is an IgG2b antibody. In certain embodiments, the antibody described herein is an IgG3 antibody. In certain embodiments, the antibody described herein is an IgG4 antibody. In certain embodiments, the antibody described herein is a mixture of multiple antibody types or a mixture of multiple subclasses. In certain embodiments, the antibody described herein is a mixture of IgG2a and IgG2b antibodies. In certain embodiments, the antibody is a humanized version of a rodent monoclonal antibody. The antigen-binding fragment of the anti-LGALS3 antibody is a Fab fragment, an F(ab')2 fragment, or a portion of the anti-LGALS3 antibody, the portion containing amino acid residues that confer its specificity to the antigen to the anti-LGALS3 antibody (e.g., a complementarity-determining region (CDR)). The anti-LGALS3 antibody can be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters).

[0039] As used herein, the terms “variable region” or “variable domain” are interchangeable and common in the industry. A variable region typically refers to a portion of an antibody, generally the light or heavy chain portion, typically the approximately 110–120 amino acids at the amino-terminus of a mature heavy chain and approximately 90–100 amino acids at the amino-terminus of a mature light chain, where the sequence of the aforementioned portion varies widely among antibodies and is used for the binding and specificity of the antibody to the relevant antigen. Sequence variability is concentrated in a region called the complementarity-determining region (CDR), while the more highly conserved region in the variable domain is called the framework region (FR). CDRs flank with FRs. Generally, the spatial orientation of CDRs and FRs is FR1–CDR1–FR2–CDR2–FR3–CDR3–FR4 from the N-terminus to the C-terminus. While not bound by any specific mechanism or theory, the CDRs of the light and heavy chains are essentially necessary for antibody-antigen interaction and specificity. In certain embodiments, the variable region is a rodent (e.g., mouse or rat) variable region. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region includes a rodent (e.g., mouse or rat) CDR and a human framework region (FR). In individual embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region includes a rodent or mouse CDR and a primate (e.g., non-human primate) framework region (FR).

[0040] CDRs are bounded in various ways within the industry. These methods include Kabat, Chothia, and IMGT, as well as exemplary bounding. Kabat bounding is based on sequence variability (Kabat, Elvin A. et al., Sequences of Proteins of Immunological Interest. Bethesda: National Institutes of Health, 1983). In Kabat's numbering system, (i) VH CDR1 is typically located at amino acid positions 31 to 35 of the heavy chain (sometimes including one or two additional amino acids after amino acid position 35 (referred to as 35A and 35B in Kabat's numbering proposal)); (ii) VH CDR2 is typically located at amino acid positions 50 to 65 of the heavy chain; and (iii) VH CDR2 is typically located at amino acid positions 95 to 102 of the heavy chain (Kabat, Elvin A. et al., Sequences of Proteins of Immunological Interest. Bethesda: National Institutes of Health, 1983). In Kabat's numbering system, (i) VL CDR1 is located at amino acid positions 24 to 34 of the light chain; (ii) VL CDR2 is typically located at amino acid positions 50 to 56 of the light chain; and (iii) VL CDR3 is typically located at amino acid positions 89 to 97 of the light chain (Kabat, Elvin A. et al., Sequences of Proteins of Immunological Interest. Bethesda: National Institutes of Health, 1983). As is well known to those skilled in the art, when using Kabat's numbering system, the actual linear amino acid sequence of the antibody's variable domain may contain fewer or additional amino acids due to shortening or elongation of FR and / or CDR, and therefore the Kabat number of an amino acid is not necessarily the same as its linear amino acid number.

[0041] Chothia's boundary determination is based on the location of structural loop regions (Chothia et al. (1987) J Mol Biol 196: 901-917; and US Patent No. 7,709,226). The term “Chothia’s CDR” and similar terms are recognized in the industry and refer to the antibody CDR sequence determined according to the method of Chothia and Lesk (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917), which will be referred to herein as “Chothia’s CDR” (see, for example, US Patent No. 7,709,226 and Martin, A., “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin, 2001). In the Chothia numbering system, when the Ksbat numbering system is used for numbering amino acid residues in the VH region, (i) VH CDR1 is typically located at amino acid positions 26 to 32 of the heavy chain; (ii) VH CDR2 is typically located at amino acid positions 53 to 55 of the heavy chain; and (iii) VH CDR3 is typically located at amino acid positions 96 to 101 of the heavy chain. In specific embodiments, when the Ksbat numbering system is used for numbering amino acid residues in the VH region in the Chothia numbering system, (i) VH CDR1 is typically located at amino acid positions 26 to 32 or 34 of the heavy chain; (ii) VH CDR2 is typically located at amino acid positions 52 to 56 of the heavy chain (in one embodiment, CDR2 is located at positions 52A-56, where 52A follows position 52); and (iii) VH CDR3 is typically located at amino acid positions 95 to 102 of the heavy chain (in one embodiment, there are no amino acids at the positions numbered 96-100).In the Chothia numbering system, when the Ksbat numbering system is used for numbering amino acid residues in the VL region, (i) VL CDR1 is typically located at amino acid positions 26 to 33 of the light chain; (ii) VL CDR2 is typically located at amino acid positions 50 to 52 of the light chain; and (iii) VL CDR3 is typically located at amino acid positions 91 to 96 of the light chain. In specific embodiments, when the Ksbat numbering system is used for numbering amino acid residues in the VL region of the Chothia numbering system, (i) VL CDR1 is typically located at amino acid positions 24 to 34 of the light chain; (ii) VL CDR2 is typically located at amino acid positions 50 to 56 of the light chain; and (iii) VL CDR3 is typically located at amino acid positions 89 to 97 of the light chain (in some embodiments, there are no amino acids at positions numbered 96-100). The arrangement of these Chothia CDRs may vary depending on the antibody and can be determined according to methods known in the art.

[0042] IMGT boundary determination originates from EVIGT (IMGT®; the international ImMunoGeneTics information system® website imgt.org (Founder and Director: Marie-Paule Lefranc, Montpellier, France) (see, for example: Lefranc, M.-P., 1999, The Immunologist, 7: 132-136; and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212; (both references are incorporated herein by reference in their entirety)). In the IMGT numbering system, (i) VH CDR1 is typically located at amino acid positions 25 to 35 of the heavy chain; (ii) VH CDR2 is typically located at amino acid positions 51 to 57 of the heavy chain; (iii) VH CDR3 is typically located at amino acid positions 93 to 102 of the heavy chain. In the IMGT numbering system, (i) VL (ii) VL CDR1 is typically located at amino acid positions 27 to 32 of the light chain; (ii) VL CDR2 is typically located at amino acid positions 50 to 52 of the light chain; (iii) VL CDR3 is typically located at amino acid positions 89 to 97 of the light chain.

[0043] Sequence and structure of LGALS3 antibody In certain embodiments, what is provided herein is an anti-LGALS3 antibody or its antigen-binding fragment, which includes, for example, the VH CDRs of any of the anti-LGALS3 antibodies provided herein, as shown in Figures 12a, 12b, and 13A-O. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment provided herein includes VH CDR1 of the anti-LGALS3 antibody shown in Figure 12a. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment provided herein includes VH CDR2 of the anti-LGALS3 antibody shown in Figure 12a. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment provided herein includes VH CDR3 of the anti-LGALS3 antibody shown in Figure 12a. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment provided herein includes one, two, or all three of the VH CDRs of the anti-LGALS3 antibodies shown in the table in Figure 12a. In certain embodiments, what is provided herein is an anti-LGALS3 antibody or its antigen-binding fragment, which includes, for example, the VL CDRs of any of the anti-LGALS3 antibodies provided herein, as shown in Figures 12 and 13. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment provided herein includes VL CDR1 of the anti-LGALS3 antibody shown in Figure 12b. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment provided herein includes VL CDR2 of the anti-LGALS3 antibody shown in Figure 12b. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment provided herein includes VL CDR3 of the anti-LGALS3 antibody shown in Figure 12b. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment provided herein includes one, two, or all three of the VL CDRs of the anti-LGALS3 antibody shown in Figure 12b.

[0044] In certain embodiments, what is provided herein is an anti-LGALS3 antibody or its antigen-binding fragment, which includes, for example, a VH CDR of any of the anti-LGALS3 antibodies provided herein, as shown in Figures 13A-C. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment provided herein includes VH CDR1 of the anti-LGALS3 antibody shown in Figures 13A-C. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment provided herein includes VH CDR2 of the anti-LGALS3 antibody shown in Figures 13A-C. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment provided herein includes VH CDR3 of the anti-LGALS3 antibody shown in Figures 13A-C. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment provided herein includes one, two, or all three of the VH CDRs of the anti-LGALS3 antibodies shown in Figures 13A-C. In certain embodiments, what is provided herein is an anti-LGALS3 antibody or its antigen-binding fragment, comprising, for example, a VL CDR of any of the anti-LGALS3 antibodies provided herein, as shown in Figures 13A-C. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment provided herein comprises VH CDR1 of the anti-LGALS3 antibody shown in Figures 13A-C. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment provided herein comprises VH CDR2 of the anti-LGALS3 antibody shown in Figures 13A-C. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment provided herein comprises VH CDR3 of the anti-LGALS3 antibody shown in Figures 13A-C. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment provided herein comprises one, two, or all three VL CDRs of the anti-LGALS3 antibody shown in Figures 13A-C.

[0045] In individual embodiments, the anti-LGALS3 antibody or antigen-binding fragment described herein comprises VH, wherein the VH comprises (a) VH CDR1 comprising the amino acid sequence SYGVH (SEQ ID NO: 5); (b) VH CDR2 comprising the amino acid sequence VIWSDGSTTYNSTLKS (SEQ ID NO: 6); and (c) VH CDR3 comprising the amino acid sequence HISNYGTMDY (SEQ ID NO: 7). In individual embodiments, the anti-LGALS3 antibody or antigen-binding fragment described herein comprises VH, wherein the VH comprises VH CDR1 comprising the amino acid sequence of SEQ ID NO: 11 or 17, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 12 or 18, and VH CDR3 comprising the amino acid sequence of SEQ ID NO: 13 or 19. In individual embodiments, the anti-LGALS3 antibody or antigen-binding fragment described herein comprises a VL, the VL comprising (a) VL CDR1 comprising the amino acid sequence RASQDIRNYLN (SEQ ID NO: 8); (b) VL CDR2 comprising the amino acid sequence YTSRLHS (SEQ ID NO: 9); and (c) VL CDR3 comprising the amino acid sequence QHFNTLPPT (SEQ ID NO: 10). In individual embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein comprises a VL, the VL comprising VL CDR1 comprising the amino acid sequence of SEQ ID NO: 14 or 120, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 15 or 21, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 16 or 22.

[0046] In individual embodiments, the anti-LGALS3 antibody or antigen-binding fragment described herein comprises: (i) VH comprising (a) VH CDR1 comprising amino acid sequence SYGVH (SEQ ID NO: 5), (b) VH CDR2 comprising amino acid sequence VIWSDGSTTYNSTLKS (SEQ ID NO: 6), and (c) VH CDR3 comprising amino acid sequence HISNYGTMDY (SEQ ID NO: 7); and (ii) VL comprising (a) VL CDR1 comprising amino acid sequence RASQDIRNYLN (SEQ ID NO: 8), (b) VL CDR2 comprising amino acid sequence YTSRLHS (SEQ ID NO: 9), and (c) VL CDR3 comprising amino acid sequence QHFNTLPPT (SEQ ID NO: 10). In individual embodiments, the anti-LGALS3 antibody or antigen-binding fragment described herein comprises a VH region, the VH region comprising QVQLKESGPGLVAPSQSLSITCTISGFSLSSYGVHWVRQPPGKGLEWLVVIWSDGSTTYNSTLKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARHISNYGTMDYWGQGTSVTVS (SEQ ID NO: 24). In individual embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein comprises a VL region, the VL region comprising DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGSIKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIRNLEQEDIATYFCQHFNTLPPTFGGGTKLEIK (Sequence ID: 26). In individual embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein includes a VH region containing a heavy chain as shown in either Figure 13D-O. In individual embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein includes a VL region containing a light chain as shown in either Figure 13D-O.

[0047] In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein may be represented by its VH domain alone, or its VL domain alone, or its three VH CDRs alone, or its three VL CDRs alone. See, for example, Rader et al. (Rader C et al. (1998) PNAS 95: 8910-8915) (the entire paper is incorporated herein by reference). The paper describes the humanization of a mouse anti-avP3 antibody by identifying complementary light chains or heavy chains from human light chain or heavy chain libraries, respectively, and obtaining a humanized antibody variant having the same or higher affinity as the original antibody. See also Clackson et al. (Clackson T et al., (1991) Nature 352: 624-628) (the entire paper is incorporated herein by reference). The aforementioned paper describes a method for generating antibodies that bind to specific antigens by using a specific VH domain (or VL domain) to screen a library for complementary variable domains. See also the paper by Kim and Hong (Kim and Hong (2007) J Microbiol 45: 572-577) (the aforementioned paper is incorporated herein by reference in its entirety). The aforementioned paper describes a method for generating antibodies that bind to specific antigens by using a specific VH domain to screen a library (e.g., a human VL library) for complementary VL domains, and then using a selected VL domain to guide the selection of additional complementary (e.g., human) VH domains.

[0048] In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein may be a humanized antibody, such as a humanized rodent antibody. Humanized antibodies can be prepared using a variety of techniques known in the art. The aforementioned technologies include, but are not limited to, the following: CDR transplantation (European Patent EP 239,400; International Publication WO 91 / 09967; and US Patents Nos. 5,225,539, 5,530,101 and 5,585,089), chain shuffling (US Patent No. 5,565,332), overlay or surface reconstruction (European Patents EP 592,106 and EP 519,596; Padlan (1991) Molecular Immunology 28(4 / 5):489-498; Studnicka et al. (1994) Protein Engineering 7(6):805-814; and Roguska et al. (1994) PNAS 91:969-973), and the technologies disclosed below (US Pat. No. 6,407,213, US Pat. No. 5,766,886, WO 9317105; Sandhu (1994) Gene 150(2):409-10; Pedersen et al. (1994) J. Mol. Biol. 235(3):959-73; Couto et al. (1995) Cancer Res. 55(8): 1717-22; Roguska et al. (1996) Protein Eng. 9(10):895 904;Baca et al. (1997) J. Biol. Chem. 272(16): 10678-84;Couto et al. (1995) Cancer Res. 55 (23 Supp):5973s- 5977s, Caldas et al. (2000) Protein Eng. 13(5):353-60;Morea See also U.S. Patent Publication U.S. 2005 / 0042664 Al (February 24, 2005) (each of which is incorporated herein by reference in its entirety).

[0049] In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein is a synthetic human antibody. A synthetic human antibody can be produced, for example, by designing a variable region sequence from fragments of multiple human antibody variable region sequences in a manner that avoids T cell epitopes, thereby minimizing the immunogenicity of the resulting antibody (see, for example, Baker et al. (2010) Self Nonself l (4):314-322; Bryson et al. (2010) BioDrugs 24(l): l-8; and Jones et al. (2009) Methods Mol Biol. 525:405-23). ​​Such an antibody may contain a human constant region sequence (e.g., human light chain and / or heavy chain constant regions). In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein may be a deimmunized antibody. A deimmunized antibody is an antibody from which the T cell epitope has been removed. Methods for producing deimmunized antibodies are described. See, for example, Jones et al. Methods Mol Biol. 2009;525:405-23, xiv; and De Groot et al. (2006) Cell. Immunol. 244: 148-153.

[0050] In specific embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein is a humanized immunoglobulin and includes three VH CDRs and three VL CDRs (i.e., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) of either of the antibodies in Figures 12 and 13, a human-derived framework region, and a human-derived constant region. Non-limiting examples of human framework regions have been described in the art. See, for example, Kabat et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein includes a framework region (e.g., a framework region of the VL domain and / or VH domain), said framework region is either a human framework region or derived from a human framework region. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein comprises a framework region (e.g., a framework region of the VL domain and / or VH domain), wherein the framework region is a framework region of a primate (e.g., a non-human primate) or is derived from a framework region of a primate (e.g., a non-human primate). For example, an antigen-specific non-human antibody, typically of rodent origin (e.g., mouse or rat), CDR is implanted in homologous human or non-human primates (e.g., Old World apes, e.g., chimpanzees (Pan troglodytes), bonobos (Pan paniscus), or gorillas, Old World monkeys, e.g., Macaca, or cynomolgus macaques (Macaca cynomolgus)). The non-human primate framework region sequence is described in U.S. Patent Application Publication US 2005 / 0208625.

[0051] In specific embodiments, the positions of VH CDR1, VH CDR2 and / or VH CDR3 in the VH region and / or VL CDR1, VL CDR2 and / or VL CDR3 in the VL region of the anti-LGALS3 antibody or its antigen-binding fragment described herein may vary by 1, 2, 3, 4, 5, 6 amino acids, or more than 6 amino acids, provided that immunospecific binding to LGALS3 or the portion containing the carbohydrate-binding domain is maintained (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In another embodiment, the lengths of VH CDR1, VH CDR2 and / or VH CDR3 in the VH region and / or VL CDR1, VL CDR2 and / or VL CDR3 in the VL region of the anti-LGALS3 antibody or its antigen-binding fragment described herein may vary by more than 1, 2, 3, 4, 5, 6 amino acids, or 6 amino acids (e.g., shorter or longer), provided that immunospecific binding to LGALS3 or the portion containing the carbohydrate-binding domain is maintained (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In another embodiment, the amino and / or carboxyl termini of the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 described herein are elongated or shortened by more than 1, 2, 3, 4, 5, 6 amino acids, or more than 6 amino acids compared to one or more of the CDRs described herein, provided that immunospecific binding to LGALS3 or the portion containing the carbohydrate-binding domain is maintained (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). Whether or not immunospecific binding to LGALS3 is maintained can be confirmed using any method known in the industry, such as an ELISA binding assay, SPR analysis, or FACS analysis.

[0052] In terms of specific features, what is provided herein is an anti-LGALS3 antibody or its antigen-binding fragment, which includes the antibody heavy chain and / or light chain, for example, the heavy chain only, the light chain only, or both the heavy chain and the light chain. In some embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein comprises a heavy chain, wherein the amino acid sequence of the variable region of the heavy chain comprises VH CDR1, VH CDR2, and VH CDR3 of any one of the antibodies listed in Figure 12 or 13, and the constant region of the heavy chain is the human heavy chain constant region. In individual embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein comprises a heavy chain, wherein the amino acid sequence of the variable region of the heavy chain comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively, and the constant region of the heavy chain is the human heavy chain constant region. In specific embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein comprises a heavy chain, wherein the amino acid sequence of the variable region of the heavy chain comprises the amino acid sequence of SEQ ID NO: 24, and the constant region of the heavy chain is the human heavy chain constant region. Non-restrictive examples of human constant region sequences have been described in this field; see, for example, the book cited above (Kabat EA et al., 1991).

[0053] In some embodiments, the heavy chain of the anti-LGALS3 antibody or its antigen-binding fragment described herein may be an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In another specific embodiment, the heavy chain of the anti-LGALS3 antibody or its antigen-binding fragment described herein may include a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein comprises a heavy chain, wherein the amino acid sequence of the variable region of the heavy chain comprises VH CDR1, VH CDR2, and VH CDR3 of any one of the antibodies listed in Figure 12 or 13, wherein the constant region of the heavy chain is an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In individual embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein comprises a heavy chain, wherein the amino acid sequence of the variable region of the heavy chain comprises VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively, and the constant region of the heavy chain is a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In specific embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein comprises a heavy chain, wherein the amino acid sequence of the variable region of the heavy chain comprises the amino acid sequence of SEQ ID NO: 24, and the constant region of the heavy chain is a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain.

[0054] In individual embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein includes a light chain, wherein the amino acid sequence of the variable region of the light chain includes VL CDR1, VL CDR2, and VL CDR3 of any one of the antibodies listed in Figure 12 or 13, and the constant region of the light chain is the human light chain constant region. In individual embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein includes a light chain, wherein the amino acid sequence of the variable region of the light chain includes VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of antibody SEQ ID NO: 8, SEQ ID NO: 8, and SEQ ID NO: 10, respectively, and the constant region of the light chain is the human light chain constant region. In specific embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein includes a light chain, wherein the amino acid sequence of the variable region of the light chain includes the amino acid sequence of SEQ ID NO: 26, and the constant region of the light chain is the human light chain constant region. In some embodiments, the light chain of the anti-LGALS3 antibody or its antigen-binding fragment described herein is a kappa light chain. In another specific embodiment, the light chain of the anti-LGALS3 antibody or its antigen-binding fragment described herein is a lambda light chain. In yet another specific embodiment, the light chain of the anti-LGALS3 antibody or its antigen-binding fragment described herein is a human kappa light chain or a human lambda light chain. In individual embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein comprises a light chain, where the amino acid sequence of the variable region of the light chain comprises VL CDR1, VL CDR2, and VL CDR3 of any one of the antibodies listed in Figure 12 or 13, and further, the constant region of the light chain is a kappa (κ) or lambda (λ) light chain constant region. In individual embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein comprises a light chain, wherein the amino acid sequence of the variable region of the light chain comprises VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of antibody SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively, and further comprises a kappa or lambda light chain constant region. In specific embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein comprises a light chain, wherein the amino acid sequence of the variable region of the light chain comprises the amino acid sequence of SEQ ID NO: 26, and further comprises a kappa or lambda light chain constant region.

[0055] In a specific embodiment, the anti-LGALS3 antibody or its antigen-binding fragment described herein comprises a heavy chain variable region (VH) and a light chain variable region (VL) described herein, wherein the constant region is an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or a type found in human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecules. In another specific embodiment, the anti-LGALS3 antibody or its antigen-binding fragment described herein comprises VH and VL containing any amino acid sequence described herein, wherein the constant region is a type found in an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b). In individual embodiments, the constant region is a type found in human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecules, any class of immunoglobulin molecules (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2).

[0056] In certain embodiments, the antibody epitope can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assay, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., MALDI mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be achieved using any method known in the art (e.g., Giege R et al. (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303). Antibody: Antigen crystals can be tested by well-known X-ray diffraction techniques and further improved by computer software, such as X-PLOR (Yale University, 1992 (distributed by Molecular Simulations, Inc.); see, for example, Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.: U.S. Patent Publication 2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW; Roversi P et al. (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323). Mutagenesis mapping can be achieved using any method known to those skilled in the art. For a description of mutagenesis techniques (including alanine scan mutagenesis), see, for example, Champe M et al., 1995; and Cunningham BC & Wells JA, 1989.In addition, antibodies that recognize and bind to the same or overlapping epitopes can be identified using routine techniques such as immunoassays, for example, by demonstrating the ability of one antibody to block the binding of another antibody to the target antigen (i.e., competitive binding assays). Competitive binding assays can also be used to determine whether two antibodies have similar binding specificity to a particular epitope. Competitive binding can be determined by assays in which the test immunoglobulin inhibits the immunospecific binding of the reference antibody to the common antigen. A variety of competitive binding assays are known (e.g., solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay (Stahli C et al. (1983) Methods Enzymol 9: 242-253), solid-phase direct biotin-avidin EIA (Kirkland TN et al. (1986) J Immunol 137: 3614-9), solid-phase direct label assay, solid-phase direct label sandwich assay (Harlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press), solid-phase direct label RIA using I-125 labeling (Morel GA et al. (1988) Mol Immunol 25(1): 7-15), solid-phase direct biotin-avidin EIA (heung RC et al. (1990) Virology 176: 546-52), and directly labeled RIA (Moldenhauer G et al. (1990) Scand J Immunol 32: 77-82)). Typically, such assays involve the use of purified antigen bound to a solid surface, or cells holding either unlabeled test immunoglobulin and labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of test immunoglobulin. Typically, there is an excess of test immunoglobulin.When competitive antibodies are present in excess, they typically inhibit the immunospecific binding of the reference antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more. Competitive binding assays can be constructed in numerous different forms using either labeled antigens or labeled antibodies. In a common version of this assay, the antigen is immobilized in a 96-well plate. Subsequently, the ability of an unlabeled antibody to block the binding of the labeled antibody to the antigen is measured using radiolabeling or enzymatic labeling. For further details, see, for example, Wagener C et al. (1983) J Immunol 130: 2308-2315; Wagener C et al. (1984) J Immunol Methods 68: 269-274; Kuroki M et al. (1990) Cancer Res 50: 4872-4879; Kuroki M et al. (1992) Immunol Invest 21: 523-538; Kuroki M et al. (1992) Hybridoma 11: 391-407; and Antibodies: A Laboratory Manual, Ed Harlow E & Lane D editors (ibid., pp. 386-389).

[0057] In certain characteristics, competitive binding assays can be used to determine whether one antibody is competitively inhibited by another antibody, for example, in a dose-dependent manner. For example, when two antibodies recognize the same or sterically overlapping epitopes in a competitive binding assay (e.g., an ELISA assay), the antibodies essentially bind to the same or overlapping epitopes as the reference antibody. The assay can be constructed using either a labeled antigen or a labeled antibody in any of the following forms. In individual embodiments, antibodies can be tested in a competitive binding assay using the anti-LGALS3 antibody or its antigen-binding fragment (e.g., a mouse IgG antibody containing the variable region of 14D11) described herein. Another feature is that the antibodies provided herein compete (e.g., in a dose-dependent manner) for the binding of LGALS3 to the anti-LGALS3 antibody or its antigen-binding fragment (e.g., a mouse IgG antibody containing the variable region of 14D11) as determined by assays known to those skilled in the art or described herein (e.g., ELISA). Another feature is that the antibodies provided herein competitively inhibit the binding of the anti-LGALS3 antibody or its antigen-binding fragment (e.g., a mouse IgG antibody containing the variable region of 14D11) to LGALS3 (e.g., in a dose-dependent manner) as determined by assays known to those skilled in the art or described herein (e.g., ELISA).

[0058] In certain embodiments, what is provided herein is an antibody that competes with the antibody described herein for binding to the same extent as the anti-GALS3 antibody or its antigen-binding fragment described herein self-competes for binding to LGALS3. In certain embodiments, what is provided herein is a first antibody that competes with the anti-LGALS3 antibody or its antigen-binding fragment described herein for binding to LGALS3, where such competition is indicated by a reduction in binding between the first antibody and the epitope of more than 80% (e.g., 85%, 90%, 95%, or 98%, or 80% to 85%, 80% to 90%, 85% to 90%, or 85% to 95%). In particular, what is provided herein is an anti-LGALS3 antibody or its antigen-binding fragment, which competes (for example, in a dose-dependent manner) with an anti-LGALS3 antibody or its antigen-binding fragment (including VH, which includes VH CDR1, VH CDR2 and / or VH CDR3, which have the amino acid sequence described in Figure 12 or 13, and / or VL, which includes VL CDR1, VL CDR2 and / or VL CDR3, which have the amino acid sequence described in Figure 12 or 13). In particular, what is provided herein is an anti-LGALS3 antibody or its antigen-binding fragment, which competes (for example, in a dose-dependent manner) with an anti-LGALS3 antibody or its antigen-binding fragment (including VH domain having the amino acid sequence described in Figure 12a and / or VL described in Figure 12b) for immune-specific binding to LGALS3. In particular, what is provided herein is an anti-LGALS3 antibody or its antigen-binding fragment, which binds to the same or overlapping epitopes as the antibody (an antibody containing VH CDR1, VH CDR2 and / or VH CDR3 containing the amino acid sequence described in Figure 12 or 13, and / or VL CDR1, VL CDR2 and / or VL CDR3 containing the amino acid sequence described in Figure 12 or 13). In particular, what is provided herein is an anti-LGALS3 antibody or its antigen-binding fragment, which binds to the same or overlapping epitopes as the antibody (an antibody containing a VH domain having the amino acid sequence described in Figure 12a and / or VL as described in Figure 12b).

[0059] Whether two antibodies bind to the same epitope can be determined using assays known to those skilled in the art or described herein (e.g., X-ray crystallography, ELISA assays, surface plasmon resonance (SPR) assays). Affinity can be measured or expressed by a number of methods known in the art, including, but not limited to, the equilibrium dissociation constant (KD) and the equilibrium binding constant (KA). The KD can be determined by techniques known to those skilled in the art, such as biolayer interference assays. In certain embodiments, antibodies can be generated using an anti-LGALS3 antibody described herein or an epitope to which its antigen-binding fragment binds as an immunogen. In some embodiments, the immunogen includes all or part of the carbohydrate-binding domain of LGALS3.

[0060] Functional characteristics of anti-LGALS3 antibodies The anti-LGALS3 antibody or its antigen-binding fragment described herein is approximately 0.5x10 -3 / s, lxl0 -3 / s, 1.5xl0 -3 / s, 2xl0 -3 / s, 2.5xl0 -3 / s, 3xl0 -3 / s, 4xl0 -3 / s, 5xl0 -3 / s, 6xl0-3 / s, 7xl0 -3 / s, 8xl0 -3 / s, 9xl0 -3 / s, lxl0 -4 / s, 2xl0 -4 / s, 3xl0 -4 / s, 4xl0 -4 / s, 5xl0 -4 / s, 6xl0 -4 / s, 7xl0 -4 / s or 8xl0 -4 It binds to LGALS3 in about 0.5xl0 kd or less than the aforementioned kd. In some embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein is about 0.5xl0 -3 / s to 8xl0 -4 Connect with LGALS3 using the / s kd. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein is at least, or about 2.5x10 4 / s, 3xl0 4 / s, 3.5xl0 4 / s, 5xl0 4 / s, 5.5xl0 4 / s, 6xl0 4 / s, 6.5xl0 4 / s, 7xl0 4 / s, 7.5xl0 4 / s, 8xl0 4 / s, 9xl0 4 / s, or 9.5xl0 4 It binds to LGALS3 with a ka of / s. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein has at least or about 2.5xl0 4 / s to 9xl0 5 It binds to LGALS3 using the / s ka.

[0061] In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein binds to LGALS3 with a KD of about 1000 nM, 500 nM, 100 nM, 50 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, or 0.05 nM or less. In some embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein binds to LGALS3 with a KD of about 10 nM to about 1000 nM. In some embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein binds to LGALS3 with a KD of about 100 nM to about 1000 nM. In some embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein binds to LGALS3 with a KD of about 500 nM to about 1000 nM. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment provided herein binds to LGALS3 at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 250, 500, or 1000 times more than the isotype control. Isotype control antibody is an industry-recognized term, and the foregoing corresponds to a class and type of primary antibody (e.g., the anti-LGALS3 antibody or its antigen-binding fragment provided herein) that lacks specificity for the target. Isotype controls are used as negative controls to help discriminate nonspecific background signals from specific antibody signals. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein binds to LGALS3 with an affinity 100–1,000 times greater than that of innate ligand binding.

[0062] Assays for determining anti-LGALS3 antibody or antigen-binding fragment-mediated Matrigel invasive inhibition are known to those skilled in the art. For example, BD BioCoat Matrigel invasive insertions TM Matrisol TMInvasion Inserts) or invasion chambers (catalog # 354480 (present in 24-well plates)) and control inserts (catalog # 354578 (present in 24-well plates)) can be purchased from BD Biosciences, MA. The Matrigel invasion assay can be performed according to the manufacturer's protocol. Briefly, the Matrigel chambers (stored at -20°C) and control inserts (stored at 4°C) in 24-well plates are brought to room temperature. Both inserts, including the inside of the inserts and the outside of the wells of the 24-well plate, are rehydrated with 0.5 mL of serum-free culture medium in a 37°C 5% CO2 humidified incubator for 2 hours. Cultured SKOV3 cells are trypsinized and washed with culture medium. One million cells are separated into a separate centrifuge tube and washed three times with serum-free culture medium. These cells are later prepared to yield 5000 cells in 0.5 mL of serum-free culture medium. Remove the culture medium from the rehydrated insert and transfer it to a new 24-well plate containing 0.75 mL of 10% fetal bovine serum (FBS) culture medium (the insert acts as a chemoattractant). Immediately add 5000 cells from 0.5 mL of serum-free culture medium to the insert. Observe carefully to ensure that no air bubbles are trapped inside or outside the insert. Incubate the 24-well plate in a 5% CO2 humidified incubator at 37°C for 48 hours. After incubation, remove non-invasive cells from the upper surface of the membrane by inserting a cotton tip swab into the Matrigel insert or control insert and gently pressing and "scraping" across the membrane surface while moving the tip of the swab. Repeat scraping with a second swab moistened with culture medium. Subsequently, stain the insert for 30 minutes in a new 24-well plate containing 0.5 mL of 0.5% crystal violet dye in distilled water. Following staining, rinse the inserts in three beakers of distilled water to remove excess dye. Air dry the inserts in a new 24-well plate. Manually count the invasive cells under an inverted microscope at 200x magnification. Count and record the number of fields of view of three membranes in each field.

[0063] In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein can inhibit or reduce metastasis, inhibit tumor growth, or induce tumor regression in mouse model studies. For example, a tumor cell line can be introduced into athymic nude mice, and these athymic mice can be administered one or more doses of the anti-LGALS3 antibody described herein, and the tumor regression of the injected tumor cells can be monitored for several weeks and / or months. In some cases, the administration of the anti-LGALS3 antibody or its antigen-binding fragment to athymic nude mice can be carried out before the introduction of the tumor cell line. In certain embodiments, SKOV3 cells are used for the mouse xenograft model described herein. In specific embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein inhibits tumor growth or induces tumor regression in a mouse model by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% compared to a simulated treatment mouse, when evaluated by the method described herein or a method known to those skilled in the art. In specific embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein inhibits tumor growth or induces tumor regression in a mouse model by at least about 25% or 35%, and optionally about 75%, compared to a simulated treatment mouse, when evaluated by the method described herein or a method known to those skilled in the art. In specific embodiments, the anti-LGALS3 antibodies or their antigen-binding fragments described herein inhibit tumor growth or induce tumor regression in mouse models by at least about 1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times compared to simulated treatment mice, when evaluated by the methods described herein or by methods known to those skilled in the art. Simulated treatment mice may be treated with, for example, phosphate-buffered saline or a control (e.g., anti-IgG antibody).

[0064] The determination of tumor growth inhibition or tumor regression can be evaluated, for example, by monitoring tumor size over a period of time, for example, by physical measurement of palpable tumors or other visual detection methods. For example, this can be done by creating tumor cell lines and recombinantly expressing a visualization agent (e.g., green fluorescent protein (GFP) or luciferase), followed by in vivo visualization of GFP by microscopic examination, or by administering a luciferase substrate to xenograft mice and detecting the luminescence resulting from luciferase enzymatic treatment of the luciferase substrate. The degree or level of GFP or luciferase detection correlates with the tumor size in the xenograft mice. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein can increase the survival of animals in a tumor xenograft model compared to simulated treatment mice. In specific embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein, when evaluated by the method described herein or by a method known to those skilled in the art, increases the survival of mice in a tumor xenograft model by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to simulated treatment mice. In specific embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein, when evaluated by the method described herein or by a method known to those skilled in the art, increases the survival of mice in a xenograft model by at least about 25% or 35%, and optionally about 75%, compared to simulated treatment mice. In specific embodiments, the anti-LGALS3 antibodies or their antigen-binding fragments described herein increase the survival of mice in tumor xenograft models by at least about 1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times compared to simulated treatment mice, when evaluated by the methods described herein or by methods known to those skilled in the art. Survival can be determined, for example, by plotting a survival curve of the number of surviving mice against time (e.g., days or weeks) after injection of tumor cell lines. Simulated treatment mice may be treated, for example, with phosphate-buffered saline or a control (e.g., anti-IgG antibody).

[0065] antibody conjugate In some embodiments, the anti-LGALS3 antibody or its antigen-binding fragment provided herein can be complexed with another molecule, such as an organic moiety, a detectable label, and / or an isotope. In certain embodiments, what is provided herein is a complex of an anti-LGALS3 antibody or its antigen-binding fragment, wherein the anti-LGALS3 antibody or its antigen-binding fragment is complexed with one or more drugs, such as imaging agents or cytotoxic agents. Furthermore, what is provided herein is also a bispecific antibody complex, wherein the bispecific antibody is complexed with one or more drugs, such as imaging agents or cytotoxic agents. Furthermore, what is provided herein is also an antibody heavy chain complex, wherein the antibody heavy chain is complexed with one or more drugs, such as imaging agents or cytotoxic agents. Furthermore, what is provided herein is also an antibody light chain complex, wherein the antibody light chain is complexed with one or more drugs, such as imaging agents or cytotoxic agents. Furthermore, what is provided herein is also a fusion protein complex, wherein the fusion protein is complexed with a drug, such as an imaging agent or cytotoxic agent. In certain embodiments, the drugs are complexed covalently or non-covalently. In certain embodiments, the imaging agent is a detectable label, such as a chromogenic, enzymatic, radioisotope, isotopic, fluorescent, damaging, or chemiluminescent label, a nuclear magnetic resonance contrast agent, or other label. Non-limiting examples of suitable colorimetric labels include diaminobenzidine and 4-hydroxyazo-benzene-2-carboxylic acid.

[0066] Non-limiting examples of appropriate enzyme labeling include: malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. Non-limiting examples of appropriate radioisotope labeling include: 3 H, 111 In, 125 I, 131 I, 32 P,35 S, 14 C, 51 Cr, 57 To, 58 Co, 59 Fe, 75 Se, 152 Eu, 90 Y, 67 Cu, 217 Ci, 211 At, 212 Pb, 47 Sc, 223 Ra, 224 Ra, 89 Zr, 177 Lu and 109 Pd are included. In certain embodiments, 125 I or 131 I-labeled anti-LGALS3 antibody or an antigen-binding fragment thereof avoids the problem of dehalogenation in the liver, so 111 In is used for in vivo imaging. In addition, for imaging 111 In has more favorable gamma emission energy (Perkins et al. (1985) Eur. J. Nucl. Med. 70:296 - 301; Carasquillo et al., (1987) J. Nucl. Med. 25:281 - 287). For example, 111 In-bound monoclonal antibodies by 1-(P-isothiocyanatobenzyl)-DPTA are hardly taken up by non-tumor tissues (especially the liver), and thus the tumor localization specificity is enhanced (Esteban et al., (1987) J. Nucl. Med. 28:861 - 870). Non-limiting examples of suitable non-radioactive isotope labels include 157 Gd, 55 Mn, 162 Dy, 52 Tr and 56 Fe are included.

[0067] Non-limiting examples of suitable fluorescent labels include 152This includes Eu labeling, fluresane labeling, isothiocyanate labeling, rhodamine labeling, phycoerythrin labeling, allophycocyanin labeling, green fluorescent protein (GFP), o-phthalaldehyde labeling, and fluoresamine labeling. Non-limiting examples of chemiluminescent labels include luminol labels, isoluminol labels, aromatic acridinium ester labels, imidazole labels, acridinium salt labels, oxalate ester labels, luciferin labels, luciferase labels, and aequorin labels. Non-limiting examples of nuclear magnetic resonance contrast agents include heavy metal nuclei (e.g., Gd, Mn, and iron). Techniques known to those skilled in the art for complexing the above-mentioned labels with the anti-LGALS3 antibody or its antigen-binding fragment, bispecific antibody, antibody heavy chain, antibody light chain, and fusion protein are described, for example, in the following literature: Kennedy et al. (1976) Clin. Chm. Acta 70: 1-31; and Schurs et al. (1977) Clin. Chm. Acta 81: 1-40. The conjugation techniques described in the latter include the glutaraldehyde method, the periodate method, the dimaleimide method, and the m-maleimidebenzyl-N-hydroxy-succinimide ester method, any of which are incorporated herein by reference. Non-exclusive examples of cytotoxic agents include cell proliferation inhibitors or cytotoxic agents, radioactive metal ions (e.g., alpha emitters), and toxins (e.g., Pseudomonas exotoxin, abrin, cholera toxin, lysine A, and diphtheria toxin).

[0068] In certain embodiments, the agent is a diagnostic agent. A diagnostic agent is an agent useful for diagnosing or detecting a disease by determining the location of cells containing an antigen. Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (e.g., with biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and magnetic resonance imaging (MRI) enhancers (e.g., paramagnetic ions). U.S. Patent No. 6,331,175 describes MRI technology and the preparation of antibodies complexed with MRI enhancing agents, which are incorporated herein by reference. In some embodiments, the diagnostic agent is selected from the group consisting of radioisotopes, enhancing agents used in magnetic resonance imaging, and fluorescent compounds. To load a radioactive metal or paramagnetic ion onto an anti-LGALS3 antibody or its antigen-binding fragment, it is necessary to react the antibody with a reagent having a long tail, to which multiple chelate groups can be attached for ionic bonding. Such tails are polymers, e.g., polylysine, polysaccharides, or other derivative or derivable chains, which have a pendant group to which a chelating group, e.g., ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrin, polyamine, crown ether, bis-thiosemicarbazone, polyoxime, and similar groups that have proven useful for this purpose may be conjugated. The chelate is conjugated to the antibody using standard chemical reactions. The chelate is typically linked to the antibody molecule by a group that enables conjugation with the antibody molecule, resulting in minimal reduction of immunoreactivity and minimal aggregation and / or internal crosslinking. Other less common methods and reagents for conjugating chelates with antibodies are disclosed in US Pat. No. 4,824,659 (Hawthorne; issued April 25, 1989; title: “Antibody Conjugates” (the above disclosure is incorporated herein by reference in its entirety)). Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogues, which are used with diagnostic isotopes for radiographic imaging.The same chelates are useful for MRI when compounded with non-radioactive metals (e.g., manganese, iron, and gadolinium) and used with the anti-LGALS3 antibodies or their antigen-binding fragments provided herein. Macrocyclic chelates (e.g., NOTA, DOTA, and TETA) are particularly useful when used with a variety of metals and radioactive metals, such as gallium, yttrium, and copper, respectively. Such metal-chelate complexes can be highly stabilized by adjusting the ring size relative to the target metal. Other cyclic chelates, such as macrocyclic polyethers (of interest for stably binding nuclides), such as 223Ra for RAIT, are included herein.

[0069] In certain embodiments, the drug is an organic factor. Such organic factors can give rise to complexes having improved pharmacokinetic properties (e.g., increased in vivo blood half-life). The organic moiety may be a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. As used herein, the term “fatty acid” includes monocarboxylic acids and dicarboxylic acids. As used herein, “hydrophilic polymer group” is an organic polymer that is more soluble in water than octane, such as polylysine. Suitable hydrophilic polymers for modifying the anti-LGALS3 antibody or its antigen-binding fragment provided herein may be linear or branched and include, for example,: polyalkane glycols (e.g., polyethylene glycol (PEG), monomethoxy-polyethylene glycol, and polypropylene glycol), carbohydrates (e.g., dextran, cellulose, oligosaccharides, and polysaccharides), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, and polyaspartic acid), polyalkane oxides (e.g., polyethylene oxide and polypropylene oxide), and polyvinylpyrrolidone. In certain embodiments, the hydrophilic polymers used to modify the anti-LGALS3 antibody or its antigen-binding fragment, bispecific antibody, antibody heavy chain, antibody light chain, or fusion protein provided herein have molecular weights ranging from about 800 to about 150,000 daltons as separate molecular entities. For example, PEG 5000 and PEG 20,000(The subscript indicates the average molecular weight of the polymer according to Dalton.) can be used. Hydrophilic polymer groups can be substituted with one to about six alkyl, fatty acid, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups can be prepared using appropriate methods. For example, polymers containing amine groups can be bonded to carboxylic acid groups of fatty acids or fatty acid esters, and activated carboxylic acid groups on fatty acids or fatty acid esters (e.g., activated with N,N-carbonyldiimidazole) can be bonded to hydroxyl groups on the polymer.

[0070] Fatty acids and fatty acid esters suitable for modifying anti-LGALS3 antibodies or their antigen-binding fragments, bispecific antibodies, antibody heavy chains, antibody light chains, or fusion proteins provided herein may be saturated or may contain one or more unsaturated units. Suitable fatty acids for modifying anti-LGALS3 antibodies or their antigen-binding fragments, bispecific antibodies, antibody heavy chains, antibody light chains, or fusion proteins provided herein include, for example: n-dodecanoic acid, n-tetradecanoic acid, n-octadecanoic acid, n-eicosanoic acid, n-docosanoic acid, n-triacontanoic acid, n-tetracontanoic acid, cis-delta-9-octadecanoic acid, all cis-delta-5,8,1,14-eicosatetraenoic acid, octanedioic acid, tetradecanediic acid, octadecanediic acid, decosanedioic acid, etc. Suitable fatty acid esters include monoesters of dicarboxylic acids containing linear or branched lower alkyl groups. The lower alkyl group may contain one to about 12, preferably one to about 6, carbon atoms. The complexes provided herein can be prepared by appropriate methods, for example, by reaction with one or more modified agents. As used herein, an "activating group" is a chemical moiety or functional group that can react with a second chemical group under appropriate conditions, thereby forming a covalent bond between the modified agent and the second chemical group. For example, amine-reactive activating groups include electrophiles such as tosylates, mesylates, halo(chloro, bromo, fluoro, iodine), and N-hydroxysuccinimidyl esters (NHS). Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acryloryl, pyridyl disulfide, and 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol). Aldehyde functional groups can be bonded to amine- or hydrazide-containing molecules, and the azide group can be reacted with a phosphorus group to form a phosphoramidate or phosphorimide bond. Appropriate methods for introducing activating groups into molecules are well known in the industry (see, for example, Hernanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif., 1996). Activating groups can be directly bonded to organic groups (e.g., hydrophilic polymers, fatty acids, fatty acid esters) or bonded via a linker moiety (e.g., divalent C1-C). 12The linker moiety is a group in which one or more carbon atoms are replaced by heteroatoms (e.g., oxygen, nitrogen, or sulfur). Suitable linker moieties include, for example, tetraethylene glycol, (CH2)3, and NH. Modifiers containing linker moieties can be produced, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine or mono-Boc-diaminohexane) with a fatty acid in the presence of 1-ethyl-3-(3-dimethylaminoprolyl)carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate. The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid to expose the primary amine (which can be bonded to another carboxylate described), or by reacting with maleic anhydride and cyclizing the resulting product to produce an activated maleimide derivative of the fatty acid (see, for example, Thompson, et al., WO 92 / 16221 (the full teaching thereof is incorporated herein by reference)).

[0071] The “modifier” can refer to a suitable organic group containing an activating group (e.g., hydrophilic polymers, fatty acids, and fatty acid esters). For example, the organic moiety can be conjugated to an anti-LGALS3 antibody or its antigen-binding fragment in a position-nonspecific manner by utilizing an amine-reactive modifier (e.g., N-hydroxysuccinimide ester of PEG). The modified anti-LGALS3 antibody or its antigen-binding fragment can also be prepared by reduction of the disulfide bond (e.g., internal chain disulfide bond) of the anti-LGALS3 antibody or its antigen-binding fragment, bispecific antibody, antibody heavy chain, antibody light chain, or fusion protein. Subsequently, the reduced anti-LGALS3 antibody or its antigen-binding fragment, bispecific antibody, antibody heavy chain, antibody light chain, or fusion protein can be reacted with a thiol-reactive modifier to produce the complex provided herein. The anti-LGALS3 antibody provided herein, or the complex containing an organic moiety bound to a specific site of its antigen-binding fragment, can be prepared using appropriate methods, such as reverse proteolysis (Fisch et al. (1992) Bioconjugate Chem., 3: 147-153; Werlen et al. (1994) Bioconjugate Chem., 5: 411-417; Kumaran et al. (1997) Protein Sci. 6(10): 2233-2241; Itoh et al. (1996) Bioorg. Chem., 24(1): 59-68; Capellas et al. (1997) Biotechnol. Bioeng. 56(4): 456-463), and methods described in the literature (Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif., 1996).

[0072] Antibody production Antibody production and screening Another feature provided herein is a method for preparing anti-LGALS3 antibodies or their antigen-binding fragments. The anti-LGALS3 antibodies or their antigen-binding fragments described herein can be prepared by any method known in the art for antibody synthesis, for example, by chemical synthesis or by recombinant expression techniques. Unless otherwise specified, the methods described herein utilize common techniques of molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields of the art. These techniques are described and fully explained in the references cited herein, for example.For example, see the following: Maniatis T et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al. (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al. Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annually updated editions); Current Protocols in Immunology, John Wiley & Sons (1987 and annually updated editions); Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al. (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0073] In specific embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein is an antibody (e.g., a recombinant antibody) prepared, expressed, produced, or isolated by any means including, for example, synthesis, or production by genetic manipulation of DNA sequences. In certain embodiments, such an antibody contains a sequence encoded by a DNA sequence that is not naturally present in the repertoire of in vivo antibody germline cells of animals or mammals (e.g., humans). In specific embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein is produced by a method that includes the use of the carbohydrate-binding domain of LGALS3 or a portion thereof. For example, details of exemplary methods for producing the antibodies described herein are described in Example 3. In certain respects, what is provided herein is an immunogenic peptide containing CNTKLDNNWGREERQSVFPFESG (SEQ ID NO: 2). In certain characteristics, what is provided herein is an immunogenic peptide containing an Fc fusion protein comprising amino acids 117-224 of LGALS3 and a hinge region linked to the Fc region (CH2 and CH3 domains) of human IgG1 heavy chain. Amino acids 117-224 of LGALS3 are represented by the following amino acid sequence: PYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTS (Sequence ID: 27). In some embodiments, amino acids 117-224 of LGALS3 may be encoded by the following exemplary nucleotide sequence: CCTTATAACCTGCCTTTGCCTGGGGGAGTGGTGCCTCGCATGCTGATAACAATTCTGGGCACGGTGAAGCCCAATGCAAACAGAATTGCTTTAGATTTCCAAAGAGGGAATGATGTTGCCTTCCACTTTAACCCACGCTTCAATGAGAACAACAGGAGAGTCATTGTTTGCAATACAAAGCTGGATAATAACTGGGGAAGGGAAGAAAGACAGTCGGTTTTCCCATTTGAAAGTGGGAAACCATTCAAAATACAAGTACTGGTTGAACCTGACCACTTCAAGGTTGCAGTGAATGATGCTCACTTGTTGCAGTACAATCATCGGGTTAAAAAACTCAATGAAATCAGCAAACTGGGAATTTCTGGTGACATAGACCTCACCAGT (Sequence ID: 28).

[0074] In certain embodiments, immunogenic peptides are complexed with immunogenic carrier proteins. In most cases, small antigens (e.g., short peptides or small haptens) are not complex enough to elicit antibody production. Immunogenic carrier proteins, due to their large size and complex structure, can confer immunogenicity to the small antigens they complex, resulting in the production of antibodies against the epitopes on the small antigens and the immunogenic carrier proteins. Therefore, small antigens are always chemically complexed with immunogenic carrier proteins, enhancing the immune response so that antibodies are successfully produced. Commonly used immunogenic carrier proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), Concholepas concholepas hemocyanin (CCH), bovine serum albumin (BSA), and ovalbumin (OVA). In specific embodiments, immunogenic peptides are complexed with KLH. KLH is a copper-containing polypeptide belonging to a group of non-heme proteins called hemocyanins, found in arthropods and mollusks. KLH is isolated from the keyhole limpet (Megathura crenulata). Due to its evolutionary divergence from mammals, high molecular weight, complex structure, and broad surface containing hundreds of lysine groups (providing primary amines as targets for complexation), KLH is a highly immunogenic and effective carrier protein in mammals. In certain embodiments, the immunogenic peptide is 10 to 60 amino acid residues long. In some embodiments, the immunogenic peptide is 10 to 30 amino acid residues long. In some embodiments, the immunogenic peptide is 15 to 25 amino acid residues long. In some embodiments, the immunogenic peptide is 15 to 20 amino acid residues long. In a specific embodiment, the immunogenic peptide is 15 to 18 amino acid residues long.

[0075] In certain embodiments, the immunogenic peptide comprises at least 10 amino acid segments of the LGALS3 CBD domain. In certain embodiments, the immunogenic peptide comprises at least 10 amino acid segments of the amino acid sequence of SEQ ID NO: 27. In certain embodiments, the immunogenic peptide comprises at least 15, 20, 25, or 30 amino acid segments of the amino acid sequence of SEQ ID NO: 27. In specific embodiments, the immunogenic peptide consists of 15 to 30 consecutive amino acid residues of SEQ ID NO: 27. In another feature, what is provided herein is a method for producing an antibody or antigen-binding fragment thereof that immunospecifically binds to LGALS3, the method comprising the step of immunizing a target animal with the immunogenic peptide described above. The target animal immunized according to the method described herein may be, but is not limited to, a goat, sheep, donkey, chicken, guinea pig, rat, rabbit, or mouse. In some embodiments, the target animal immunized according to the method described herein is a rat, rabbit, or mouse. In specific embodiments, the target animal immunized according to the method described herein is a mouse. In some embodiments, the target animal immunized according to the method described herein is a transgenic mouse expressing chimeric forms of diverse immunoglobulin chains. In some embodiments, transgenic mice express a human variable region linked to a constant region of the mouse (see, for example, U.S. Patents 8502018, 9580491, U.S. Patent Publications 20170218090, 20160222093, and 20130167256, each of which is incorporated herein by reference in whole, or see the AlivaMab mouse platform (Ablexis)).

[0076] Immunization of target animals can be carried out by any method known in the art, for example, by administering immunogenic peptides and adjuvants to the target animals as described in Example 3. In another feature, what is provided herein is also a method for preparing immunogenic peptides as described herein. In certain embodiments, the method for preparing immunogenic peptides includes a step of synthesizing the peptide portion. The peptide portion of the immunogenic peptide can be synthesized by any method known in the art, for example, by Fmoc solid-phase peptide synthesis. Methods for producing anti-LGALS3 antibodies or their antigen-binding fragments as described herein are known to those skilled in the art and include, for example, chemical synthesis, purification from biological sources, or recombinant expression techniques (including, for example, those using mammalian cells or transgenic preparations). Unless otherwise specified, the methods described herein utilize the common techniques of molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and the relevant fields of the art. These techniques are described and fully explained in the references cited herein, for example.For example, see the following: Maniatis T et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al. (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al. Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annually updated editions); Current Protocols in Immunology, John Wiley & Sons (1987 and annually updated editions); Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al. (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0077] A variety of methods exist in the art for producing the anti-LGALS3 antibodies or their antigen-binding fragments described herein. For example, the anti-LGALS3 antibodies or their antigen-binding fragments can be produced by recombinant DNA methods (e.g., the method described in U.S. Patent No. 4,816,567). One or more DNAs encoding the anti-LGALS3 antibodies or their antigen-binding fragments provided herein can be readily isolated and sequenced using common procedures. These procedures use, for example, oligonucleotide probes that can specifically bind to genes encoding the heavy or light chain of a mouse antibody (or such chains from human, humanized, or other sources). Once isolated, the DNA is placed in an expression vector. Subsequently, host cells (e.g., NSO cells, monkey COS cells, Chinese hamster ovary (CHO) cells, yeast cells, algal cells, or myeloma cells (which in other embodiments do not produce immunoglobulin proteins)) are transformed with the expression vector to synthesize the anti-LGALS3 antibodies or their antigen-binding fragments in the recombinant host cells. The DNA can also be modified, for example, by substituting the heavy and light chain sequences of a desired species for homologous human sequences (U.S. Patent No. 4,816,567; cited above (Morrison et al.)), or by covalently binding all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide can be substituted for the constant domain of the anti-LGALS3 antibody or its antigen-binding fragment provided herein. In certain embodiments, the DNA encoding the anti-LGALS3 antibody or its antigen-binding fragment provided herein can also be prepared by providing a transgenic animal or mammal (e.g., goats, dairy cows, horses, sheep, etc. (which produce such antibodies in their milk)) using a polynucleotide encoding at least one anti-LGALS3 antibody or its antigen-binding fragment. Such animals can be provided by known methods.See, for example, US Pat. Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; 5,304,489, etc. (not limited to those listed above) (each of the above is included herein in its entirety by reference).

[0078] In certain embodiments, the anti-LGALS3 antibodies or their antigen-binding fragments provided herein may also be prepared by providing transgenic plants and cultured plant cells (e.g., tobacco and maize, but not limited to these) using polynucleotides encoding at least one of the anti-LGALS3 antibodies or their antigen-binding fragments provided herein. The transgenic plants or cultured plant cells produce such antibodies, specific parts or variants in the plant parts or in cell cultures derived from the plant. As a non-limiting example, large quantities of recombinant proteins have been provided using inductive promoters by successfully utilizing transgenic tobacco leaves expressing recombinant proteins. See, for example, the following paper (Cramer et al. (1999) Curr. Top. Microbol. Immunol. 240:95-118, 1999) and the literature cited therein. Furthermore, mammalian proteins have been expressed at commercial production levels using transgenic maize, and these proteins have equivalent biological activity to those produced by other recombinant systems or from natural sources. For example, see the following paper (Hood et al. (1999) Adv. Exp. Med. Biol. 464: 127-147) and the references cited therein. Antibodies have also been produced in large quantities from the seeds of transgenic plants (including tobacco seeds and potato tubers) containing antibody fragments (e.g., scFv). For example, see the following paper (Conrad et al. (1998) Plant Mol. Biol. 38: 101-109) and the references cited therein. Therefore, anti-LGALS3 antibodies or their antigen-binding fragments can also be produced using transgenic plants according to known methods.For example, please also refer to the following papers (Fischer et al. (1999) Biotechnol. Appl. Biochem. 30:99-108; Ma et al. (1995) Trends Biotechnol. 13:522-7; Ma et al, (1995) Plant Physiol. 109:341-6; Whitelam et al. (1994) Biochem Soc. Trans. 22:940-944) and the references cited therein (each of the above references is included in this specification by reference).

[0079] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments provided herein may be prepared by providing a bacterium that produces such anti-LGALS3 antibodies or antigen-binding fragments using a polynucleotide encoding at least one anti-LGALS3 antibody or antigen-binding fragment provided herein. As a non-limiting example, large quantities of recombinant proteins have been provided by successfully utilizing E. coli expressing recombinant proteins. See, for example, the following paper (Verma et al. (1998) 216(1-2): 165-181) and the references cited therein. For methods to achieve multispecificity (e.g., bispecific antibodies), see, for example, U.S. Patent Nos. 7,951,917; 7,183,076; 8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992; and 8,586,713. In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment provided herein is used to create a bispecific antibody. A bispecific antibody can be produced by fusing two hybridomas to create a hybrid immunoglobulin molecule having two binding sites. The bispecific antibody not only binds tumors to T cells, but also cross-links with CD3 on T cells, initiating an activation cascade. In this way, T cell receptor-mediated cytotoxicity is redirected to the desired tumor target, bypassing MHC constraint. Arming polyclonally activated T cells (ATCs) with the anti-CD3-anti-LGALS3 bispecific binding molecule links the target specificity of the anti-LGALS3 antibody to the non-MHC-restricted perforin / granzyme-mediated cytotoxicity of T cells. The bispecific binding molecule BsAb or BiTE can be used to arm T cells that have been proliferated and activated ex vivo before being administered to the patient. This strategy converts all ATCs into specific CTLs (Thakur and Lum (2010) Curr Opin Mol Ther 12, 340-349; Grabert et al. (2006) Clin Cancer Res 12, 569-576).

[0080] The bispecific binding molecule can consist of an anti-LGALS3 antibody, where the anti-LGALS3 antibody is an immunoglobulin, and each light chain of the immunoglobulin is a fusion protein, which is an immunoglobulin light chain linked to a CD3-targeting scFv via a peptide linker. The N297A mutation in the CH2 domain results in glycosylation that leads to FcR or C1q unbound. CARs can be produced using the anti-LGALS3 antibodies or their antigen-binding fragments provided herein. Most commonly, a CAR consists of: a single-chain variable fragment length antibody (scFv) (e.g., derived from a monoclonal antibody targeting a given tumor-associated antigen and / or a variant thereof), a transmembrane main (e.g., a transmembrane main derived from a T cell surface molecule, e.g., co-stimulatory molecules, e.g., CD8, CD28, OX-40, and 4-1BB), and a signaling portion of the TCR complex (e.g., the intracellular domain and / or additional portion of the TCR zeta (ζ) chain, e.g., its cytoplasmic signaling domain).

[0081] In specific embodiments, the heavy and light chain variable regions of the monoclonal anti-LGALS3 antibody described herein are isolated from a hybridoma cell line that produces the monoclonal anti-LGALS3 antibody. For example, RNA is extracted from the hybridoma cell line, and cDNA is generated from the RNA by reverse transcription PCR. The VH and VL chain variable regions are cloned by standard PCR using primers specific to such variable regions. The resulting VH and VL fragments are subcloned and sequenced using a shuttle vector (e.g., TopoTA PCR2.1 cloning vector (Invitrogen)). Next, the VH and VL fragments are linked to the (Gly4Ser)3 spacer domain to produce an anti-LGALS3 antibody scFv, which is then fused to the human CD8 leader peptide (CD8L) by overlap PCR (CD8L-anti-LGALS3 antibody scFv) (see, for example, Maher et al. (2002) Nat Biotechnol 20(l):70-5; and Gong et al. (1999) Neoplasia 1(2):123-7). The coding region of the CD8L-anti-LGALS3 antibody scFv is fused with the human CD8 hinge and transmembrane main, or separately with the CD28 transmembrane main and cytoplasmic signaling domain, and then fused with the T cell receptor 0)3-ζ signaling domain (see, for example, Maher et al. (2002) Nat Biotechnol 20(l):70-5; Brentjens et al. (2003) Nat Med 9(3):279-86; and Brentjens et al. (2007) Clin Cancer Res 13(18 Pt l):5426-35).

[0082] Also provided herein are T cells expressing the CAR described herein. Methods for producing CAR-expressing T cells are known in the art. For example, CAR constructs can be subcloned with the modified MMLV retroviral vector SFG (see, e.g., Riviere et al. (1995) Proc Natl Acad Sci USA 92(15):6733-7) or other suitable retroviral vectors. In some embodiments, the retroviral vector is a lentiviral vector (e.g., an HIV-type vector). A stable PG13 gibbon leukemia virus (GaLV) envelope-pseudoretrovirus-producing cell line can be constructed using the VSV-G pseudoretroviral supernatant obtained from gpg29 transduced fibroblasts (see, e.g., Gong et al. (1999) Neoplasia 1(2):123-7). By activating isolated mononuclear cells (PBMCs) from the peripheral blood of healthy donors with 2 μg / mL phytohemagglutinin (PHA) (Sigma. St. Louis, MO) and then transducing them with a retrovirus on a retronectin-coated non-tissue culture plate (Quintas-Cardama A, et al. (2007) Hum Gene Ther 18(12): 1253-60), T cells expressing CAR recombinantly can be produced. Gene transfer of CAR into T cells can be evaluated by FACS. Monodomain antibodies (e.g., antibodies lacking a light chain) can be prepared by methods well known in the art. For example, see: Riechmann and Muyldermans (1999) J Immunol 231: 25-38; Nuttall SD et al. (2000) Curr Pharm Biotechnol 1(3): 253-263; Muyldermans S, (2001) J Biotechnol 74(4): 277-302; U.S. Patent No. 6,005,079; and International Publications WO 94 / 04678, WO 94 / 25591 and WO 01 / 44301 (each of which is incorporated herein by reference in its entirety). In individual embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein (which binds to the same or overlapping epitope as the anti-LGALS3 antibody described herein) is a human anti-LGALS3 antibody or its antigen-binding fragment. In individual embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein (which competitively inhibits any one of the antibodies described herein from binding to LGALS3 (e.g., in a dose-dependent manner)) is a human anti-LGALS3 antibody or its antigen-binding fragment.

[0083] Human antibodies can be produced using any method known in the art. For example, transgenic mice can be used (these mice cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes). In individual embodiments, the human heavy-chain and light-chain immunoglobulin gene complex can be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to the human heavy-chain and light-chain genes, the human variable region, constant region, and diversity region can be introduced into mouse embryonic stem cells. The mouse heavy-chain and light-chain immunoglobulin genes can be made non-functional separately or simultaneously by homologous recombination of the human immunoglobulin locus. In particular, homozygous deletion of the JH region prevents endogenous antibody production. Modified embryonic stem cells are grown and microinjected into blastocysts to produce chimeric mice. Subsequently, the chimeric mice are mated to produce homozygous offspring that express human antibodies. These transgenic mice are immunized in the usual manner with a selected antigen (e.g., all or part of the antigen). These antibodies can be obtained from transgenic mice immunized with monoclonal antibodies against the antigen using conventional hybridoma technology. The human immunoglobulin transgenes held by these transgenic mice undergo rearrangement during B cell differentiation, followed by class switching and somatic mutation. Therefore, it is possible to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies using such technology. For an overview of this technology for human antibody production, see, for example, the literature (Lonberg and Huszar (1995) Int Rev Immunol 13:65-93). For a detailed discussion of this technique concerning the production of human antibodies and human monoclonal antibodies, and protocols for the production of such antibodies, see, for example, International Publications WO 98 / 24893, WO 96 / 34096 and WO 96 / 33735; and U.S. Patents 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318 and 5,939,598. Examples of mice capable of producing human antibodies include: Xenomouse TM(Abgenix, Inc.; U.S. Patents 6,075,181 and 6,150,184), HuAb-Mouse TM (Mederex, Inc. / Gen Pharm; U.S. Patents No. 5,545,806 and 5,569,825), Trans Chromo Mouse™ (Kirin) and KM Mouse TM (Medarex / Kirin).

[0084] Human antibodies that bind immunospecifically to LGALS3 can be prepared by a variety of methods known in the art. These methods include the phage display method described above, which uses an antibody library derived from human immunoglobulin sequences. See also: U.S. Patents 4,444,887, 4,716,111, and 5,885,793; and International Publications WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741. In some embodiments, human antibodies can be produced using mouse-human hybridomas. For example, mouse-human hybridomas that secrete human monoclonal antibodies can be created by fusing human peripheral blood lymphocytes transformed with Epstein-Barr virus (EBV) to mouse myeloma cells. These mouse-human hybridomas can be screened to determine which secrete human monoclonal antibodies that immunospecifically bind to target antigens. Such methods are known and described in the art (see, for example, Shinmoto H et al. (2004) Cytotechnology 46: 19-23; Naganawa Y et al. (2005) Human Antibodies 14: 27-31).

[0085] Exemplary methods for producing antibodies or antigen-binding fragments that immunospecifically bind to LGALS3, and methods for screening and selecting antibodies or antigen-binding fragments that immunospecifically bind to LGALS3, are described in Example 3. In the exemplary embodiment, a mouse-human hybridoma is prepared from a mouse immunized with an immunogenic peptide or chimeric fusion protein of LGALS3, as described in Example 3. In the exemplary embodiment, a monoclonal antibody secreted by the hybridoma is screened for binding to the LGALS3 carbohydrate-binding domain peptide (CBD) and the full-length LGALS3 protein. In some embodiments, the LGALS3 N-terminus (including the polymerization domain) is used as a control. In some embodiments, the monoclonal antibody is further screened against mouse Gal3 CBD (mCBD3) or other members of the galectin family (e.g., LGAL1, LGAL7, LGAL8, and LGAL9). Once the anti-LGALS3 antibody or its antigen-binding fragment described herein has been produced, it can be purified by any method known to those skilled in the art for the purification of immunoglobulin molecules. Purification may be performed, for example, by chromatography (e.g., ion exchange, affinity (particularly by affinity for specific antigens after protein A), and size-based column chromatography), centrifugation, discriminative solubility, or any other standard technique for protein purification. Furthermore, the antibodies described herein can be fused to heterologous polypeptide sequences known in the art to facilitate purification as described herein or otherwise.

[0086] In specific embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein is isolated or purified. Generally, the isolated antibody is substantially free of other antibodies with various antigen specificities other than the isolated antibody. For example, in individual embodiments, the preparations of the anti-LGALS3 antibody or its antigen-binding fragment described herein are substantially free of cellular material and / or chemical precursors. The phrase "substantially free of cellular material" includes preparations of the anti-LGALS3 antibody or its antigen-binding fragment in which the antibody is isolated or recombinantly isolated from the cellular components of the cell from which it is produced. Therefore, an anti-LGALS3 antibody or its antigen-binding fragment that is substantially free of cellular material includes an antibody preparation containing less than 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred herein as “contaminating proteins”) and / or variants of the anti-LGALS3 antibody or its antigen-binding fragment (e.g., various post-translational modifications of the anti-LGALS3 antibody or its antigen-binding fragment, or various other versions of the anti-LGALS3 antibody or its antigen-binding fragment (e.g., antibody fragments)). When the antibody is recombinantly produced, it is also generally substantially free of culture medium; that is, the culture medium is less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When the antibody is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals; that is, it is isolated from chemical precursors or other chemicals involved in protein synthesis. Therefore, such preparations of antibodies contain (by dry weight) about 30%, 20%, 10%, or less than 5% of chemical precursors or compounds other than the antibody in question. In specific embodiments, the antibodies described herein are isolated or purified.

[0087] Polynucleotides In certain embodiments, what is provided herein is a polynucleotide comprising a nucleotide sequence encoding an anti-LGALS3 antibody or its antigen-binding fragment. What is provided herein is also a vector comprising such a polynucleotide. What is provided herein is also a polynucleotide encoding the antigen of the anti-LGALS3 antibody or its antigen-binding fragment described herein. What is provided herein is also a polynucleotide that hybridizes with the polynucleotide encoding the anti-LGALS3 antibody or its antigen-binding fragment described herein under stringent conditions or lower stringency conditions. The term “purified” includes preparations of polynucleotides or nucleic acid molecules having about 15%, 10%, 5%, 2%, 1%, 0.5%, or less than 0.1% (particularly less than about 10%) of other substances (e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals). In specific embodiments, the nucleic acid molecules encoding the anti-LGALS3 antibody or its antigen-binding fragment described herein are purified or isolated. The nucleic acid molecules provided herein may be in the form of RNA (e.g., mRNA, hnRNA, tRNA, or any other form), or in the form of DNA (cDNA and genomic DNA obtained by cloning or produced by synthesis), or any combination thereof. The DNA may be triple-stranded, double-stranded, single-stranded, or any combination thereof. Any portion of at least one strand of DNA or RNA may be a coding strand (also known as a sense strand) or a non-coding strand (also referred to as an antisense strand).

[0088] In certain embodiments, what is provided herein is a polynucleotide comprising a nucleotide sequence encoding an anti-LGALS3 antibody or its antigen-binding fragment as described herein. In individual embodiments, what is provided herein is also a polynucleotide comprising a nucleotide sequence encoding an anti-LGALS3 antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment binds immunospecifically to LGALS3 and further comprises the amino acid sequences described herein, and the antibody competes with such anti-LGALS3 antibody or its antigen-binding fragment for binding to LGALS3, or binds to the same epitope as such antibody. The polynucleotides provided herein can be obtained by any method known in the art. For example, if the nucleotide sequence encoding the anti-LGALS3 antibody or its antigen-binding fragment described herein is known, the polynucleotide encoding the anti-LGALS3 antibody or its antigen-binding fragment can be assembled from chemically synthesized oligonucleotides, for example, as described in the literature (Kutmeier et al. (1994) BioTechniques 17:242). Briefly, this involves the synthesis of overlapping oligonucleotides containing portions of the antibody-coding sequence, annealing and ligation of those oligonucleotides, and subsequent PCR amplification of the ligated oligonucleotides.

[0089] Alternatively, polynucleotides encoding anti-LGALS3 antibodies or their antigen-binding fragments can be generated from nucleic acids derived from a suitable source. While clones containing the nucleic acids encoding individual anti-LGALS3 antibodies or their antigen-binding fragments are not available, if the sequences of the anti-LGALS3 antibodies or their antigen-binding fragments are known, the nucleic acids encoding the anti-LGALS3 antibodies or their antigen-binding fragments can be obtained by chemical synthesis, PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of the sequence from a suitable source, or by cloning using oligonucleotide probes specific to individual gene sequences, for example, by identifying cDNA from an antibody-encoding cDNA library. Such suitable sources are, for example, antibody cDNA libraries, or cDNA libraries or nucleic acids, preferably poly(A+RNA), generated or isolated from any tissue or cells expressing the antigen (e.g., hybridoma cells selected for expression of the anti-LGALS3 antibodies or their antigen-binding fragments provided herein). Subsequently, the amplified nucleic acids generated by PCR are cloned in a replicable cloning vector using any method well known in the art.In such embodiments, a polynucleotide encoding such an anti-LGALS3 antibody or its antigen-binding fragment can be manipulated using methods well known in the art for manipulating nucleotide sequences, such as recombinant DNA technology, site-directed mutagenesis, PCR, etc., to produce anti-LGALS3 antibodies or their antigen-binding fragments having various amino acid sequences, for example, by creating amino acid substitutions, deletions, and / or insertions (see, for example, the techniques described in the following literature: Sambrook et al. (1990) Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; and Ausubel et al., eds. (1998) Current Protocols in Molecular Biology, John Wiley & Sons, NY (both of the aforementioned literatures are incorporated herein by reference in their entirety)). For example, such manipulation can be performed to deglycosylate the coding amino acid or to disrupt the antibody's ability to bind to the C1q, Fc receptor or its complement system activating ability.

[0090] The isolated nucleic acid molecules provided herein may include: nucleic acid molecules comprising an open reading frame (ORF) (with one or more introns as may be included), for example, at least one specific portion of at least one complementarity-determining region (CDR), for example, at least one heavy or light chain CDR1, CDR2 and / or CDR3 (but not limited to the foregoing); nucleic acid molecules comprising an anti-LGALS3 antibody or a coding sequence of a variable region; and nucleic acid molecules comprising a nucleotide sequence substantially different from those described above, but which, due to the degeneracy of the genetic code, still encode at least one anti-LGALS3 antibody or its antigen-binding fragment as described herein. The provided material is also an isolated nucleic acid that hybridizes with the polynucleotides disclosed herein under selective hybridization conditions. Therefore, the polynucleotides of this embodiment can be used to isolate, detect, and / or quantify nucleic acids containing such polynucleotides. For example, the polynucleotides provided herein can be used to identify, isolate, or amplify partial-length or full-length clones in a deposit library. In some embodiments, the polynucleotides are isolated genomic sequences or cDNA sequences, or are complementary to cDNA derived from a human or mammalian nucleic acid library.

[0091] Conveniently, the nucleic acid may contain multiple sequences in addition to the polynucleotide provided herein. For example, a multicloning site containing one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in the isolation of the polynucleotide. In addition, a translatable sequence can be inserted to aid in the isolation of the translated polynucleotide provided herein. For example, a 6-histidine marker sequence provides a convenient means for the purification of the polypeptide provided herein. The nucleic acid provided herein (excluding the coding sequence) may optionally be a vector, adapter, or linker for the cloning and / or expression of the polynucleotide provided herein. Additional sequences can also be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, assist in the isolation of polynucleotides, or improve the introduction of polynucleotides into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the industry (see, for example, Ausubel's or Sambrook's). In specific embodiments, one or more CDRs of the anti-LGALS3 antibody or its antigen-binding fragment described herein can be inserted into a known framework region using routine recombinant DNA techniques. The framework region may be naturally occurring or a consensus framework region, preferably a human framework region (see, for example, Chothia et al., (1998) J. Mol. Biol. 278: 457-479 for a list of human framework regions). In some embodiments, the polynucleotide produced by the combination of the framework region and the CDR encodes an anti-LGALS3 antibody or its antigen-binding fragment that binds immunospecifically to LGALS3. One or more amino acid substitutions can be carried out within the framework region, preferably, these amino acid substitutions improve the binding of the antibody to its antigen. In addition, using such methods, amino acid substitutions or deletions of one or more variable region cysteine ​​residues involved in intrachain disulfide bonds can be carried out to produce antibody molecules lacking one or more intrachain disulfide bonds. Other modifications to the polynucleotide are also provided herein and are within the scope of the art.

[0092] In certain embodiments, isolated or purified nucleic acid molecules or fragments thereof can be linked with another nucleic acid molecule to encode a fusion protein. The preparation of fusion proteins is within the scope of the art of the art and includes the use of restriction enzymes or recombinant cloning techniques (e.g., Gateway). TM (See Invitrogen). See also U.S. Patent No. 5,314,995. In certain embodiments, the polynucleotides provided herein are in the form of a vector (e.g., an expression vector). In certain features, what is provided herein is a polynucleotide comprising a nucleotide sequence encoding an anti-LGALS3 antibody described herein that binds immunospecifically to LGALS3 or its antigen-binding fragment (e.g., a variable light chain region and / or a variable heavy chain region), and a vector (e.g., a vector containing such a polynucleotide for the efficient expression of the polynucleotide in host cells (e.g., E. coli and mammalian cells)). In some embodiments, the polynucleotide is isolated or purified. In terms of specific features, what is provided herein is a polynucleotide comprising a nucleotide sequence encoding an anti-LGALS3 antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment comprises not only the amino acid sequence described herein, but also competes with such antibody for binding to LGALS3 (e.g., in a dose-dependent manner), or binds to the same or overlapping epitopes as such antibody.

[0093] In certain features, what is provided herein is a polynucleotide comprising a nucleic acid sequence encoding the light chain or heavy chain of the anti-LGALS3 antibody or its antigen-binding fragment as described herein. The polynucleotide may include a nucleotide sequence encoding the heavy chain comprising the VH CDR as described herein (see, for example, Figures 12 and 13). The polynucleotide may also include a nucleotide sequence encoding the light chain comprising the VL CDR as described herein (see, for example, Figures 12 and 13). In individual embodiments, what is provided herein is a polynucleotide comprising a nucleotide sequence encoding an anti-LGALS3 antibody containing three VH CDRs (e.g., VH CDR1, VH CDR2, and VH CDR3 as shown in Figures 12 and 13), wherein the antibody binds immunospecifically to LGALS3. In specific embodiments, the polynucleotide described herein encodes VH CDR1, VH CDR2, and VH CDR3 of the 14D11 antibody (i.e., SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively), wherein the antibody binds immunospecifically to LGALS3. In certain embodiments, the polynucleotide described herein comprises a nucleotide sequence encoding an anti-LGALS3 antibody comprising a heavy chain variable region (e.g., SEQ ID NO: 24) containing the amino acid sequence described herein, wherein the antibody binds immunospecifically to LGALS3. In individual embodiments, what is provided herein is a polynucleotide comprising a nucleotide sequence encoding an anti-LGALS3 antibody containing three VL CDRs (e.g., VL CDR1, VL CDR2, and VL CDR3 as shown in Figures 12 and 13), wherein the antibody binds immunospecifically to LGALS3. In specific embodiments, the polynucleotide described herein encodes VL CDR1, VL CDR2, and VL CDR3 of the 14D11 antibody (i.e., SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively), wherein the antibody binds immunospecifically to LGALS3. In certain embodiments, the polynucleotide described herein comprises a nucleotide sequence encoding an anti-LGALS3 antibody comprising a light chain variable region (e.g., SEQ ID NO: 26) containing the amino acid sequence described herein, wherein the antibody binds immunospecifically to LGALS3. In certain embodiments, the polynucleotide described herein comprises a nucleotide sequence encoding an anti-LGALS3 antibody described herein, comprising a heavy chain variable region (e.g., SEQ ID NO: 24) comprising the amino acid sequence described herein and a light chain variable region (e.g., SEQ ID NO: 26) comprising the amino acid sequence described herein, wherein the antibody binds immunospecifically to LGALS3. In specific features, what is provided herein is a polynucleotide comprising a nucleotide sequence encoding an anti-LGALS3 antibody or its antigen-binding fragment, comprising a light chain and a heavy chain (e.g., separate light and heavy chains). With respect to the heavy chain, in specific embodiments, the polynucleotide provided herein comprises a nucleotide sequence encoding an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. With respect to the light chain, in specific embodiments, the polynucleotide provided herein comprises a nucleotide sequence encoding a kappa (κ) or lambda (λ) light chain.

[0094] Cells and vectors In certain embodiments, what is provided herein is a cell (e.g., isolated cell or ex vivo cell) expressing one or more anti-LGALS3 antibodies or their antigen-binding fragments (e.g., by recombination). Also provided herein is a vector (e.g., expression vector) containing a nucleotide sequence encoding the anti-LGALS3 antibody or its antigen-binding fragment described herein for expressing recombinants in host cells, preferably mammalian cells. Also provided herein is a cell (e.g., isolated cell or ex vivo cell) containing such a vector or nucleotide sequence for recombinant expression of the anti-LGALS3 antibody or its antigen-binding fragment described herein. Also provided herein is a method for producing the anti-LGALS3 antibody or its antigen-binding fragment described herein, the method comprising the step of expressing such anti-LGALS3 antibody or its antigen-binding fragment from cells (isolated cell or ex vivo cell).

[0095] A vector (e.g., an expression vector) is a DNA molecule containing a gene to be expressed in a cell (e.g., an ex vivo cell). Typically, gene expression is under the control of certain regulatory elements (including constitutive or inductive promoters, tissue-specific regulatory elements, and enhancers). Such a gene is said to be "conjugated to work" with the regulatory element (e.g., the promoter). A recombinant host can be any prokaryotic or eukaryotic cell containing either a cloning vector or an expression vector. The term also includes transgenic animals along with those prokaryotic or eukaryotic cells. The aforementioned are genetically engineered to contain the cloned gene in the chromosome or genome of the host cell or in the host cell (e.g., an ex vivo cell). In some embodiments, the promoter is a CMV promoter. In certain embodiments, what is provided herein is one or more polynucleotides described herein. In certain embodiments, the polynucleotides described herein can be cloned in a suitable vector and used for transformation or transfection of any suitable host. Vectors and methods for constructing such vectors are known to those skilled in the art and are described in general technical references (see, for example, “Recombinant DNA Part D” Methods in Enzymology, Vol. 153, Wu and Grossman, eds., Academic Press, 1987). In certain embodiments, the vector includes regulatory sequences (e.g., transcription and translation start and stop codons) as appropriate and taking into consideration whether the vector is DNA or RNA. The regulatory sequences are specific to the type of host into which the vector is introduced (e.g., bacteria, fungi, plants, insects, or mammals). In certain embodiments, the vector includes regulatory sequences specific to the genus of the host. In certain embodiments, the vector includes regulatory sequences specific to the species of the host. In certain embodiments, the vector comprises one or more marker genes, which enable the selection of a transformed or transfected host. Non-limiting examples of marker genes include biocide resistance (e.g., antibiotic resistance, heavy metal resistance, etc.) and complementation of a trophic-requiring host that provides protrophotrophy. In individual embodiments, the vector comprises ampicillin and hygromycin selection markers.

[0096] In certain embodiments, the expression vector includes a native or normative promoter ligated to the polynucleotides described herein. The choice of promoter (e.g., virulent, weak, inducible, tissue-specific, and developmental-specific) is within the scope of the art. Similarly, the binding of the nucleic acid molecules or fragments thereof described above to the promoters is also within the scope of the art. A non-limiting example of a suitable vector includes vectors designed for growth, amplification, expression, or both. For example, cloning vectors can be selected from the group consisting of the pUC series, pBluescript series (Stratagene, LaJolla, Calif), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif). Bacteriophage vectors, such as lambda-GTIO, lambda-GTl 1, lambda-ZapII (Stratagene), lambda-EMBL4, and lambda-NMl 149, can also be used. A non-limiting example of a plant expression vector includes pBIl 10, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). A non-limiting example of an animal expression vector includes pEUK-Cl, pMAM, and pMAMneo (Clontech). The TOPO cloning system (Invitrogen, Carlsbad, Calif.) can also be used according to the manufacturer's instructions.

[0097] In certain embodiments, the vector is a mammalian vector. In certain embodiments, the mammalian vector includes at least one promoter element (mediating the initiation of mRNA transcription), an anti-LGALS3 antibody or its antigen-binding fragment coding sequence, and signals necessary for transcription termination and transcriptional polyadenylation. In certain embodiments, the mammalian vector includes additional components (e.g., enhancers, Kozak sequences, and intervening sequences flanked into donor and acceptor sites for RNA splicing). In certain embodiments, highly efficient transcription can be achieved, for example, with the early and late promoters of SV40, the long terminal repeats (LTRs) of retroviruses (e.g., RSV, HTLVI, HIVI), and the early promoter of cytomegalovirus (CMV). However, cellular elements (e.g., human actin promoters) can also be used. Non-exclusive examples of mammalian expression vectors include, for example, the following vectors: pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, Calif), pcDNA3.1(+ / -), pcDNA / Zeo(+ / -) or pcDNA3.1 / Hygro(+ / -) (Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), and pBC12MI (ATCC 67109). Non-limiting examples of mammalian host cells that can be used in combination with such mammalian vectors include: human Hela293, H9, and Jurkat cells; mouse NIH3T3 and C127 cells; Cos 1, Cos 7, and CV 1 cells; quail QCl-3 cells; mouse L cells; and Chinese hamster ovary (CHO) cells.

[0098] In certain embodiments, the vectors are viral vectors, such as retroviral vectors, parvovirus vectors, such as adenovirus-associated virus (AAV) vectors, AAV-adenovirus chimeric vectors, and adenovirus vectors, as well as lentiviral vectors, such as herpes simplex virus (HSV) vectors. In certain embodiments, the viral vectors are manipulated to be deficient in viral replication. In certain embodiments, the viral vectors are manipulated to remove toxicity to the host. These viral vectors can be prepared using standard recombinant DNA techniques, for example, as described in the following literature: Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY, 1994. In certain embodiments, the vector or polynucleotide described herein can be introduced into cells (e.g., ex vivo cells) by conventional techniques, and the resulting cells can be cultured by conventional techniques to produce the anti-LGALS3 antibody or its antigen-binding fragment described herein. Therefore, provided herein are cells containing a polynucleotide, the polynucleotide encoding the anti-LGALS3 antibody or its antigen-binding fragment, its heavy or light chain, or its light chain fusion polypeptide, and ligated to a promoter for the expression of such sequences in a host cell. In certain embodiments, a vector encoding a heavy chain ligated to a promoter and a vector encoding a light chain ligated to a promoter can be co-expressed in a cell for the expression of the entire anti-LGALS3 antibody. In certain embodiments, a vector encoding a heavy chain ligated to a promoter and a vector encoding a light chain ligated to a promoter can be co-expressed in a cell for the expression of the entire bispecificity binding molecule. In certain embodiments, the cell contains a vector containing a polynucleotide (ligated to a promoter) encoding both the heavy and light chain polypeptides of the anti-LGALS3 antibody described herein. In certain embodiments, the cell comprises a vector containing a polynucleotide (linked to a promoter so as to actuate) encoding both the heavy chain and light chain fusion polypeptide of the bispecific binding molecule described herein. In certain embodiments, the cell comprises two different vectors, the first vector containing a polynucleotide encoding a heavy chain linked to a promoter so as to actuate, and the second vector containing a polynucleotide encoding a light chain polypeptide linked to a promoter so as to actuate. In certain embodiments, the cell comprises two different vectors, the first vector containing a polynucleotide encoding a heavy chain linked to a promoter so as to actuate, and the second vector containing a polynucleotide encoding a light chain fusion polypeptide linked to a promoter so as to actuate.In certain embodiments, a first cell comprises a nucleotide sequence encoding the heavy chain of the anti-LGALS3 antibody described herein, and a second cell comprises a polynucleotide encoding the light chain polypeptide of the anti-LGALS3 antibody described herein. In certain embodiments, what is provided herein is a cell mixture comprising such first and second cells. In certain embodiments, a first cell comprises a first vector comprising a polynucleotide encoding the heavy chain of the bispecific binding molecule described herein, and a second cell comprises a second vector comprising a polynucleotide encoding the light chain fusion polypeptide of the bispecific binding molecule described herein. In certain embodiments, what is provided herein is a cell mixture comprising such first and second cells. In individual embodiments, the cell expresses the one or more vectors such that the polynucleotides are efficiently transcribed and translated within the cell. In some embodiments, cells express the vector such that polynucleotides or their fragments are efficiently transcribed and translated within the cell.

[0099] In certain embodiments, the cells reside within host cells and may be animal cells (e.g., mammalian). Examples of cells include, but are not limited to, human cells, human cell lines, Escherichia coli (e.g., E. coli TB-1, TG-2, DH5a, XL-Blue MRF (Stratagene), SA2821, and Y1090), Bacillus subtilis, Pseudomonas aeruginosa, S. cerevisiae, N. crassa, insect cells (e.g., Sf9, Ea4), and other cells listed below. In certain embodiments, the cells are CHO cells. In other distinct embodiments, the cells are CHO-S cells. In certain embodiments, the polynucleotides described herein can be expressed in stable cell lines containing polynucleotides integrated into chromosomes by introducing the polynucleotides into cells. In certain embodiments, the polynucleotides are introduced into cells, for example, by electroporation. In certain embodiments, the polynucleotides are introduced into cells, for example, by transfection of a vector containing the polynucleotides. In certain embodiments, the vector is co-transfected with a selectable marker (e.g., DHFR, GPT, neomycin, or hygromycin) to enable identification and isolation of transfected cells. In certain embodiments, the transfected polynucleotides can also be amplified to express large amounts of encoded anti-LGALS3 antibodies or their antigen-binding fragments. For example, using the DHFR (dihydrofolate reductase) marker, it is possible to develop cell lines that hold hundreds or thousands of copies of the polynucleotide in question. Another example of a selection marker is the enzyme glutamine synthase (GS) (Murphy, et al., Biochem. J. 227:277-279, 1991; Bebbington, et al., Bio / Technology 10: 169-175). Using these markers, cells are grown in a selective medium and selected for those with the best resistance. These cell lines contain amplified genes integrated into the chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for antibody production. In some embodiments, the vector comprises (i) a first polynucleotide ligated to act on a first promoter encoding an immunoglobulin light chain that binds to LGALS3, and (ii) a second polynucleotide ligated to act on a second promoter encoding an immunoglobulin heavy chain that binds to LGALS3. In certain embodiments, the vector is a viral vector. In some embodiments, the vector comprises (i) a first polynucleotide linked to a first promoter so as to actuate with a first promoter, encoding a light chain fusion polypeptide containing an immunoglobulin light chain fused to scFv via a peptide linker (where the light chain binds to LGALS3 and the scFv binds to CD3); and (ii) a second polynucleotide linked to a second promoter so as to actuate with a second promoter, encoding an immunoglobulin heavy chain that binds to LGALS3. In certain embodiments, the vector is a viral vector.

[0100] Pharmaceutical composition In certain embodiments, what is provided herein is a composition (e.g., a pharmaceutical composition) and a kit comprising one or more pharmaceutically effective anti-LGALS3 antibodies or their antigen-binding fragments. In certain embodiments, the pharmaceutical composition comprises immune cells (e.g., T cells) which recombinantly express the antibodies, their antigen-binding fragments, and / or CARs described herein. The composition can be used to prepare individual single-unit dosage forms. The compositions provided herein may be formulated for parenteral, subcutaneous, intramuscular, intravenous, intraarterial, intrabronchial, intraperitoneal, intrasacral, intracartilaginous, intracavitary, intracellular, intracerebellar, intraventricular, intracranial, intraocular, intravitreous, intracolon, intracervical, intrastomical, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intraneural, intraretinal, intraspinal cord, intraarticular, intrajoint capsule, intrathoracic, intrauterine, intrabladder, bolus, vagina, rectum, cheek, sublingual, nasal, intrathecal, intraventricular, intracerebral parenchymal, or transdermal administration. In certain embodiments, what is provided herein is a composition comprising one or more polynucleotides comprising a nucleotide sequence encoding the anti-LGALS3 antibody or its antigen-binding fragment described herein. In certain embodiments, what is provided herein is a composition comprising cells, wherein the cells comprise one or more polynucleotides comprising a nucleotide sequence encoding the anti-LGALS3 antibody or its antigen-binding fragment described herein. In certain embodiments, what is provided herein is a composition comprising a vector, wherein the vector comprises one or more polynucleotides comprising a nucleotide sequence encoding the anti-LGALS3 antibody or its antigen-binding fragment described herein. In certain embodiments, what is provided herein is a composition comprising cells, wherein the cells comprise a vector, and the vector comprises one or more polynucleotides comprising a nucleotide sequence encoding the anti-LGALS3 antibody or its antigen-binding fragment described herein.

[0101] In certain embodiments, the compositions described herein are stable or preservative formulations. In certain embodiments, the stable formulations include phosphate buffer along with saline or a selected salt. In certain embodiments, the compositions described herein are versatile preservative formulations suitable for pharmaceutically or veterinary use. In certain embodiments, the compositions described herein include preservatives. Preservatives are known to those skilled in the art. Non-limiting examples of preservatives include phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercury nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, and sodium dehydroacetate and thimerosal, or mixtures thereof in aqueous dilutions. Any suitable concentration or mixture known in the art may be used. The concentration is, for example, 0.001-5% or any range or value in between, such as 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 , 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9%, or any range or value in between.Non-limiting examples include: no preservatives, 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01%), and 0.001-2.0% phenol (e.g., 0.05%). This includes 0.25%, 0.28%, 0.5%, 0.9%, and 1.0%, and 0.0005-1.0% of (one or more types) alkylparabens (e.g., 0.00075%, 0.0009%, 0.001%, 0.002%, 0.005%, 0.0075%, 0.009%, 0.002%, 0.005%, 0.075%, 0.09, 0.1%, 0.2%, 0.3%, 0.5%, 0.75%, 0.9%, and 1.0%).

[0102] The compositions provided herein may be desired to be delivered to target animals over a long period, for example, from one week to one year or more than one year, in a single dose. A variety of sustained-release, depot, or implantation formulations can be utilized. For example, the formulation may include pharmaceutically acceptable nontoxic salts of compounds having low solubility in body fluids. The salts may be, for example, (a) acid addition salts with polybasic acids (e.g., phosphoric acid, sulfuric acid, citrate, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or di-sulfonic acid, polygalacturonic acid, etc.); (b) salts with polyvalent metal cations (e.g., zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc.) or organic cations formed from, for example, N,N'-dibenzyl-ethylenediamine or ethylenediamine; or (c) a combination of (a) and (b), for example, zinc tartrate. In addition, the compositions provided herein, preferably relatively insoluble salts (e.g., the salts described above), can be formulated in a gel, such as an aluminum monostearate gel, with sesame oil suitable for injection, for example. Particularly suitable salts include zinc salts, zinc tannate salts, pamoates, etc. Another type of sustained-release depot formulation for injection may involve compounds or salts dispersed and encapsulated in a slowly degradable, non-toxic, non-antigenic polymer (e.g., polylactic acid / polyglycolic acid polymer, e.g., described in US Pat. No. 3,773,919). The compounds or preferably relatively insoluble salts (e.g., those described above) can also be formulated in a cholesterol matrix, silusic pellets (especially used in animals). Additional sustained-release, depot, or implantation compositions, e.g., gaseous or liquid liposomes, are known in the literature (US Pat. No. 5,770,222; and “Sustained and Controlled Release Drug Delivery Systems”, JR Robinson ed., Marcel Dekker, Inc., NY, 1978).

[0103] The range of compositions of at least one anti-LGALS3 antibody or its antigen-binding fragment provided herein includes amounts that yield approximately 1.0 microgram / mL to approximately 1000 mg / mL upon reconstitution in wet / dry systems, although lower and higher concentrations are also available and depend on the intended delivery vehicle (for example, solution formulations may differ for transdermal patches, lung, transmucosal, osmotic, or micropump methods). In certain embodiments, the compositions provided herein include at least one of any suitable adjuvants (e.g., diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, etc.). In certain embodiments, pharmaceutically acceptable adjuvants are applicable. Non-limited examples of such sterile solutions and methods for preparing them are well known in the art, for example, (but not limited to) Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990. A pharmaceutically acceptable carrier suitable for the administration method, solubility, and / or stability of the anti-LGALS3 antibody or its antigen-binding fragment described herein can be routinely selected.

[0104] In certain embodiments, the compositions provided herein include one or more pharmaceutical excipients and / or additives. Non-limiting examples of pharmaceutical excipients and additives include proteins, peptides, amino acids, lipids, and carbohydrates, such as sugars (including monosaccharides, di, tri, tetrasaccharides, and oligosaccharides; derived sugars, such as alditol, aldonic acid, esterified sugars, etc.; and polysaccharides or sugar polymers), which may exist alone or in combination, and which may constitute 1-99.99% by weight or volume. Non-limiting examples of protein excipients include serum albumin (e.g., human serum albumin (HAS)), recombinant human albumin (rHA), gelatin, casein, etc. Non-limiting examples of amino acid / antibody components (which may also function as buffering capacity) include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. In certain embodiments, the amino acid is glycine. Non-limiting examples of carbohydrate excipients include monosaccharides, e.g., fructose, maltose, galactose, glucose, D-mannose, sorbose; disaccharides, e.g., lactose, sucrose, trehalose, cellobiose; polysaccharides, e.g., raffinose, melegitose, maltodextrin, dextran, starch; and algitols, e.g., mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), myo-inositol, and the like. In certain embodiments, the carbohydrate excipient is mannitol, trehalose, or raffinose.

[0105] In certain embodiments, the compositions provided herein include one or more buffers or pH adjusters, typically the buffer being a salt prepared from an organic acid or base. Non-limiting examples of buffers include organic acid salts, e.g., salts of citric acid, ascorbic acid, gluconic acid, carbonate, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers. In certain embodiments, the buffer is an organic acid salt (e.g., citrate). Other excipients, e.g., isotonic agents, buffers, antioxidants, and preservative enhancers, are optionally and preferably added to diluents. Isotonic agents (e.g., glycerin) are used in generally known concentrations. Physiologically acceptable buffers are preferably added to provide improved pH control. The compositions can cover a wide range of pH, e.g., pH about 4 to pH about 10, particularly pH about 5 to pH about 9, and most specifically pH about 6.0 to about 8.0. In some embodiments, the compositions provided herein have a pH of about 6.8 to about 7.8. Preferred buffers include phosphate buffers, most preferably sodium phosphate, and especially phosphate-buffered saline (PBS). The compositions provided herein include: one or more polymer excipients / additives, e.g., polyvinylpyrrolidone, Ficol (polymerized sugars), dextran (e.g., cyclodextrin, e.g., 2-hydroxypropyl-beta-cyclodextrin), polyethylene glycol, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates, e.g., "TWEEN20" and "TWEEN80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and / or chelating agents (e.g., EDTA).

[0106] Other additives may be added to the composition as needed to prevent aggregation. These include, for example: pharmaceutically acceptable solubilizers, such as Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block copolymer), and PEG (polyethylene glycol) or nonionic surfactants, such as polysorbate 20 or 80 or poloxamer 184 or 188, Pluronic® polyols, other block copolymers, and chelators, such as EDTA and EGTA. These additives are particularly useful when dispensing the composition using a pump or plastic container. The presence of pharmaceutically acceptable surfactants mitigates the tendency of proteins to aggregate. Appropriate additional pharmaceutical excipients and / or additives for use in the compositions provided herein are known to those skilled in the art, and are, for example, referred to: “Remington: The Science & Practice of Pharmacy”, 19th ed., Williams & Williams, 1995; and “Physician's Desk Reference”, 52nd ed., Medical Economics, Montvale, NJ, 1998 (the foregoing is incorporated herein in its entirety by reference). In certain individual embodiments, the carrier or excipient material is a carbohydrate (e.g., sugars and algitols) and a buffer (e.g., citric acid) or polymer agent. In some embodiments, the aqueous diluent may optionally further contain pharmaceutically acceptable preservatives. Exemplary preservatives include phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof. The concentration of the preservative used in the composition is sufficient to produce an antimicrobial effect. Such a concentration depends on the preservative selected and can be readily determined by those skilled in the art.

[0107] The compositions provided herein can be prepared by a process comprising mixing at least one anti-LGALS3 antibody or its antigen-binding fragment described herein with phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal or a mixture thereof in an aqueous diluent. The step of mixing at least one anti-LGALS3 antibody or its antigen-binding fragment and preservative described herein in an aqueous diluent is carried out using a normal dissolution and mixing procedure. To prepare a suitable composition, a measured amount of at least one anti-LGALS3 antibody or its antigen-binding fragment in a buffer solution is combined with the desired preservative in a buffer solution in an amount sufficient to provide the desired concentration of the anti-LGALS3 antibody or its antigen-binding fragment and preservative described herein. The compositions provided herein can be prepared by a process comprising mixing at least one anti-LGALS3 antibody or its antigen-binding fragment described herein with a phosphate buffer containing a selected buffer, preferably saline or a selected salt. The step of mixing the at least one anti-LGALS3 antibody or its antigen-binding fragment and the buffer in an aqueous diluent is carried out using a standard dissolution and mixing procedure. To prepare a suitable composition, a measured amount of the at least one anti-LGALS3 antibody or its antigen-binding fragment described herein in water or buffer, together with the desired buffer in water, is added in an amount sufficient to provide the desired concentration of protein and buffer. Those skilled in the art will recognize the variety of these processes. For example, the order in which the compositions are added, whether or not additional additives are used, and the temperature and pH at which the compositions are prepared are all factors that can be optimized for the concentration and means of administration used.

[0108] Parenteral prescriptions In certain embodiments, the compositions provided herein are formulated for parenteral injectable administration. As used herein, the term “parenteral” includes intravenous, intravascular, intramuscular, intradermal, subcutaneous, and intraocular administration. For parenteral administration, the compositions are provided as solutions, suspensions, emulsions, or lyophilized powders, either conjugated to or separately from a pharmaceutically acceptable parenteral vehicle. Non-limiting examples of such vehicles include water, saline, Ringer's solution, dextrose solution, glycol, ethanol, and 1-10% human serum albumin. Liposomes and non-aqueous vehicles (e.g., non-volatile oils) can also be used. The vehicle or lyophilized powder may contain additives to maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers or preservatives). The formulations are stabilized by known or suitable techniques. Appropriate drug carriers are described in the latest edition of Remington's book, *Remington's Pharmaceutical Sciences*, by A. Osol, which is a standard reference in this field. Parenteral formulations may include sterile water or saline, polyalkylene glycol (e.g., polyethylene glycol), plant-derived oils, hydrogenated naphthalene, etc., as common excipients. Aqueous or oily suspensions for injection can be prepared according to known methods using appropriate emulsifiers or humectants and suspensions. Injectable drugs may be non-toxic parenterally administered diluents, such as aqueous solutions or suspensions in sterile injectable solutions or solvents. Useful vehicles or solvents include water, Ringer's solution, isotonic saline, etc. Sterile non-volatile oils can be used as solvents or suspension solvents. Any type of non-volatile oil and fatty acid may be used for these purposes, including natural, synthetic, or semi-synthetic fatty oils or fatty acids; natural, synthetic, or semi-synthetic mono-, di-, or triglycerides. Parenteral administration is known in the industry and includes, but is not limited to, conventional injection methods, gas-pressurized needleless injectors (as described in U.S. Patent No. 5,851,198) and laser puncture devices (as described in U.S. Patent No. 5,839,446) (the aforementioned patents are incorporated herein by reference in their entirety).

[0109] Lung prescriptions In certain embodiments, compositions comprising the anti-LGALS3 antibody or its antigen-binding fragment described herein are formulated for pulmonary administration. For pulmonary administration, the composition is delivered in a particle size effective for reaching the lungs or the lower airways of the sinuses. Compositions for pulmonary administration may be delivered by any of the various inhalation or nasal devices known in the art for the administration of therapeutic agents by inhalation. These devices, which can deposit aerosolized formulations into the patient's sinuses or alveoli, include dose-measuring inhalers, nebulizers, dry powder generators, and sprayers. Other suitable devices for pulmonary or nasal administration of the anti-LGALS3 antibody or its antigen-binding fragment described herein are also known in the art. Any such device uses a suitable formulation for a device that dispenses the aerosol of the anti-LGALS3 antibody or its antigen-binding fragment described herein. Such aerosols may consist of either a solution (both aqueous and non-aqueous) or solid particles. Dose-measuring inhalers, e.g., Ventolin® dose-measuring inhalers typically use a propellant gas and include inhalation-driven operation (see, e.g., WO 94 / 16970, WO 98 / 35888). Dry powder inhalers, e.g., Turbuhaler TM Devices such as Astra, Rotahaler (trademark) (Glaxo), and Diskus (trademark) (Glaxo) (for example, devices marketed by Inhale Therapeutics) utilize exhalation to drive a mixture of powders (US Pat. No. 4,668,218 (Astra), EP 237507 (Astra), WO 97 / 25086 (Glaxo), WO 94 / 08552 (Dura), US Pat. No. 5,458,135 (Inhale), WO 94 / 06498 (Fisons) (the above are included in their entirety herein by reference)).

[0110] Nebulizers, such as the Ultravent® nebulizer (Mallinckrodt) and the Acorn II® nebulizer (Marquest Medical Products) (US Pat. No. 5,404,871 (Aradigm), WO 97 / 22376 (the aforementioned reference is incorporated herein by reference in its entirety)), produce aerosols from solutions, while dose-measuring inhalers, dry powder inhalers, etc., produce fine particle aerosols. Such examples of inhalers available on the market are non-limiting and are not intended to limit the scope. In certain embodiments, a spray containing the anti-LGALS3 antibody or its antigen-binding fragment described herein can be produced by extruding a suspension or solution of at least one of the anti-LGALS3 antibody or its antigen-binding fragment described herein from a pressurized nozzle. The desired output and particle size can be achieved by selecting the nozzle size and structure, the applied pressure, and the liquid feed rate. Electrospray can be produced, for example, by an electric field connected to a capillary or nozzle feed. Advantageously, the particles of the composition containing at least one of the anti-LGALS3 antibody or its antigen-binding fragment described herein, delivered by the spraying device, have a particle size in the range of less than about 10 μm, e.g., from about 1 μm to about 5 μm, e.g., from about 2 μm to about 3 μm.

[0111] Formulations of compositions suitable for use with a spray device, comprising at least one anti-LGALS3 antibody or its antigen-binding fragment as described herein, typically contain at least one anti-LGALS3 antibody or its antigen-binding fragment as described herein in an aqueous solution at any concentration ranging from about 0.1 mg to about 100 mg (or mg / gm) per 1 mL of solution, or values ​​in between (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg / mL (or mg / gm) (but not limited to the foregoing)). The formulation contains pharmaceuticals (e.g., excipients, buffers, isotonic agents, preservatives, surfactants, and preferably zinc). The formulation may also contain excipients or pharmaceuticals for stabilizing the composition of the anti-LGALS3 antibody or its antigen-binding fragment, such as buffers, reducing agents, bulk proteins, or carbohydrates. Useful bulk proteins when formulating such compositions include albumin, protamine, etc. Typical carbohydrates useful when formulating antibody composition proteins include sucrose, mannitol, lactose, trehalose, glucose, etc. The composition may also contain surfactants, which can reduce or prevent surface-induced aggregation of the composition caused by atomization of the solution during aerosol formation. A variety of common surfactants, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters, can be used. The amount will generally range from 0.001 to 14% by weight of the formulation. Preferred surfactants include polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, etc.

[0112] In certain embodiments, the composition is administered by a nebulizer (e.g., a jet nebulizer or an ultrasonic nebulizer). Typically, a jet nebulizer uses a compressed air source to create a high-speed air jet through a nozzle. As the gas expands beyond the nozzle, a low-pressure region is created, which draws the solution of antibody composition protein from a capillary tube connected to a liquid reservoir. The liquid flow from the capillary tube diffuses into an unstable filament, and as it exits the tube, droplets form an aerosol. Using a range of structures, flow rates, and baffle types, desired performance characteristics can be achieved from a given jet nebulizer. An ultrasonic nebulizer uses high-frequency electrical energy, typically utilizing a piezoelectric transducer to generate vibratory mechanical energy. This energy is transferred directly to the antibody composition protein or via a coupling fluid to create an aerosol containing the antibody composition protein. Advantageously, the particles of antibody composition protein delivered by the nebulizer have a particle size in the range of less than about 10 μm, preferably about 1 μm to about 5 μm, and most preferably about 2 μm to about 3 μm. In certain embodiments, the composition is administered by a dose-dose inhalation device (MDI), in which the propellant, at least one anti-LGALS3 antibody or its antigen-binding fragment as described herein, and any excipients or other additives are contained as a mixture in a canister containing liquefied compressed gas. The metering valve is driven to release the die mixture as an aerosol, which preferably contains particles in a size range of less than about 10 μm, preferably from about 1 μm to about 5 μm, and most preferably from about 2 μm to about 3 μm. The desired aerosol particle size can be obtained by using formulations of antibody composition proteins produced by various methods known to those skilled in the art (including jet milling, spray drying, critical point condensation, etc.). Preferred dose metering inhalers include those manufactured by 3M or Glaxo using hydrofluorocarbon propellants.

[0113] The formulations of anti-LGALS3 antibodies or their antigen-binding fragments described herein, used in dose-measuring inhalation devices, generally comprise a finely divided powder containing at least one anti-IL-6 antibody as a suspension in an aqueous medium (suspended in a propellant aided by a surfactant). The propellant may be any common material used for this purpose, such as chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons, or hydrocarbons, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, HFA-134a (hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227), etc. In some embodiments, the propellant is a hydrofluorocarbon. The surfactant may be selected to stabilize the at least one anti-LGALS3 antibody or its antigen-binding fragment as a suspension in the propellant, for example, to protect the active agent against chemical degradation. Suitable surfactants include sorbitan trioleic acid, soy lectin, and oleic acid. In some cases, the solution aerosol uses a solvent such as ethanol. Additional drugs known in the industry for the purpose of formulating proteins may also be included in the said formulation.

[0114] Oral prescriptions In certain embodiments, the compositions provided herein are formulated for oral administration. In certain embodiments, compositions and methods for administering at least one anti-LGALS3 antibody or its antigen-binding fragment described herein for oral administration include co-administration of an adjuvant (e.g., resorcinol and a nonionic surfactant, e.g., polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether) for artificial enhancement of intestinal wall permeability, and co-administration of an enzyme inhibitor (e.g., pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DFF) and tracylol) for enzymatic degradation inhibition. The active ingredient compound in a solid-type dosage form for oral administration can be mixed with at least one additive. The additives include sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, acacia gum, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, and glycerides. These dosage forms may also contain other types of additives, such as inert diluents, lubricants (e.g., magnesium stearate, parabens), preservatives (e.g., sorbic acid, ascorbic acid, alpha-tocopherol), antioxidants (e.g., cysteine), disintegrants, binders, leavening agents, buffers, sweeteners, flavoring agents, and fragrances. In certain embodiments, oral tablets and pills can be further processed into enteric-coated preparations. In certain embodiments, oral liquid preparations include, for example, emulsions, syrups, elixirs, suspensions, and solution preparations that are medically usable. These preparations may contain inert diluents commonly used in the art, such as water. Liposome preparations can be used for oral preparations, for example, as described for insulin and heparin (U.S. Patent No. 4,239,754). In addition, microspheres of artificial polymers (proteinoids) of mixed amino acids can be used for the oral administration of pharmaceuticals, for example, as described for U.S. Patent No. 4,925,673. Furthermore, carrier compounds (for example, those described for U.S. Patents No. 5,879,681 and 5,871,753) are used for the oral administration of biologically active drugs.

[0115] Mucosal prescriptions In certain embodiments, the compositions provided herein are formulated for absorption through mucosal surfaces. In certain embodiments, for absorption through mucosal surfaces, compositions and methods for administering at least one anti-LGALS3 antibody or its antigen-binding fragment described herein include an emulsion, the emulsion comprising a plurality of submicron particles, mucosal-adherent macromolecules, bioactive peptides, and an aqueous continuous phase (which facilitates absorption through mucosal surfaces by achieving mucosal adsorption of the emulsion particles (U.S. Patent No. 5,514,670)). Suitable mucosal surfaces for application of the emulsions provided herein may include, for example, the cornea, conjunctiva, cheeks, sublingual, nose, lungs, stomach, intestines, and colonic administration routes. Formulations for vaginal or rectal administration (e.g., suppositories) may include, for example, polyalkylene glycol, petrolatum, cocoa butter, etc., as excipients. Formulations for intranasal administration may be solids and may contain, for example, lactose as an excipient, or may be aqueous or oily nasal solutions. For gus administration, excipients include, for example, sugars, calcium stearate, magnesium stearate, and alpha-starch (U.S. Patent No. 5,849,695).

[0116] Transdermal prescriptions In certain embodiments, the compositions provided herein are formulated for transdermal administration. In certain embodiments, for transdermal administration, the compositions comprise at least one anti-LGALS3 antibody or its antigen-binding fragment as described herein, wherein the antibody is encapsulated in a delivery device (e.g., liposomes or polymer nanoparticles, microparticles, microcapsules, microspheres (collectively referred to as microparticles unless otherwise specified)). For transdermal administration, several suitable devices comprising microparticles are known, and the microparticles are made from: synthetic polymers, e.g., polyhydroxy acids, e.g., polylactic acid, polyglycolic acid and copolymers thereof, polyoltoesters, polyacid anhydrides, and polyphosphazenes; and natural polymers, e.g., collagen, polyamino acids, albumin and other proteins, alginates and other polysaccharides; and combinations thereof (U.S. Patent No. 5,814,599).

[0117] kit What is provided herein is also a kit, which comprises one or more antibodies or their antigen-binding fragments as described herein, or the composite thereof. In specific embodiments, what is provided herein is a pharmaceutical pack or kit, which comprises one or more containers filled with one or more components of the pharmaceutical composition described herein (e.g., one or more antibodies or their antigen-binding fragments as described herein). In some embodiments, the kit comprises the pharmaceutical composition described herein and a prophylactic or therapeutic agent. In some cases, such containers may be accompanied by a notice in a prescribed format from a government agency regulating the manufacture, use, or sale of a pharmaceutical or biological product or dosage form, and / or instructions for its use. In certain embodiments, instructions included in the kit provide instructions regarding dosage and / or dosage regimens for the administration of the pharmaceutical composition. Examples of packaging materials for pharmaceuticals include, but are not limited to, blister packs, bottles, boxes, sachets, tubes, inhalers, pumps, bags, vials, containers, syringes, and any packaging materials appropriate for the selected pharmaceutical composition and intended mode of administration and treatment. The kits provided herein may further include devices used for administering the active ingredient. Examples of such devices include, but are not limited to, syringes, needleless injectors, drip bags, patches, and inhalers. The kits provided herein may further include pharmaceutically acceptable vehicles that can be used to administer the components. For example, if the component is in solid form and needs to be reconstituted for parenteral administration, the kit may include a suitable vehicle enclosure in which the component can be dissolved to form a particulate-free solution suitable for parenteral administration. Alternatively, the solution may be reconstituted as a suspension for oral administration. Examples of pharmaceutically acceptable vehicles include (but are not limited to): USP injection water, sodium chloride injection, Ringer's injection, dextrose injection, dextrose sodium chloride injection, lactated Ringer's injection, water-miscible vehicles (including, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol), and non-aqueous vehicles (including, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate).

[0118] Usage and Method Therapeutic use and methods In certain embodiments, what is provided herein is a method for treating LGALS3-related diseases or conditions, the method comprising the step of administering a therapeutically effective amount of an anti-LGALS3 antibody or its antigen-binding fragment to a target animal in need. Exemplary LGALS3-related diseases or conditions include, but are not limited to, cancer, tumor metastasis, tumor vascularization, heart failure, pulmonary fibrosis, renal glomerular disease, inflammatory diseases, and rheumatic diseases. In specific embodiments, an anti-LGALS3 antibody or its antigen-binding fragment is administered in combination with one or more additional therapeutic agents. In some embodiments, one or more additional therapeutic agents suppress or treat one or more symptoms of LGALS3-related disease or symptoms. In certain embodiments, what is provided herein is a method for treating cancer in a subject animal, in particular LGALS3-positive cancer in a subject animal, the method comprising the step of administering a therapeutically effective amount of an anti-LGALS3 antibody or its antigen-binding fragment to a subject animal in need. In specific embodiments, LGALS3-positive cancer is ovarian cancer, lung cancer, pancreatic cancer, breast cancer, fallopian tube cancer, uterine (e.g., endometrial) cancer, primary peritoneum cancer, or cancer of any other tissue that expresses LGALS3. To use an anti-LGALS3 antibody or its antigen-binding fragment in a specific target animal species, an anti-LGALS3 antibody or its fragment that binds to LGALS3 of that specific species is used. For example, to treat a human, an anti-LGALS3 antibody or its antigen-binding fragment that binds to human LGALS3 is used. In specific embodiments, the anti-LGALS3 antibody or its antigen-binding fragment is an immunoglobulin. In addition, in certain embodiments, for the use of an anti-LGALS3 antibody or its fragment in a specific target animal species, the constant region of the anti-LGALS3 antibody or its antigen-binding fragment is derived from that specific species. For example, to treat a human, the anti-LGALS3 antibody or its fragment comprises an immunoglobulin, the anti-LGALS3 antibody or its antigen-binding fragment, where the immunoglobulin includes a human constant region. In specific embodiments, the target animal is a human.

[0119] In specific embodiments, treatment may be performed to achieve beneficial or desired clinical outcomes. These outcomes include, but are not limited to, symptom relief, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, relief or reduction of the disease state, and remission (whether partial or complete), whether detectable or undetectable. In specific embodiments, “treatment” may also extend survival compared to survival expected without treatment.In specific embodiments, administration of the anti-LGALS3 antibody or its antigen-binding fragment described herein, or the pharmaceutical composition described herein, to a target animal having cancer (e.g., ovarian cancer, lung cancer, pancreatic cancer, breast cancer, fallopian tube cancer, uterine (e.g., endometrial) cancer, or primary peritoneal cancer, or cancer of any other tissue expressing LGALS3) achieves at least one, two, three, four, or five or more of the following effects: (i) reduction or mitigation of the severity of one or more symptoms of cancer; (ii) reduction of the duration of one or more symptoms associated with cancer; (iii) prevention of recurrence of symptoms associated with cancer; (iv) reduction of hospitalization of the target animal; (v) reduction of hospitalization period; (vi) increase in survival of the target animal; (vii) enhancement or improvement of the therapeutic effect of another therapy; (viii) suppression of the development or onset of one or more symptoms associated with cancer; (ix) reduction of the number of symptoms associated with cancer; (x) improvement of quality of life as assessed by methods well known in the industry; (x) tumor recurrence (xi) suppression of development; (xii) regression of one or more symptoms associated with the tumor; (xiii) reduction of tumor growth; (xiv) reduction of tumor size (e.g., volume or diameter); (xv) reduction of the formation of new tumors; (xvi) prevention, eradication, removal or control of primary, regional and / or metastatic tumors; (xvii) reduction of the number or size of metastases; (xviii) reduction of mortality; (xix) increase in recurrence-free survival; (xx) maintenance or no increase in tumor size, or less than the increase in tumor size after standard treatment, as assessed by conventional methods available to those skilled in the art (e.g., magnetic resonance imaging (MRI), dynamic contrast-enhanced MRI (DCE-MRI), X-ray and computed tomography (CT) scans, or positron emission tomography (PET) scans); and / or (xxi) increase in the patient's remission period. Treatment may achieve one or more of the above.

[0120] Diagnostic use In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment described herein can be used for diagnostic purposes to detect, diagnose, or monitor the symptoms described herein (e.g., symptoms involving LGALS3-positive cancer cells). In certain embodiments, the anti-LGALS3 antibody or its antigen-binding fragment used for diagnostic purposes is labeled. In certain embodiments, what is provided herein is a method for detecting the symptoms described herein, the method comprising: (b) assaying the expression of LGALS3 or its fragments in a cell or tissue sample of a subject animal using one or more anti-LGALS3 antibodies or their antigen-binding fragments described herein; and (b) comparing the LGALS3 or its fragment expression level to a control level, for example, a normal tissue sample (for example, from a subject animal without the symptoms described herein, or from the same patient before the onset of symptoms), wherein an increase or decrease in the assayed level of LGALS3 or its fragment expression compared to the control level of LGALS3 or its fragment expression serves as an indicator of the symptoms described herein. The antibodies described herein can be used to assay the levels of LGALS3 or its fragments in biological samples using classical immunohistochemical methods. These immunohistochemical methods are described herein or are known to those skilled in the art (see, for example, Jalkanen et al., 1985, J. Cell. Biol. 101:976-985; and Jalkanen et al. (1987) J. Cell. Biol. 105:3087-3096). Other antibody-based methods useful for detecting protein gene expression include immunoassays (e.g., enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA)). Suitable antibody assay labels are known in the art, including enzyme labels (e.g., glucose oxidase); radioisotopes, e.g., iodine. 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In), and technetium (99 Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine; and biotin are also included. In certain embodiments, monitoring of the symptoms described herein (e.g., LGALS3-positive cancer) is carried out by repeating the diagnostic method for a period of time from the initial diagnosis. The presence of labeled molecules can be detected in the target animal using methods known in the art for in vivo scanning. Those skilled in the art will be able to determine appropriate methods for detecting individual labels. Methods and apparatus that can be used in the diagnostic methods of the present invention include, but are not limited to, computed tomography (CT), whole-body scans such as positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasound.

[0121] Dosage and regimen The anti-LGALS3 antibody or its antigen-binding fragment or composition described herein, or cells expressing such antibody or its antigen-binding fragment, can be delivered to target animals via a variety of routes. These routes include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, percutaneous, intravenous, intratumoral, and subcutaneous routes. Lung administration is also available, for example, through formulations with aerosolizing agents for use in inhalers or nebulizers and sprays. In some embodiments, the anti-LGALS3 antibody or its antigen-binding fragment or composition described herein is administered parenterally to the target animal. In specific embodiments, such parenteral administration is intravenous, intramuscular, or subcutaneous. The amount of anti-LGALS3 antibody or its antigen-binding fragment, or composition effective in treating and / or preventing symptoms depends on the nature of the disease and can be determined by standard clinical techniques. The precise dose to be used in the composition also depends on the route of administration and the type of cancer, and should be determined according to the physician's judgment and the environment of each target animal. For example, the effective dose may also vary depending on the means of administration, the target site, the patient's physiological state (including age, weight, and health status), whether the patient is human or animal, other medicines being administered, or whether the treatment is preventive or therapeutic. Therapeutic dosages may be titrated as needed to optimize safety and efficacy. In certain embodiments, in vitro assays are used to help identify the optimal dosage range. Effective doses can be extrapolated from dose-response curves obtained from in vitro or animal model testing systems. For anti-LGALS3 antibodies or their antigen-binding fragments, the dosage ranges from approximately 0.0001 to 100 mg / kg patient body weight, more typically from 0.01 to 15 mg / kg. For example, the dosage is in the range of 1 mg / kg body weight, 10 mg / kg body weight, or 1-10 mg / kg; in other words, for a 70 kg patient, this would be in the range of 70 mg, 700 mg, or 70-700 mg, respectively. Generally, due to the immune response to exogenous polypeptides, human antibodies have a longer half-life in the human body than antibodies from other species. Therefore, lower dosages and less frequent administration are often possible with human antibodies.

[0122] In certain embodiments, for example, administration of an antibody or its antigen-binding fragment, or manipulated cells expressing a CAR, the target animal is divided into approximately 1 million to 100 billion cells, for example, in the range of approximately 1 million to 50 billion cells (e.g., approximately 5 million cells, approximately 25 million cells, approximately 500 million cells, approximately 1 billion cells, approximately 5 billion cells, approximately 20 billion cells, approximately 30 billion cells, approximately 40 billion cells, or a range specified by any two of the aforementioned values), for example, approximately 10 million to 100 billion cells (e.g., approximately 20 million cells, approximately 30 million cells, approximately 40 million cells, approximately 600 The total cell dose and / or individual partial cell population dose is 100 million cells, approximately 70 million cells, approximately 80 million cells, approximately 90 million cells, approximately 10 billion cells, approximately 25 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells, or a range specified by any two of the aforementioned values), and in some cases, approximately 100 million to approximately 50 billion cells (e.g., approximately 120 million cells, approximately 250 million cells, approximately 350 million cells, approximately 450 million cells, approximately 650 million cells, approximately 800 million cells, approximately 900 million cells, approximately 3 billion cells, approximately 30 billion cells, approximately 45 billion cells) or any value between these ranges. In some embodiments, the total cell dose and / or individual partial cell population dose is 1000 million cells per kg of body weight. 4 Or about 10 4 from 10 9 Or about 10 9 Within the range of cells, for example 10 5 from 10 6 Cells / kg body weight, e.g., 1 x 10⁻¹⁶ 5 cells / kg or approximately 1 x 10⁶ 5 cells / kg, 1.5x10 5 Cells / kg or approximately 1.5 x 10⁻⁶ 5 cells / kg, 2x10 5 cells / kg or approximately 2 x 10⁻⁶ 5 cells / kg, 1x10 6 cells / kg or approximately 1 x 10⁶ 6 cells / kg, 2x10 6 cells / kg or approximately 2 x 10⁻⁶ 6 cells / kg, 5x10 6 cells / kg or approximately 5 x 10 6 cells / kg, or 10x10 6 Cells / kg body weight or approximately 10 x 10 6The value is cells / kg body weight. For example, in some embodiments, the cells are 10 within a certain error range or within an error range. 4 Or about 10 4 from 10 9 Or about 10 9 For example, 10 T cells / kg body weight 5 from 10 7 It is administered at a rate of T cells / kg body weight. Anti-LGALS3 antibodies or their antigen-binding fragments can be administered at multiple time points. The intervals between doses may be, for example, one week, two weeks, three weeks, four weeks, one month, two months, three months, six months, one year, or two years.

[0123] Combination therapy In specific embodiments, a method for treating cancer in a target animal (e.g., ovarian cancer, lung cancer, pancreatic cancer, breast cancer, fallopian tube cancer, uterine (e.g., endometrial) cancer, or primary peritoneal cancer) provided herein, comprising the step of administering a pharmaceutical composition comprising the anti-LGALS3 antibody or its antigen-binding fragment described herein to a target animal in need, further comprising the step of administering one or more additional therapeutic agents to the target animal. In specific embodiments, the additional therapeutic agents are for treating cancer in the target animal (e.g., ovarian cancer, lung cancer, pancreatic cancer, breast cancer, fallopian tube cancer, uterine (e.g., endometrial) cancer, and primary peritoneal cancer). In specific embodiments, the additional therapeutic agents are for treating any side effects of treatment with the anti-LGALS3 antibody or its antigen-binding fragment described herein. In specific embodiments, the additional drug is used to treat ovarian cancer. In specific embodiments, the additional drug is used to treat pancreatic cancer. In specific embodiments, the additional drug is used to treat lung cancer. In specific embodiments, the additional drug is used to treat breast cancer. In specific embodiments, the additional drug is used to treat fallopian tube cancer. In specific embodiments, the additional drug is used to treat uterine (e.g., endometrial) cancer. In specific embodiments, the additional drug is used to treat primary peritoneal cancer. The anti-LGALS3 antibody or its antigen-binding fragment described herein may be administered simultaneously or sequentially (before and / or after) with additional therapeutic agents. The antibody or its antigen-binding fragment and the additional therapeutic agent may be administered as the same or different compositions, and further via the same or different routes of administration. The first treatment (the anti-LGALS3 antibody or its antigen-binding fragment described herein, or additional therapeutic agent) may be administered before the administration of the second treatment (the anti-LGALS3 antibody or its antigen-binding fragment described herein, or additional therapeutic agent) (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks) It may be administered to target animals having cancer (e.g., ovarian cancer, lung cancer, pancreatic cancer, breast cancer, fallopian tube cancer, uterine (e.g., endometrial) cancer, and primary peritoneal cancer) in conjunction with or following administration (e.g., 8 weeks or 12 weeks prior to administration). In certain embodiments, additional therapeutic agents administered to target animals in combination with the anti-LGALS3 antibody or its antigen-binding fragment described herein are administered in the same composition (pharmaceutical composition). In other embodiments, additional therapeutic agents administered in combination with the anti-LGALS3 antibody or its antigen-binding fragment described herein are administered to target animals in different compositions other than the anti-LGALS3 antibody or its antigen-binding fragment described herein (e.g., two or more pharmaceutical compositions are used).

[0124] Patient group The animals treated according to the methods provided herein may be any mammal, such as rodents, cats, dogs, horses, dairy cows, pigs, monkeys, primates, or humans. In one particular embodiment, the animal is a human. In another particular embodiment, the animal is a dog. As used herein, “animal” and “patient” are interchangeable. In certain embodiments, the animals treated according to the methods provided herein are diagnosed with LGALS3-positive cancer (including, but not limited to, ovarian cancer, lung cancer, pancreatic cancer, breast cancer, uterine cancer, fallopian tube cancer, or primary peritoneal cancer, or cancer of any other tissue that expresses LGALS3). The following examples are provided for illustrative purposes only and are not intended as limitations. [Examples]

[0125] Production of galectin-3-Fc domain fusion proteins Galectin-3 (LGALS3)-Fc domain fusion proteins were created for use as antigens and for LGALS3 function research. Previously, mucin-16 (MUC16) ectodomain monoclonal antibodies and fusion proteins were constructed using the pFUSE-hIgG1-Fc2 vector (InvivoGen). This vector functions as a 'dummy' antibody with a human IgG1-Fc backbone (Rao et al. (2015) PLoS One 10, e012663; Rao et al. (2017) ACS Chem. Biol. 12: 2085-2096 (each of which is incorporated herein by reference in its entirety)). The pFUSE-hIgG1-Fc2 vector enables the construction of fusion proteins containing the Fc region (CH2 and CH3 domains) of the human IgG1 heavy chain and a select antigen (e.g., LGALS3) linked to the hinge region. The hinge acts as a flexible spacer between the two parts of the Fc fusion protein, allowing each part of the molecule to function independently. A 20-amino acid IL-2 signal sequence is ligated to the N-terminus of a selected antigen, enabling the secretion of an antigenic fusion protein. The IL-2 signal peptide is cleaved behind Ser20 within the cell. PCR primers were designed using the restriction enzyme site EcoRV as the forward primer and NcoI as the reverse primer, and DNA encoding the LGALS3 fragment (amino acids 117-244 of SEQ ID NO: 1, underlined below) was extracted. The fragment contains the LGALS3 sugar-binding domain (Figure 4, bolded below). Human galectin-3

[0126] JPEG2026048820000001.jpg41156 (Array ID: 1)

[0127] The template used for PCR amplification was the LGALS3 cDNA clone BC001120.2 (MGC:2058 IMAGE:3050135 GenBank:AAH01120.1) obtained from ATCC (Manassas, VA). The BC001120.2 galectin-3 cDNA clone has the following nucleotide sequence (the complete coding sequence of LGALS3 is italicized, and the amplified portion is underlined).

[0128] JPEG2026048820000002.jpg101157 (Sequence ID: 29)

[0129] The amplified portion encodes 117-244 LGALS3 having the following amino acid sequence: PYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTS(Sequence ID: 27) 117-244 The PCR product encoding LGALS3 was subsequently purified on a 1% agarose gel, sequenced, and inserted into the pFUSE-IgG1-Fc2 vector (Invivogen). 117-244LGALS3-pFUSE-hIgG1-Fc2 was prepared. While it mimics the galectin-3 sugar binding site, it lacks the ability to form galectin-3 pentamers and thus hinders the formation of a stable galectin-3 gel / matrix. The pFUSE-IgG1-Fc2 vector (Invivogen) has the following nucleic acid sequence (EcoRI and NcoI cloning sites are underlined):

[0130]

[0131] The fusion protein was expressed in human 293 cells and purified using a protein A column for immunization. The expressed fusion protein has the following sequence: MYRMQLLSCIALSLALVTNSPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID: 31) As a negative control possessing the sugar-binding domain of galectin-1, a second sugar-binding fusion protein ( 111-119 We fabricated LGALS1-pFUSE. [Examples]

[0132] Inhibition of galectin-3 suppresses oncogene activation, cell invasion, and tumor growth. Our previous research, as described in the paper by Rao et al., established the role of LGALS3-MUC16 interaction in ovarian tumor growth and invasion (Rao et al. (2017) ACS Chem. Biol. 12: 2085-2096 (the aforementioned paper is included in its entirety herein by reference)). Selected experiments from this study are important herein and support LGALS3 as a target for therapeutic drug development. Figure 2A shows LGALS3 shRNAs that inhibit MUC16 oncogene activation (assessed by inhibition of phosphorylation of several oncogenes (including EGFR, AKT, ERK, and SRC) (P-EGFR, P-AKT, P-ERK, and P-SRC, respectively)). MUC16 expression in SKOV3 cells increased phosphorylation of pERK1 / 2, pSRC, and EGFR. However, shRNA knockdown of MGAT5 (shMGAT5), galectin-3 (shLGALS3), and the N->A mutation of N30 in MUC16 all impair MUC16c114-induced oncogene activation. MGAT5 (mannosyl(alpha-1,6-)-glycoprotein beta-1,6-N-acetyl-glucosaminyltransferase) catalyzes the formation of 4-branched N-glycans (which bind to galectin-3 with high affinity). Similar effects occurred in A2780-MUC16 cells. Therefore, the elimination of LGALS3 significantly inhibits MUC16 activation (MUC16c344) in various oncogenes in the two ovarian models. Figure 2B shows that competition with LGALS3, rather than LGAL1 (galectin-1), inhibits Matrigel invasion in ovarian cancer cell lines (OVCAR3, OVCA-432, OVCA-433, and CAOV3). These cell lines expressed full-length MUC16 with multiple tandem repeats and released the CA125 antigen into the cell culture supernatant. The fusion protein contains the carbohydrate-binding domain of LGALS3 linked to the IgG1 Fc domain. 117-244 LGAL3-pFUSE significantly inhibited ovarian cancer cell invasion, while fusion proteins containing the N-terminal protein of LGALS1 were not. 11-119 LGAL1-pFUSE did not inhibit invasiveness. Both swainsonin and kifunensin inhibited the synthesis of LGALS3 ligand (polylactosamine) and also substantially reduced Matrigel invasiveness. Figure 2C shows the suppression of A2780 ovarian cancer in a tumor xenograft model by an inhibitor of LGALS3 function. The cell line was introduced into the flanks of 10 female athymic nude mice per group. Loss of LGALS3 expression due to shLGALS3 inhibition is highly inhibitory to tumor growth. 117-244Mild inhibition of tumor growth by LGAL3-pFUSE was also observed. Figure 2D shows that antisense knockdown of MGAT5 and LGALS3 inhibits tumor growth in a SKOV3 ovarian cancer xenograft model. In SKOV3, MUC16 overexpression (c114) enhanced tumor growth (top line). In contrast, loss of the N-glycosylation site (N1-N24-N13-mut c114) or shRNAs that counteract MGAT5 or LGALS3 reduced growth to control levels. [Examples]

[0133] Production of galectin-3 antibodies Several strategies were employed in separate campaigns to produce high-affinity anti-galectin-(LGALS3) antibodies. In the first immunization campaign (Campaign 1), five female BALB / c mice were immunized with a series of immunogens at a dose of 50 μg / mouse via intraperitoneal (IP) injection every three weeks for a total of seven times. The first five immunizations were: 117-244LThe procedure was performed using a fusion protein generated by the GALS3-pFUSE-hIgG1-Fc2 construct (Example 1). The last two immunizations were performed using human LGALS3 protein (OriGene: Rockville, MD). Mouse immunoserum was collected at each immunization time point and screened for reactivity to LGALS4 by enzyme-linked immunosorbent assay (ELISA). After the last immunization, two mice were selected from five that showed high ELISA titers for the antigen and were intravenously boosted (IV) with human LGALS3 protein at a dose of 10 μg / mouse (one mouse was planned to be sacrificed, and the other was selected as a backup). 24 hours after IV boosting, the mice were sacrificed, spleen cells were collected and fused with SP2 / mIL6 hybridoma fusion partners, and plated in 96-well plates at 10, 1, 0.3, or 0.1 cells / well. Subsequently, the hybridoma supernatant was collected and screened for LGALS3 binding. The supernatant-positive hybridomas were expanded from one 96-well plate to multiple 96-well plates, and then to 24-well plates. ELISA reactivity to the antigen was tested after each passage of hybridoma cells to avoid false positives. To increase antibody yield, a second immunization campaign (Campaign 2) was conducted. A 23-amino acid custom peptide was synthesized from the sugar-binding domain of LGALS3, CNTKLDNNWGREERQSVFPFESG (SEQ ID NO: 2) (referred to as Peptide 1). This peptide sequence was selected because it is conserved across various species and therefore is thought to represent an important region of the protein. Subsequently, the peptide was complexed with keyhole limpet hemocyanin (KLH) using the Imject Maleimide-Activated mcKLH Kit (Rockford, IL) to create KLH-peptide 1. Next, the remaining four mice were immunized with KLH-peptide 1 at 50 μg / mouse via IP injection every three weeks for a total of three times. Immunoses were collected and screened for responsiveness to LGALS3 and peptide 1 by ELISA. After the final immunization, two mice were selected and IV-boiled with KLH-peptide 1 at 10 μg / mouse, and one mouse was sacrificed 24 hours later. The spleen cells of this mouse were fused with a hybridoma fusion partner as described above. Hybridoma supernatants were screened for immunoglobulin (Ig) isotypes, and IgG1, IgG2a, and IgG2b isotypes were selected. The supernatants were also screened against mouse LGALS3 (mLGALS3), human LGALS1, human LGALS7, and human LGALS9. Hybridoma cells producing the desired antibodies were selected for purification (using Bio X Cell (West Lebanon, NH)). Additional antibodies were also generated using the AlivaMab mouse platform (Ablexis). AlivaMab mice produce chimeric antibodies containing human variable domains. [Examples]

[0134] Functional assays characterizing galectin-3 antibodies Once purified anti-LGALS3 antibodies were obtained, their activity was screened using binding assays, invasive assays, and antitumor assays. method ELISA: Purified antibodies that bind to Gal3 were identified by ELISA. Two strategies were used for this. The first strategy utilized a 96-well plastic microplate coated with LGALS3 protein and an isotype-specific secondary antibody (conjugated with horseradish peroxidase (HRP) for detection). Because there were concerns that LGALS3 did not retain its natural structure, the second strategy involved creating an LGALS3 construct with a polyhistidine tag at the N-terminus (Abcam, ab89487). Nickel-coated 96-well microplates were used with polyhistidine-tagged LGALS3 as the primary protein. Similarly, an isotype-specific secondary antibody was used for detection. Antibody binding to SPR:LGALS3 was confirmed by surface plasmon resonance (SPR) assay. Antibodies were captured on anti-mouse Fc surface and 8-pt. Serial 2-fold dilutions of LGALS3 were started at 500 nM and flowed onto the antibody. Antibodies that tested positive by nickel-plate ELISA and SPR were then used for further experiments. Laminin binding inhibition: An ELISA assay was performed to check whether LGALS3 binding to laminin was inhibited by the antibody. Various antibody concentrations were used relative to fixed laminin concentrations. A Maxisorb plate was coated with 50 μL of laminin (Sigma) (10 μg / mL in PBS) by orbital shaking at 4°C overnight. The following morning, the plate was equilibrated to room temperature, washed three times with PBS-Tween20 (PBS-T), and blocked for 2 hours with SuperBlock (Thermo Scientific). The wells were incubated with galectin::biotin at room temperature for 1 hour. Subsequently, the wells were washed three times with PBS-T and incubated for 1 hour with either 50 μL of antibiotin-HRP (1:5000) or streptavidin-HRP (1:1000). The plate was then washed three times and incubated with 100 μL of TMB-Ultra for 30 minutes, after which 100 μL of 2N H2SO4 was added and read with a plate reader at a wavelength of 450 nm. Matrigel Invasive Assay: Following the previous description by Rao et al., antibody inhibition of basement membrane invasion was determined in a Matrigel invasion chamber (Rao, et al. (2017) ACS Chem. Biol. 12 (8): 2085-2096 (the above literature is included in its entirety herein by reference)). Briefly, MUC16-expressing cell lines were created by transfecting MUC16-negative human cell lines (SKOV3 and A2780) with the C-terminal MUC16 sequence element (essential for tumorigenic activity). The transfection was performed using a phrGFP vector (A2780-phrGFP-MUC16c 344 and SKOV3-phrGFP-MUC16c 344 ) was used. Transfected cells and wild-type MUC16-expressing cells (OVCAR3) were pretreated with selected antibodies before exposure to a Matrigel invasion chamber. The number of invasive cells was counted. Mouse model: Next, the effectiveness of the antibody was evaluated in a transplanted ovarian tumor growth graft model using the lead antibody from the in vitro experiment. A2780-MUC16c was found in the flank region of 16 female athymus nude mice. 344 Cells were introduced at a rate of 2 million cells / mouse. Similarly, control A2780-phrGFP cells were introduced at a rate of 2 million cells / mouse into the flanks of six female athymoid nude mice. A2780-MUC16c 344 Eight mice transplanted with transfected cells received antibody injections twice a week (50 μg / mouse, intravenous (iv)). Routine animal care was provided by the MSKCC Antitumor Assessment Core Facility. Tumor measurements were performed twice a week and tumor growth was recorded according to the guidelines of the MSKCC Research Animal Resource Center. 2000 mm 3 The animals were sacrificed after reaching their maximum tumor volume. Statistical analysis: To compare studies of proliferation and invasiveness, data were expressed as mean ± standard error (SE), and statistical significance was analyzed using independent Student t-tests. Kaplan-Meier curves were constructed for survival and log-rank tests and used to determine significance (Stata 14, StataCorp 2015; College Station, TX). result: Two antibodies were produced from the first immunization campaign, and an additional 12 antibodies were produced from the second immunization campaign (Table 1). Table 1 provides a summary of the selected antibodies produced, their immunoglobulin isotypes, and their antigenic targets. In the table below, LGALS3 is human galectin-3, peptide-1 is a 23-amino acid peptide constructed from the conserved region of the carbohydrate-binding domain of LGALS3, mLGALS3 is mouse galectin-3, and LGALS1 is human galectin-1.

[0135] Table 1: Summary of selected antibodies, immunoglobulin isotypes, and antigenic targets of the antibodies. JPEG2026048820000003.jpg145163 Figure 5 shows a summary of the binding characteristics of the Campaign 2 antibody. Figure 6 shows the ELISA results for the binding of the Campaign 2 antibody to laminin. Achieving high-affinity antibodies against naturally occurring LGALS3 proved particularly challenging. While antibodies from Campaigns 1 and 2 were screened positive for LGALS3 binding by ELISA assays, only 112516.14D11.2D2 demonstrated affinity for naturally occurring LGALS3 via SPR (Figures 7 and 8). Therefore, only this single antibody was screened using functional assays. 112516.14D11.2D2 is also referred to herein as 14D11.2D2. Figure 7 shows that 14D11.2D2 binds to the LGALS3 protein in an ELISA assay. Figure 8 shows that 14D11.2D2 binds to LGALS3 in an SPR assay. The antibody dissociation constant was 14.6 nM in SPR. Figure 9 shows that 14D11.2D2 inhibits the binding of LGALS3 to laminin. At a concentration of 150 nM, 14D11.2D2 reduced the binding of galectin-3 to laminin by 36.6% compared to the untreated control. Figure 10 shows that in the Matrigel assay, 14D11.2D2 inhibited invasion of SKOV3-MUC16c344 (SKOV3c344), A2780-MUC16c344 (A2780c344), and OVCAR3 cell lines compared to untreated cells. It was noted that significantly fewer MUC16-transfected SKOV3 cells invaded the Matrigel membrane when treated with the antibody (p=0.001). Similar results were observed in antibody-treated OVCAR3 cells (p=0.0009). Figure 11 shows that mice immunized with the 14D11.2D2 antibody were more likely to survive than mice that were not administered the antibody in the ovarian tumor xenograft mouse model described above. 14D11.2D2 was administered at a dose of 50 μg / mouse twice a week for a total of eight doses. After 28 days, the survival rate of animals not administered the antibody was statistically significantly lower than that of animals administered the antibody (p=0.012). In addition, MUC16-expressing A2780 tumors showed a tendency toward reduced tumor growth in mice treated with Mab mice. In summary, these data suggest that the 14D11.2D2 antibody binds to undenatured LGALS3 and exhibits antitumor activity. [Examples]

[0136] Loss of metastatic activity in tumor cells lacking galectin-3 expression. To demonstrate the role of LGALS3 in in vivo tumor metastasis, we investigated lung metastasis of breast cancer cell lines in a mouse xenograft model. For this experiment, we used an induced strain of the MDA-MB-231 cell line (epithelial MUC16+ human breast cancer cell line). MDA-MB-231-TGL expresses luciferase and GFP, enabling non-invasive bioluminescence analysis (Minn et al. (2005) Nature 436:518-524). MDA-MB-231-TGL shGAL is a further induced cell line in which LGALS3 is silenced using hairpin RNA. At the start of the experiment, 6-8 week old female athymoid nude mice were divided into two groups (Group 1 (10 mice) and Group 2 (10 mice)), and 1 x 10 units each of MDA-MB-231-TGL wt or MDA-MB-231-TGL shGAL3 were administered via tail vein injection. 6 Cells were administered. Figure 14A shows dorsal images of injected mice at 1 and 11 weeks post-injection. Figure 14B shows the survival curve for this experiment. Mice injected with LGALS3-deficient cells showed a significant reduction in lung metastasis even after tumor establishment (compared to Group 1 at 1 to 11 weeks). In several mice, the tumors appeared to be completely eradicated (Figure 14A). Mice injected with LGALS3-deficient cells also showed significantly longer survival (Figure 14A). Based on the above results, the effects of 14D11 and 2D2 anti-LGALS3 antibodies on metastasis were tested. As expected, administration of anti-LGALS3 antibodies replicated the effects of LGALS3 siRNA on lung metastases. As shown in Figure 14, administration of the 14D11 antibody enhanced survival and further eliminated cancer metastases in one-third of the mice. Taken together, these data support the use of anti-LGALS3 antibody therapy to inhibit LGALS3 function for the treatment of metastatic and metastatic cancers. All publications, patents, and patent applications referenced herein are incorporated herein by reference in their entirety, as if each individual publication or patent application were presented specifically and individually. While the invention described herein has been described in some detail by illustration and examples for clarity, it will be readily apparent to those skilled in the art that certain changes and modifications can be made in light of the teachings of the invention without departing from the scope or spirit of the appended claims.

Claims

1. An antibody or its antigen-binding fragment that binds immunospecifically to the galectin-3 (LGALS3) carbohydrate-binding domain (CBD).

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising LGALS3 CBD, sequence number:

27.

3. The antibody or antigen-binding fragment thereof according to claim 1 or claim 2, wherein the antibody or antigen-binding fragment thereof inhibits in vitro invasion of tumor cells in a Matrigel invasion assay.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein the tumor cells are ovarian tumor cells.

5. An antibody or antigen-binding fragment thereof according to any one of claims 1-4, which inhibits the binding of LGALS3 to glycosylated cell surface proteins.

6. An antibody or antigen-binding fragment thereof according to any one of claims 1-4, which inhibits the binding of LGALS3 to glycosylated cell surface receptors.

7. An antibody or antigen-binding fragment thereof according to any one of claims 1-4, which inhibits the binding of LGALS3 to the glycosylated growth factor receptor.

8. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which inhibits the binding of LGALS3 to glycosylated mucin-1 (MUC-1), mucin-4 (MUC4), mucin-16 (MUC16), disialoganglioside, GD2, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), insulin-like growth factor receptor (IGFR), integrin, and CTLA4.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein glycosylated MUC16 is N-glycosylated at Asn1800 or Asn1806.

10. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, which inhibits the proliferation of tumors expressing the glycosylated form of MUC16.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody is a monoclonal antibody.

12. The antibody or antigen-binding fragment according to any one of claims 1 to 11, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) comprising the following: (a) VH complementarity-determining region (CDR) 1 containing the amino acid sequence SYGVH (SEQ ID NO: 5); (b) VH CDR2 containing the amino acid sequence VIWSDGSTTYNSTLKS (SEQ ID NO: 6); and (c) VH CDR3 containing the amino acid sequence HISNYGTMDY (SEQ ID NO: 7).

13. (a) VH complementarity-determining region (CDR) 1 containing the amino acid sequence GFSLSSY (SEQ ID NO: 11); (b) VH CDR2 containing the amino acid sequence WSDGS (SEQ ID NO: 12); and (c) VH CDR3 containing the amino acid sequence HISNYGTMDY (SEQ ID NO: 13), An antibody or antigen-binding fragment thereof according to any one of claims 1-11, comprising VH containing the above.

14. (a) VH complementarity-determining region (CDR) 1 containing amino acid sequence GFSLSSYG (SEQ ID NO: 17); (b) VH CDR2 containing the amino acid sequence IWSDGST (SEQ ID NO: 18); and (c) VH CDR3 containing the amino acid sequence ARHISNYGTMDY (SEQ ID NO: 19), An antibody or antigen-binding fragment thereof according to any one of claims 1-11, comprising VH containing a

15. An antibody or antigen-binding fragment according to any one of claims 1-11, comprising VH containing the amino acid sequence QVQLKESGPGLVAPSQSLSITCTISGFSLSSYGVHWVRQPPGKGLEWLVVIWSDGSTTYNSTLKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARHISNYGTMDYWGQGTSVTVS (SEQ ID NO: 24).

16. (a) VL CDR1 containing the amino acid sequence RASQDIRNYLN (SEQ ID NO: 8); (b) VL CDR2 containing the amino acid sequence YTSRLHS (SEQ ID NO: 9); and (c) VL CDR3 containing amino acid sequence QHFNTLPPT (SEQ ID NO: 10), An antibody or antigen-binding fragment according to any one of claims 1 to 15, comprising a light chain variable region (VL) containing the above.

17. (a) VL CDR1 containing the amino acid sequence RASQDIRNYLN (SEQ ID NO: 14); (b) VL CDR2 containing the amino acid sequence YTSRLHS (SEQ ID NO: 15); and (c) VL CDR3 containing the amino acid sequence QHFNTLPPT (SEQ ID NO: 16), An antibody or antigen-binding fragment thereof according to any one of claims 1-15, comprising a VL containing a VL.

18. (a) VL CDR1 containing amino acid sequence QDIRNY (SEQ ID NO: 20); (b) VL CDR2 containing amino acid sequence YTS (SEQ ID NO: 21); and (c) VL CDR3 containing amino acid sequence QHFNTLPPT (SEQ ID NO: 22), An antibody or antigen-binding fragment thereof according to any one of claims 1-15, comprising a VL containing the antibody.

19. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, comprising a VL containing the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGSIKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIRNLEQEDIATYFCQHFNTLPPTFGGGTKLEIK (Sequence ID: 26).

20. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, comprising VH containing the amino acid sequence of SEQ ID NO: 24 and VL containing the amino acid sequence of SEQ ID NO:

26.

21. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, wherein the antibody comprises a human-derived heavy chain and a light chain constant region.

22. The antibody or antigen-binding fragment according to claim 21, wherein the heavy chain constant region has an isotype selected from the group consisting of gamma 1, gamma 2, gamma 3, and gamma 4.

23. The antibody or antigen-binding fragment according to claim 21 or 22, wherein the light chain constant region has an isotype selected from the group consisting of kappa and lambda.

24. A humanized antibody or antigen-binding fragment thereof according to any one of claims 1-23.

25. The antibody or antigen-binding fragment thereof according to claim 24, which is a humanized form of a rodent antibody.

26. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 25, wherein the antibody is an immunoglobulin comprising two identical heavy chains and two identical light chains.

27. The antibody or antigen-binding fragment thereof according to claim 26, wherein the immunoglobulin is IgG.

28. An antibody complex comprising an antibody according to any one of claims 1-27 or an antigen-binding fragment thereof, which is complexed with a drug.

29. The antibody complex according to claim 28, wherein the drug is an imaging agent or a cytotoxic agent.

30. The antibody or antigen-binding fragment according to any one of claims 1 to 29, wherein the antibody or its antigen-binding fragment is a bispecific antibody.

31. A bispecific antibody according to claim 30, which binds to CD3 in an immunospecific manner.

32. The bispecific antibody according to claim 30 or 31, comprising an immunoglobulin that immunospecifically binds to LGALS3, wherein the light chain of the immunoglobulin is complexed via a peptide linker with a single-chain variable fragment (scFv) that immunospecifically binds to CD3.

33. A bispecific antibody complex comprising a bispecific antibody according to any one of claims 30-32, which is conjugated with a drug.

34. The bispecific antibody complex according to claim 33, wherein the drug is an imaging agent or a cytotoxic agent.

35. The antibody or antigen-binding fragment according to any one of claims 1 to 27, wherein the antigen-binding fragment is scFv.

36. An scFv complex comprising the scFv described in claim 35, which is complexed with a drug.

37. The scFv complex according to claim 36, wherein the drug is an imaging drug or a cytotoxic drug.

38. A chimeric antigen receptor (CAR) comprising an antibody or antigen-binding fragment according to any one of claims 1-27 or an scFv according to claim 35.

39. T cells that recombinantly express the CAR described in claim 38.

40. A polynucleotide comprising an antibody heavy chain according to any one of claims 12-15, an antibody light chain according to any one of claims 16-19, an scFv according to claim 35, and / or a nucleic acid sequence encoding a CAR according to claim 38.

41. A vector comprising the polynucleotide according to claim 40, which is linked to a promoter so as to be activatable.

42. Isolated cells comprising the polynucleotide described in claim 40 or the vector described in claim 41.

43. A therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 27, the antibody complex according to claim 28 or 29, the bispecific antibody according to any one of claims 30 or 32, the bispecific antibody complex according to claim 33, the scFv according to claim 35, the scFv complex according to claim 36 or 37, the CAR according to claim 38, the polynucleotide according to claim 40, the vector according to claim 41, or the cell according to claim 39 or 42; and a pharmaceutically acceptable carrier. A pharmaceutical composition containing the following:

44. A method for treating cancer in a patient who is in need of such treatment, comprising the step of administering to the patient the pharmaceutical composition described in claim 43.

45. The method according to claim 44, wherein the cancer is ovarian cancer, lung cancer, pancreatic cancer, breast cancer, uterine cancer, fallopian tube cancer, or primary peritoneal cancer.

46. The method according to claim 44 or 45, wherein the cancer is metastatic cancer.

47. The method according to any one of claims 44-46, which inhibits metastasis in a patient.

48. The method according to any one of claims 44-47, wherein the patient is a human patient.

49. The method according to any one of claims 44-48, further comprising the step of administering to the patient an additional therapeutically effective amount of a therapeutic agent.

50. The immunogenic peptide with SEQ ID NO:

2.

51. Immunogenic peptides complexed with immunogenic carrier proteins.

52. Fusion proteins including the following: (a) LGALS3 protein or a fragment thereof containing the LGALS3 carbohydrate-binding domain; and (b) Fc domain.

53. The fusion protein according to claim 52, wherein the LGALS3 carbohydrate-binding domain comprises domain sequence number:

32.

54. The fusion protein according to claim 52, wherein the LGALS3 carbohydrate-binding domain comprises amino acids 117-244 of SEQ ID NO:

1.

55. The fusion protein according to any one of claims 52-54, wherein the Fc domain is a human IgG1 Fc domain.

56. A method for producing an antibody or antigen-binding fragment thereof that specifically binds to LGALS3 CBD, comprising the step of immunizing a target animal with the immunogenic peptide described in claim 50 or the fusion protein described in any of claims 52 or 55.

57. The method according to claim 56, wherein the target animal is a goat, sheep, donkey, chicken, guinea pig, rat, rabbit, or mouse.

58. The method according to claim 56 or 57, wherein an immunogenic peptide is complexed with an immunogenic carrier protein.

59. The method according to claim 58, wherein the immunogenicity carrier protein is keyhole limpet hemocyanin.

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