Antibodies to galectin-3 and methods of use thereof

Abstract

Provided herein are compositions, methods, and uses involving antibodies that specifically bind to the carbohydrate binding domain (CBD) of galectin-3 (LGALS3).Also provided herein are uses and methods for managing, treating, or preventing disorders, such as cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 122,714, filed December 8, 2020, the entire contents of which are incorporated herein by reference.

[0002] The present technology generally relates to the preparation and use of immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that specifically bind to galectin-3 protein. In particular, the present technology relates to the preparation of galectin-3-binding antibodies and their use in the detection and treatment of galectin-3-associated cancer, cardiovascular disease, fibrosis, and inflammation. [Background technology]

[0003] Galectins are a family of small, highly conserved eukaryotic proteins that recognize specific complex sugars on glycosylated cell surface proteins. Galectins 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 in exosomes and do not appear to be classically secreted from the ER or Golgi. Humans possess at least 12 distinct galectins, which are variably expressed in various tissues and developmental stages. Over the last decade, it has become clear that human galectins, particularly galectin-3 (LGALS3), represent a critical link between the microenvironment and tumor cells. In particular, the biological functions of glycoproteins and other surface glycans primarily depend on the specific glycans attached to the Golgi, resulting in unique galectin selectivity. Extracellularly, LGALS3 is involved in regulating plasma membrane residence time, adhesion, migration, invasion, and angiogenesis. Although LGALS3 binds to other natural ligands, its highest affinity ligand is the most proximal lactosamine disaccharide in the poly-lactosamine chains that decorate many O- and N-glycan species. By binding and polymerization, LGALS3 forms a lattice, regulating the location and residence time of growth factor receptors, particularly EGFR, PDGFR, integrins, and CTLA4 (Figures 1A-1B). Activation of downstream signaling molecules, such as SRC, ERK, AKT, and FAK, leads to the production of key molecules involved in metastasis and invasion. On the surface of T cells, CTLA4 surface concentration is stabilized by LGALS3, resulting in immunosuppression. Loss of the LGALS3 lattice inhibits the behavior of multiple cancer and immune cells. In addition to cancer, excess LGALS3 has also been associated with kidney disease, liver fibrosis, pulmonary fibrosis, heart failure, and parasitic diseases (Figure 2A). Summary of the Invention

[0004] Provided herein are compositions, methods, and uses involving antibodies that specifically bind to galectin-3 (LGALS3) and modulate the expression and / or activity of LGALS3 for managing or treating LGALS-mediated disorders, such as cancer. The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to a galectin-3 (LGALS3) polypeptide. In some embodiments, the LGALS3 polypeptide comprises SEQ ID NO: 12.

[0005] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof, comprising a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ), including (a) V H is selected from the group consisting of SEQ ID NOs: 7, 19, 29, 39, and 49 H - a CDR1 sequence and a V selected from the group consisting of SEQ ID NOs: 8, 20, 30, 40, and 50 H - a CDR2 sequence and a V selected from the group consisting of SEQ ID NOs: 9, 21, 31, 41, and 51 H and / or (b) V L is selected from the group consisting of SEQ ID NOs: 2, 14, 24, 34, and 44 L CDR1 sequence and a V selected from the group consisting of SEQ ID NOs: 3, 15, 25, 35, and 45 L CDR2 sequence and a V selected from the group consisting of SEQ ID NOs: 4, 16, 26, 36, and 46 L and a CDR3 sequence of the antibody or antigen-binding fragment thereof.

[0006] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to galectin-3 (LGALS3), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), including (a) V H V containing SEQ ID NO: 7 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 8 H CDR2 sequence and V comprising SEQ ID NO: 9 H CDR3 sequence, and / or VL V containing SEQ ID NO: 2 L CDR1 sequence and V comprising SEQ ID NO: 3 L CDR2 sequence and V comprising SEQ ID NO: 4 L and (b) a V H V comprising SEQ ID NO: 19 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 20 H CDR2 sequence and V comprising SEQ ID NO: 21 H CDR3 sequence, and / or V L V comprising SEQ ID NO: 14 L CDR1 sequence and V comprising SEQ ID NO: 15 L CDR2 sequence and V comprising SEQ ID NO: 16 L and (c) V H V comprising SEQ ID NO: 29 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 30 H CDR2 sequence and V comprising SEQ ID NO: 31 H CDR3 sequence, and / or V L V comprising SEQ ID NO: 24 L CDR1 sequence and V comprising SEQ ID NO: 25 L CDR2 sequence and V comprising SEQ ID NO: 26 L and (d) V H V comprising SEQ ID NO: 39 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 40 H CDR2 sequence and V comprising SEQ ID NO: 41 H CDR3 sequence, and / or V L V comprising SEQ ID NO: 34 L CDR1 sequence and V comprising SEQ ID NO: 35 L CDR2 sequence and V comprising SEQ ID NO: 36 L CDR3 sequence; or (e) V H V comprising SEQ ID NO: 49 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 50 H CDR2 sequence and V comprising SEQ ID NO: 51 H CDR3 sequence, and / or V L V comprising SEQ ID NO: 44L CDR1 sequence and V comprising SEQ ID NO: 45 L CDR2 sequence and V comprising SEQ ID NO: 46 L and a CDR3 sequence. The antibody or antigen-binding fragment thereof comprises a human or humanized heavy chain variable domain (V H ) and / or a human or humanized light chain variable domain (V L In some embodiments, V H comprises an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:38, and SEQ ID NO:48, and / or V L comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 33, and SEQ ID NO: 43. Additionally or alternatively, in some embodiments, the antigen-binding fragment is a Fab, F(ab')2, Fab', Fv, or scFv.

[0007] Additionally or alternatively, in certain embodiments, the antibody or antigen-binding fragment is selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:1, SEQ ID NO:18 and SEQ ID NO:13, SEQ ID NO:28 and SEQ ID NO:23, SEQ ID NO:38 and SEQ ID NO:33, and SEQ ID NO:48 and SEQ ID NO:43, respectively. H Amino acid sequence and V L In some embodiments, the antibody or antigen-binding fragment is a humanized or fully human antibody, or an antigen-binding fragment thereof.

[0008] In any of the foregoing embodiments, the antibody comprises heavy and light chain constant regions of human origin. In certain embodiments of the antibodies of the present technology, the heavy chain constant region has an isotype selected from the group consisting of gamma 1, gamma 2, gamma 3, and gamma 4, and / or the light chain constant region has an isotype selected from the group consisting of kappa and lambda. Additionally or alternatively, in some embodiments, the antibody is an immunoglobulin comprising two identical heavy chains and two identical light chains. In certain embodiments, the immunoglobulin is an IgG. In some embodiments, the antibody is a monoclonal antibody, a single-chain antibody, or any composition comprising an antigen-binding fragment thereof. Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof is produced by immunizing a transgenic mouse.

[0009] In some embodiments, the antibody or antigen-binding fragment of the present technology inhibits Gal-3-PE binding to tumor cells. In particular embodiments, the tumor cells are ovarian tumor cells (e.g., OVCAR3 cells). Additionally or alternatively, in some embodiments, the antibody or antigen-binding fragment thereof inhibits binding of LGALS3 to glycosylated cell surface proteins (e.g., glycosylated cell surface receptors). In particular embodiments, the antibody or antigen-binding fragment inhibits binding of LGALS3 to one or more of glycosylated mucin-1 (MUC1), 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), cMET / hepatocyte growth factor receptor (HGFR), integrin, or CTLA4. In some embodiments, the glycosylated MUC16 is N-glycosylated at Asn1800 or Asn1806. In embodiments of any and all of the antibodies or antigen-binding fragments disclosed herein, the antibody or antigen-binding fragment inhibits the growth of tumors expressing a glycosylated form of MUC16.

[0010] In one aspect, the present disclosure provides an antibody conjugate comprising an agent conjugated to any of the antibodies or antigen-binding fragments described herein. The agent can be an imaging agent or a cytotoxic agent. In some embodiments, the antibody or antigen-binding fragment is a bispecific antibody. In certain embodiments, the bispecific antibody specifically binds to CD3. Additionally or alternatively, in some embodiments, the bispecific antibody comprises an immunoglobulin that specifically binds to LGALS3, wherein the light chain of the immunoglobulin is conjugated via a peptide linker to a single-chain variable fragment (scFv) that specifically binds to CD3.

[0011] In another aspect, the present disclosure provides a bispecific antibody conjugate comprising an agent conjugated to any of the bispecific antibodies or antigen-binding fragments described herein. The agent can be an imaging agent or a cytotoxic agent.

[0012] Also disclosed herein are scFv conjugates comprising an agent conjugated to any of the scFvs described herein. The agent can be an imaging agent or a cytotoxic agent. Also provided herein are chimeric antigen receptors (CARs) comprising any of the antibodies or antigen-binding fragments (e.g., scFvs) described herein, and T cells recombinantly expressing the CARs.

[0013] In one aspect, the present disclosure provides a recombinant nucleic acid sequence encoding any of the antibodies or antigen-binding fragments or CARs described herein. In some embodiments, the recombinant nucleic acid sequence comprises the polynucleotide of any one of SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:47, SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:42, or SEQ ID NO:52, or a portion thereof encoding at least one CDR sequence.

[0014] In another aspect, the present disclosure provides a vector comprising any of the recombinant nucleic acid sequences disclosed herein. In some embodiments, the recombinant nucleic acid sequence is operably linked to a promoter. In yet another aspect, the present disclosure provides an isolated cell comprising any of the recombinant nucleic acid sequences or vectors disclosed herein.

[0015] In one aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of any of the antibodies, antigen-binding fragments, antibody conjugates, bispecific antibodies or antibody conjugates, scFvs, scFv conjugates, CARs, T cells, recombinant nucleic acid sequences, vectors, or isolated cells described herein, and a pharmaceutically acceptable carrier.

[0016] In another aspect, the present disclosure provides a method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of any of the pharmaceutical compositions disclosed herein. The cancer may be ovarian, lung, pancreatic, breast, uterine, fallopian tube, or primary peritoneal cancer, and / or metastatic cancer. In some embodiments, the pharmaceutical composition inhibits metastasis in the patient. Additionally, or alternatively, in some embodiments, the method further comprises administering to the patient a therapeutically effective amount of an additional therapeutic agent. In certain embodiments, the patient is a human patient.

[0017] In yet another aspect, the present disclosure provides a method for detecting LGALS3 protein expression levels in a biological sample, comprising contacting the biological sample with any of the antibodies or antigen-binding fragments disclosed herein and detecting binding to LGALS3 protein in the biological sample.

[0018] Also disclosed herein are kits for the detection and / or treatment of galectin-3-associated diseases or conditions (e.g., galectin-3-associated cancers, cardiovascular diseases, fibrosis, and inflammation), comprising at least one immunoglobulin-related composition of the present technology (e.g., any of the antibodies, antigen-binding fragments, antibody conjugates, bispecific antibodies or antibody conjugates, scFvs, scFv conjugates, CARs, T cells, recombinant nucleic acid sequences, vectors, or isolated cells described herein) and instructions for use. In certain embodiments, the immunoglobulin-related composition is linked to one or more detectable labels. In one embodiment, the one or more detectable labels comprise a radioactive label, a fluorescent label, or a chromogenic label. Additionally or alternatively, in some embodiments, the kit further comprises a secondary antibody that specifically binds to the immunoglobulin-related composition described herein. In some embodiments, the secondary antibody is linked to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, or a chromogenic label. [Brief explanation of the drawings]

[0019] [Figure 1A] The structure and activity of extracellular galectin-3 (LGALS3) is shown, which forms a pentamer to link cell surface cancer molecules, e.g., MUC16 in ovarian cancer, to signaling molecules through contact with signaling receptors, e.g., EGFR and integrins. Activation of downstream signaling molecules, such as SRC, ERK, AKT, and FAK, then leads to the production of molecules important for metastasis and invasion. [Figure 1B] We show how antibody inhibitors that bind to the LGALS3 carbohydrate-binding domain disrupt the LGALS3 surface complex and block activation of "outside-in" signaling by destabilizing the cell surface receptor. [Figure 2A] The experimental function of galectin-3 in vitro and in vivo, as well as the effect of potential blocking antibodies on treatment with the provided antibodies, are shown. [Figure 2B]Figure 1 shows the primary structure of the LGALS3 carbohydrate-binding domain (CBD). The LGALS3 CBD is divided into five subdomains, each of which binds a single sugar residue and is conventionally labeled ABCD and E. In this diagram, highly conserved amino acids are underlined, while domains C, D, and E are indicated in the boxed regions. In particular, R186 (*) is important for the function of LGALS3. The LGALS3 CBD sequence is highly homologous to mouse galectin-3 but distinct from GAL1, GAL7, and GAL9. Although each of these family members has a similar sugar-binding domain, LGALS3 is the only lectin with a polymerization domain. [Figure 3A-3B] Figure 3 shows the development and optimization of an assay for Gal-3-PE binding to OVCAR3 cells. Figure 3A shows the binding of the indicated concentrations of PE-labeled Gal-3 (Gal-3-PE) to the indicated numbers of OVCAR3 cells. The number of OVCAR3 cells bound to Gal-3-PE as detected by a Mirrorball® flow cytometer is plotted. Figure 3B shows the binding of the indicated concentrations of PE-labeled Gal-3 protein (Gal-3-PE) to the indicated numbers of MOLT-4 cells. The number of MOLT-4 cells bound to Gal-3-PE as detected by a Mirrorball® flow cytometer is plotted. [Figure 4]Figure 1 shows exemplary results of a secondary functional screen to select antibodies that inhibit Gal-3-PE binding to OVCAR3 cells. Reactions to measure Gal-3-PE binding to OVCAR3 cells were set up in the presence of PBS (negative control) or the indicated antibodies obtained by enrichment from the supernatants of hybridoma clones selected in the primary binding screen (crude antibody purification). The extent of binding, as measured by a Mirrorball® laser scanning flow cytometer, was plotted. The secondary screen showed that five antibodies subsequently blocked Gal-3-PE binding to OVCAR3 cells. Clone 14D11 did not block Gal-3-PE binding to OVCAR3 cells in this assay. [Figure 5A-5B] Species cross-reactivity of the indicated antibodies is shown. ELISA was used to detect binding to mouse Gal-3 (FIG. 5A) or human Gal-3 (FIG. 5B). OD450 values ​​for each clone are shown. As shown, clones 39F02, 38E05, and 46H02 bound to mouse Gal-3, whereas clones 20F08 and 12H07 did not. [Figures 6A-6D] Figure 6 shows the dose response of purified anti-Gal-3 antibody hits for blocking Gal-3-PE binding to OVCAR3 cells. Reactions to measure Gal-3-PE binding to OVCAR3 cells were set up in the presence of PBS (negative control) or increasing concentrations of the indicated antibodies. Purified antibodies were used in the binding reactions. The effect of antibody concentration on the extent of inhibition of Gal-3-PE binding to OVCAR3 by antibody clones 20F08 and 12H07 (Figure 6A), 46H02 (Figure 6B), clones 38E05 and 39F02 (Figure 6C), and 14D11.2D3 (Figure 6D), which have identical sequences, is shown. These data indicated that the three antibodies (46H02, 20F08, and 12H07) exhibited dose-dependent blocking of Gal-3 binding to OVCAR3 cells. [Figures 7A-7B]Figure 7A shows the binding kinetics of monoclonal antibodies (mAbs) 46H02 (Figure 7A) and 20F08 (Figure 7B) to human Gal-3 as determined by surface plasmon resonance (SPR). Line graphs show the change in resonance units (RU, which reflects the change in analyte binding capacity of the surface) as a function of time (seconds) upon addition of the indicated concentrations of mAb. These data showed that mAbs 46H02 and 20F08 bind to human Gal-3 with KDs of 10 nM and 44 nM, respectively. [Figure 8] Determination of the light chain species of the indicated antibodies is shown. The light chain species of the indicated mAbs was determined by ELISA using anti-mouse kappa, anti-mouse lambda, anti-human kappa, and anti-human lambda antibodies. The data showed that 46H02 has a fully human F(ab)2, while the other hits have a human HC and a mouse LC. [Figures 9A-9C]Figure 9A shows the results of an epitope binning assay for mAbs 46H02 and 14D11.2D2 by surface plasmon resonance (SPR). Figure 9A illustrates the premix epitope binning assay strategy used herein. In this strategy, the test antibody (analyte) is immobilized on a surface, the ligand is flowed over the analyte, and binding is detected by SPR. The ligand was either antigen alone or a premixed solution of antigen complexed with a second antibody. A premixed solution of antigen complexed with the test antibody (analyte) was used as a positive control to demonstrate a blocking reaction. As shown, antigen alone generates a distinctive SPR profile (left panel) that indicates analyte binding to the antigen. When the ligand is a premixed solution of antigen complexed with a second antibody, an SPR profile is obtained that indicates analyte binding to the antigen if the second antibody binds to a distinct epitope on the antigen (center panel). The SPR profile of the positive control showed no binding, indicating that the antigen complexed with the test antibody (analyte) blocks analyte binding (right panel). Figure 9B shows a premix epitope binning assay using mAb 2D3 as the analyte and Gal-3, Gal-3 + 2D3 complex, or Gal-3 + 46H02 complex as the ligand. Figure 9C shows a premix epitope binning assay using mAb 46H02 as the analyte and Gal-3, Gal-3 + 2D3 complex, or Gal-3 + 46H02 complex as the ligand. These data indicate that mAb 46H02 and 14D11 do not compete with each other and therefore bind to distinct epitopes. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present disclosure is not limited in terms of the specific embodiments described in this application, which are intended as single illustrations of individual aspects of the present disclosure. Not all of the various embodiments of the present disclosure are described herein. As will be apparent to those skilled in the art, many modifications and variations of the present disclosure can be made without departing from its spirit and scope. In addition to those enumerated herein, functionally equivalent methods and apparatuses within the scope of the present disclosure will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0021] It is to be understood that this disclosure is not limited to particular uses, methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0022] Additionally, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0023] As will be understood by those skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of those subranges. Any recited range can be readily recognized as fully descriptive and allowing for that same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, and upper third, etc. As will also be understood by those skilled in the art, all language such as "up to," "at least," "greater than," "less than," etc., refers to a range that is inclusive of the recited numbers and can subsequently be broken down into subranges as discussed above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1 to 5 cells refers to a group having 1, 2, 3, 4, or 5 cells, and so on.

[0024] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure belongs. The following references provide those skilled in the art with general definitions of many of the terms used in the art of the present invention: 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, the following terms have the meanings ascribed to them below, unless otherwise specified. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the disclosure.

[0025] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0026] As used herein, when used to modify a numerical value or numerical range, the term "about" indicates a variation of 5% to 10% above and below that value or range while remaining within the intended meaning of the cited value or range.

[0027] As used herein, the term "administration" of an agent to a subject includes any route of introducing or delivering an agent to a subject to perform its intended function. Administration may be by any suitable route, including but not limited to intravenous, intramuscular, intraperitoneal, subcutaneous, and other suitable routes described herein. Administration includes self-administration and administration by another.

[0028] The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics 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 pyrrolysine and selenocysteine. Amino acid analogs refer to agents that have the same basic chemical structure as naturally occurring amino acids (i.e., an alpha carbon bonded to a hydrogen atom, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, or methionine methylsulfonium). Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. In some embodiments, the amino acids that form polypeptides are in the D-form. In some embodiments, the amino acids forming the polypeptide are in the L-form. In some embodiments, a first plurality of amino acids forming the polypeptide are in the D-form and a second plurality of amino acids are in the L-form.

[0029] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted single-letter codes.

[0030] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers 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. The terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0031] As used herein, a "control" is a substitute sample used in an experiment for comparison purposes. A control can be "positive" or "negative." For example, if the purpose of an experiment is to determine the correlation of the effectiveness of a therapeutic agent for treating a particular type of disease, a positive control (a composition known to exhibit the desired therapeutic effect) and a negative control (a subject or sample that does not receive the therapy or receives a placebo) are typically used.

[0032] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to achieve a desired therapeutic effect. In the context of therapeutic applications, the amount of a therapeutic peptide administered to a subject may depend on the type and severity of infection, as well as individual characteristics such as general health, age, sex, weight, and tolerance to drugs. It may also depend on the extent, severity, and type of disease. Those skilled in the art will be able to determine appropriate dosages depending on these and other factors.

[0033] As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one embodiment, the expression level of a gene from one sample can be directly compared to the expression level of that gene from a control or reference sample. In another embodiment, the expression level of a gene from one sample can be directly compared to the expression level of that gene from the same sample after administration of a composition disclosed herein. The term "expression" also refers to one or more of the following events: (1) generation of an RNA template from a DNA sequence within a cell (e.g., by transcription), (2) processing of the RNA transcript within the cell (e.g., by splicing, editing, 5' cap formation, and / or 3' end formation), (3) translation of the RNA sequence into a polypeptide or protein within the cell, (4) post-translational modification of the polypeptide or protein within the cell, (5) presentation of the polypeptide or protein on the cell surface, and (6) secretion, presentation, or release of the polypeptide or protein from the cell.

[0034] The term "linker" refers to a synthetic sequence (e.g., an amino acid sequence) that connects or links two sequences, e.g., links two polypeptide domains. In some embodiments, the linker contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0035] As used herein, the term "antibody" refers not only to intact antibody molecules but also to fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are commonly used both in vitro and in vivo. Thus, as used herein, the term "antibody" refers not only to intact immunoglobulin molecules but also to the well-known active fragments F(ab')2 and Fab. F(ab')2 and Fab fragments, which lack the Fc fragment of intact antibodies, are cleared from the circulation more rapidly and may exhibit less nonspecific tissue binding than intact antibodies (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). The antibodies of the present technology include whole natural 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., nanobodies and single-domain camel antibodies), V NAR Antibodies include non-traditional antibodies and antigen-binding fragments, such as antibodies, fragments, bispecific T cell engager (BiTE) antibodies, minibodies, disulfide-linked Fvs (dsFvs), and anti-idiotypic (anti-Id) antibodies, intrabodies, fusion polypeptides, and any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0036] In certain embodiments, an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (referred to herein as V H ) and heavy chain constant (C H The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (referred to herein as V L ) and light chain constant C LThe light chain constant region consists of one domain, C L It consists of V H and V L The regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged in the following order from amino terminus to 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 (e.g., 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 an antibody that binds to an antigen and confers antigen specificity to the antibody. Fragments of antigen-binding proteins, e.g., antibodies, include one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a peptide / HLA complex). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding portions encompassed by the term "antibody fragment" of an antibody include V L , V H , C L and CHI domains; F(ab)2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; V H and an Fd fragment consisting of the CHI domain, a V fragment of a single arm of the antibody L and V H Fv fragment consisting of domains, V H Examples include dAb fragments consisting of domains (Ward et al., Nature 341:544-546 (1989)), as well as isolated complementarity determining regions (CDRs).

[0037] Antibodies and antibody fragments may be derived wholly or partially from mammals (e.g., humans, non-human primates, goats, guinea pigs, hamsters, horses, mice, rats, rabbits, and sheep) or from antibody-producing animals that are non-mammals (e.g., chickens, ducks, geese, snakes, and urodele amphibians). Antibodies and antibody fragments may be produced in animals or produced outside animals, for example, from yeast or phage (e.g., as single antibodies or antibody fragments or as part of an antibody library).

[0038] Furthermore, the two domains V of the Fv fragment L and V H are encoded by separate genes, but these are collectively referred to as V L and V H These antibody fragments can be joined using recombinant methods by synthetic linkers that allow them to be produced as a single protein chain that pairs to form monovalent molecules. These are known as single-chain Fvs (scFvs); see, e.g., 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 of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0039] An "isolated antibody" or "isolated antigen binding protein" is one that has been identified and separated and / or recovered from a component of its natural environment. A "synthetic antibody" or "recombinant antibody" is generally produced using recombinant techniques or peptide synthesis techniques known to those of skill in the art.

[0040] As used herein, the term "single chain variable fragment" or "scFv" refers to a V H :V L Heavy chains (V) of immunoglobulins (e.g., murine or human) covalently linked to form heterodimers H ) and light chain (V L) is a fusion protein of the variable region of the heavy chain (V H ) and light chain (V L ) are either directly connected or connected by a linker encoding a peptide (e.g., about 10, 15, 20, 25 amino acids), and V H N-terminus of V L or V H The C-terminus of V L The linker is usually rich in glycine for flexibility and rich in serine or threonine for solubility. The linker can connect the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain.

[0041] Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies can be synthesized by the VFv method, as described by Huston et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883 (1988)). H and V LThe polypeptide can be expressed from a nucleic acid containing a sequence encoding the polypeptide. See also U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778, and U.S. Patent Publication Nos. 2005 / 0196754 and 2005 / 0196754. Antagonistic scFvs with inhibitory activity have been described (e.g., Zhao et al., Hybridoma (Larchmt) 27(6):455-51 (2008); Peter et al., J Cachexia Sarcopenia Muscle (2012); Shieh et al., J Immunol 183(4):2277-85 (2009); Giomarelli et al., Thromb Haemost 97(6):955-63 (2007); Fife et al., J Clin Invst 116(8):2252-61 (2006); Brocks et al., Immunotechnology 3(3):173-84 (1997); Moosmayer et al., Ther See Immunol 2(10):31-40(1995). Agonistic scFvs with stimulatory activity have been described (see, e.g., 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)).

[0042] As used herein, "F(ab)" refers to the fragment of an antibody structure that binds to an antigen but is monovalent and does not have the Fc portion; for example, digestion of an antibody with the enzyme papain produces two F(ab) fragments and an Fc fragment (heavy (H) chain constant region, the Fc region that does not bind to antigen).

[0043] As used herein, "F(ab)2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, which fragment has two antigen-binding (ab') (bivalent) regions, each of which has a 1 The "F(ab')2" fragment can be divided into two individual Fab' fragments. The "F(ab')2" fragment contains two separate amino acid chains, a portion of the heavy chain and a light (L) chain, linked by an S-type disulfide bond to bind the antigen, and the remaining portions of the heavy chain are linked together.

[0044] As used herein, "CDR" is defined as the complementarity-determining region amino acid sequence of an antibody, which is the hypervariable region of an immunoglobulin heavy chain and light chain. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th USDapartment of Health and Human Services, National Institutes of Health (1987). Generally, an antibody contains three heavy chain and three light chain CDRs or CDR regions in the variable region. CDRs provide the majority of contact residues for antibody binding to an antigen or epitope. In certain embodiments, CDR regions are delineated using the Kabat system (Kabat, EA, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242 (1991)).

[0045] As used herein, the terms "constant region" or "constant domain" are interchangeable and have their common meaning in the art. The constant region is the portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen but can exhibit various effector functions, e.g., interaction with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence compared to the immunoglobulin variable domain.

[0046] As used herein, "epitope" is a term used in the art and can refer to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (a linear or branched epitope), or an epitope can be, for example, the combination of two or more discontinuous regions of a polypeptide or polypeptides (a conformational, nonlinear, discontinuous, or noncontinuous epitope).

[0047] As used herein, the term "ligand" refers to a molecule that binds to a receptor. In some embodiments, a ligand binds to a receptor on another cell, allowing recognition and / or interaction between the cells.

[0048] As used herein, the term "affinity" refers to a measure of binding strength. Without being bound by theory, affinity depends on the closeness of stereochemical compatibility 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., either monovalent or multivalent). Methods for calculating the affinity of an antibody for an antigen, including the use of binding experiments to calculate affinity, are known in the art. Antibody activity in functional assays (e.g., flow cytometry assays) also reflects antibody affinity. Antibodies and affinities can be characterized phenotypically and compared using functional assays (e.g., flow cytometry assays). Nucleic acid molecules useful in the presently disclosed subject matter include any nucleic acid molecule encoding a polypeptide or a fragment thereof. In certain embodiments, nucleic acid molecules useful in the presently disclosed subject matter include nucleic acid molecules encoding an antibody or an antigen-binding portion thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial homology" or "substantial identity" to an endogenous sequence is typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridizing" refers to the pairing of complementary polynucleotide sequences (e.g., genes described herein), or portions thereof, to form a double-stranded molecule under various stringency conditions. (See, e.g., Wahl, GM and SL Berger, Methods Enzymol. 152:399 (1987); Kimmel, AR Methods Enzymol. 152:507 (1987)).

[0049] As used herein, the terms "specifically bind" and "specifically recognize" are similar terms in the context of antibodies and refer to an antibody or antigen-binding fragment thereof that binds to an antigen (e.g., an epitope or immune complex) via its antigen-binding site and does not exclude cross-reactivity of the antibody or antigen-binding fragment with other antigens, as would be understood by one of skill in the art.

[0050] The terms "substantially homologous" or "substantially identical" refer to a polypeptide or nucleic acid molecule that exhibits at least 50% or greater homology or identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one 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% homologous or identical at the amino acid level or nucleic acid to a sequence used for comparison (e.g., a wild-type or naturally occurring sequence). In some embodiments, a substantially homologous or substantially identical polypeptide contains one or more amino acid substitutions, insertions, or deletions compared to the sequence used for comparison. In some embodiments, a substantially homologous or substantially identical polypeptide contains one or more unnatural amino acids or amino acid analogs, including D-amino acids and retro-inverso amino acids, to replace the homologous sequence.

[0051] Sequence homology or sequence identity is typically measured using sequence analysis software (e.g., 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 assigning degrees of homology to various substitutions, deletions, and / or other modifications. In an exemplary approach to determining the degree of identity, the BLAST program may be used, e.g., -3 ~e -100 A probability score of 0.05 indicates closely related sequences.

[0052] 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.

[0053] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the anti-LGALS3 antibody or antigen-binding fragment thereof disclosed herein, including the amino acid sequence. Conservative modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced into the human scFv of the anti-LGALS3 antibody or antigen-binding fragment thereof disclosed herein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to physicochemical properties, such as charge and polarity. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid from the same group. For example, amino acids can be classified by charge, with positively charged amino acids including lysine, arginine, and histidine, negatively charged amino acids including aspartic acid and glutamic acid, and neutrally charged amino acids including alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Additionally, amino acids can be classified by polarity, with polar amino acids including arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine, and nonpolar amino acids including alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues in a CDR region can be replaced with other amino acid residues from the same group, and the altered antibody can be tested for retained function (i.e., the functions set forth in (c)-(l) above) using the functional assays described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, or no more than five residues in a designated sequence or CDR region are altered.

[0054] As used herein, the term "heterologous nucleic acid molecule or polypeptide" refers to a nucleic acid molecule (e.g., a cDNA, DNA, or RNA molecule) or polypeptide that is not normally present in a cell or a sample obtained from a cell. The nucleic acid may be from another organism, or may be, for example, an mRNA molecule that is not normally expressed in the cell or sample.

[0055] As used herein, the term "modulate" refers to a positive or negative change. Exemplary modulations include a change of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0056] As used herein, the term "increase" refers to a positive change of at least about 5%, including but not limited to a positive change of about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or about 100%.

[0057] As used herein, the term "reduce" refers to a negative change of at least about 5%, including but not limited to a negative change of about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or about 100%.

[0058] As used herein, an "isolated" polynucleotide or nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the nucleic acid molecule's natural source (e.g., mouse or human). Furthermore, an "isolated" nucleic acid molecule, e.g., a cDNA molecule, can be substantially free of other cellular material or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. For example, the language "substantially free" includes preparations of polynucleotides or nucleic acid molecules that have less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% of other materials, e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals.

[0059] As used herein, the term "isolated cell" refers to a cell that is separated from the molecules and / or cellular components that naturally accompany the cell.

[0060] An "effective amount" (or "therapeutically effective amount") is an amount sufficient to produce a beneficial or desired clinical result upon treatment. An effective amount can be administered to a subject in one or more doses. From a therapeutic perspective, an effective amount is an amount sufficient to palliate, ameliorate, stabilize, reverse, or slow the progression of a disease (e.g., a neoplasm) or otherwise reduce the pathological consequences of a disease (e.g., a neoplasm). An effective amount is generally determined by a physician on a case-by-case basis and is within the skill of one of ordinary skill in the art. Several factors are typically considered in determining the appropriate dosage to achieve an effective amount. These factors include the age, sex, and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the engineered immune cells administered.

[0061] As used herein, the term "neoplasm" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration or invasion into other tissues or organs. Neoplastic growth is typically uncontrolled and progressive, occurring under conditions that do not induce or cause the cessation of normal cell proliferation. Neoplasms can affect various cell types, tissues, or organs, including, but not limited to, the bladder, colon, bone, brain, breast, cartilage, glia, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, nervous tissue, ovaries, pleura, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urinary tract, ureter, urethra, uterus, and vagina, or organs selected from the group consisting of these tissues or cell types. Neoplasms include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells).

[0062] As used herein, the term "treating" or "treatment" refers to clinical intervention in an attempt to alter the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. The therapeutic effect of treatment includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and remission or improved prognosis. By preventing the progression of a disease or disorder, treatment can not only prevent deterioration due to the disorder in a subject affected or diagnosed with, or suspected of having, the disorder, but also prevent the onset of the disorder or symptoms of the disorder in a subject at risk of, or suspected of having, the disorder.

[0063] As used herein, the term "subject" refers to any animal (e.g., a mammal), including but not limited to a human, non-human primate, rodent, etc. (e.g., recipient of a particular treatment or from which cells are obtained).

[0064] overview Galectin-3 (LGALS3) is a β-galactoside-binding protein secreted by many cells; however, it lacks signal sequences for translocation to the endoplasmic reticulum and Golgi compartments and entry into the classical secretory pathway. LGALS3 is found in both intracellular and extracellular locations and has pleiotropic biological functions, including cell growth, cell adhesion, and cell-cell interactions. Depending on its localization and post-translational modifications, such as cleavage and phosphorylation, it can exhibit anti- or pro-apoptotic activity. Cleavage of galectin-3 has been reported to be involved in angiogenesis and apoptosis resistance. Phosphorylation of galectin-3 regulates its carbohydrate-binding ability.

[0065] Preliminary studies by the present inventors in ovarian cancer using an artificially synthesized chimeric LGALS3 have established that LGALS3 plays an important role in the growth, spread, and invasive properties of ovarian cancer (Rao, et al. (2017) ACS Chem. Biol. 12(8):2085-2096, incorporated by reference in its entirety). This study utilized both shRNA knockdown of LGALS3 and a chimeric antibody in which the native low-affinity LGALS3 carbohydrate-binding domain was replaced with an antibody variable region. Knockdown experiments demonstrated that tumor expression of LGALS3 is required for the oncogenic effect of the ovarian cancer mucin MUC16 / CA125. Transplantation of LGALS3 knockdown cells into nude mice showed severely restricted growth, confirming the essential role of LGALS3 for tumor growth in vivo. Furthermore, the enhanced growth / invasion by LGALS3 was demonstrated extracellularly. Invasion studies demonstrated that ovarian cancer cell invasion into Matrigel gel was dependent on LGALS3. A low-affinity chimeric antibody containing a fusion protein of the unmodified carbohydrate-binding domain of LGALS3 linked to an Fc backbone can block LGALS3 function and prevent Matrigel gel invasion, whereas a chimeric blocking antibody for galectin-1 was ineffective. Additionally, the introduction of a low-affinity chimeric anti-LGALS3 blocking antibody reduced the in vivo growth of xenografts in mice. These chimeric antibodies had no significant adverse effects on host mice, consistent with the reported minimal effects of LGALS3 knockout (Wright et al., (2017) J Leukoc Biol 101(3):717-726).

[0066] Described herein is an immunization strategy for isolating inhibitory, high-affinity antibodies against LGALS3. The primary sequence of the LGALS3 carbohydrate-binding domain (CBD) is shown in Figure 2B. Boxes indicate the C, D, and E domains of the CBD, which are important for lactosamine binding. Highly conserved amino acids are underlined. R186 (*) is important for GAL3 function. The GAL3 CBD sequence is highly homologous to mouse galectin-3 but distinct from GAL1, GAL7, and GAL9. While each of these family members possesses a similar carbohydrate-binding domain, LGALS3 is the only lectin with a polymerization domain. As described in the examples herein, to generate antibodies against the LGALS3 CBD, mice were immunized with peptides and / or fusion constructs containing LGALS3, or portions thereof comprising the amino acid sequence of SEQ ID NO: 53. Anti-LGALS3 antibodies were screened using peptides and / or fusion constructs containing LGALS3, or portions thereof. The isolated antibody binds to native human LGALS3 and the mouse homologue Lgal3.

[0067] In exemplary embodiments, anti-Gal3 antibodies are produced using genetically modified animals expressing chimeric antibody genes. For example, in exemplary embodiments, the anti-Gal3 antibodies provided herein are produced using AlivaMab® Mouse Kappa mice and / or AlivaMab® Mouse Lambda mice (also referred to interchangeably herein as AlivaMab® Kappa Mice and AlivaMab® Lambda Mice, respectively). Antibodies produced by AlivaMab® Kappa Mice contain a chimeric immunoglobulin heavy (IgH) chain and a human immunoglobulin kappa (κ) light chain. Antibodies produced by AlivaMab® Lambda Mice contain a chimeric IgH chain and a human immunoglobulin lambda (λ) light chain. The chimeric IgH chain of the AlivaMab® Mouse antibody contains a human variable heavy chain (VH). H) domain, a human variable region comprising a human diversity heavy (DH) domain and a human joining heavy (JH) domain, a human constant heavy 1 (CH1) domain, a human upper hinge region (except for Cμ, which naturally lacks an upper hinge region), a mouse middle hinge region, a mouse CH2 domain, and a mouse CH3 domain. In an exemplary embodiment, as shown in Figure 8, AlivaMab® Mouse contains: (1) a fully human variable heavy domain (V H ) and human light chain domain (V L ) and (2) an antibody comprising a human variable heavy chain domain (V H ) and mouse light chain domain (V L ) to generate a mixture of antibodies containing a mixture of Gal-3-PE antibodies. In an exemplary embodiment, hits (e.g., anti-Gal3 antibodies) are identified using ELISA. In an exemplary embodiment, enriched clones are identified using an assay to measure inhibition of Gal-3-PE binding to OVCAR3 (Figure 4). In an exemplary embodiment, a mixture of antibodies containing a fully human variable heavy domain (V H ) and human light chain domain (V L Identification of antibodies containing the V) is performed using ELISA. In a further exemplary embodiment, the V of antibodies identified from AlivaMab® Mouse H and / or V L The V region of an antibody identified from AlivaMab® Mouse is used to generate additional constructs described herein. H and / or V L The CDRs of the regions are used to generate the additional constructs described herein.

[0068] Various studies have demonstrated the role of LGALS3 in tumor biology and inflammation. A partial list of the effects and predicted outcomes of targeting LGALS3 is shown in Figure 2A. Many of the predicted effects are expected to be beneficial in cancer treatment. While the effects of LGALS3 are wide-ranging, even complete loss of LGALS3 in knockout mouse models is consistent with near-normal development, and some immunosuppressive effects are not substantially different 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.

[0069] Antibodies and antigen-binding fragments thereof, as well as polypeptides, e.g., fusion proteins, conjugates, and / or chimeric antigen receptors, comprising such antibodies or fragments, and cells expressing them, are provided. Some antibodies and fragments specifically bind to epitopes of the LGALS3 protein. Such antibodies are referred to herein as "anti-LGALS3 antibodies."

[0070] In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit or treat cancer. In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit tumor cell invasion and growth. In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit tumor angiogenesis. In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit metastasis. In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit metastatic tumor growth. In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit activation of cancer-associated signaling molecules, such as AKT or ERK.

[0071] In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit the induction of T cell apoptosis by LGALS3.

[0072] In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit immunosuppression of T cells.

[0073] In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit or treat cardiovascular disease, hi some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit or treat heart failure, coronary heart disease, or myocardial infarction.

[0074] In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit or treat pulmonary fibrosis.

[0075] In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit or treat kidney disease. In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit or treat glomerulonephritis. In some embodiments, the anti-LGALS3 antibodies and antigen-binding fragments thereof described herein inhibit or treat renal cell carcinoma.

[0076] Also provided herein are nucleic acid sequences (e.g., complementary DNA (cDNA)) encoding such antibodies, and polynucleotides (e.g., isolated polynucleotides) comprising antigen-binding fragments, heavy chains, or light chains thereof. Additionally, provided are vectors (e.g., expression vectors) and cells (e.g., isolated cells or ex vivo cells) comprising polynucleotides (e.g., isolated polynucleotides) comprising nucleic acid sequences (e.g., complementary DNA (cDNA)) encoding such antibodies, and antigen-binding fragments, heavy chains, or light chains thereof. Also provided are methods of making such antibodies, antigen-binding fragments thereof, heavy chains, light chains, vectors, and cells. In other aspects, provided herein are methods and uses for anti-LGALS3 antibodies for treating or managing specific conditions or disorders described herein, such as the treatment or management of cancer. Related compositions (e.g., pharmaceutical compositions), kits, and diagnostic methods are also provided.

[0077] As used herein, the terms "LGALS3," "LGALS3 polypeptide," "LGALS3 peptide," "Gal-3," "Gal-3 polypeptide," or "Gal-3 peptide" are used interchangeably herein to refer to galectin-3, which binds to β-galactoside, and functional homologs and fragments thereof. GenBank™ Accession No. NM_002306.4 (SEQ ID NO: 11) provides an exemplary human LGALS3 nucleic acid sequence. GenBank™ Accession No. NP_002297.2 (SEQ ID NO: 12) provides an exemplary human LGALS3 amino acid sequence.

[0078] Anti-LGALS3 antibody Anti-LGALS3 antibodies or antigen-binding fragments thereof can include, for example, monoclonal antibodies, polyclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain 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), camelid antibodies, affibodies, and disulfide-linked Fvs (dsFvs), or fragments thereof. Such antibodies can be produced by methods known in the art.

[0079] A multispecific antibody or fragment thereof refers to an antibody or fragment thereof that can simultaneously bind to at least two targets with different structures, such as two different antigens, two different epitopes on the same antigen, or a hapten and an antigen or epitope. One specificity can be, for example, for an antigen or epitope of B cells, T cells, bone marrow, plasma, or mast cells, such as CD3. Another specificity can be for a different antigen on the same or a different cell type, such as LGALS3. A multispecific, multivalent antibody has more than one binding site, where the binding sites have different specificities (e.g., bispecific diabodies), and one binding site reacts with one antigen and the other binding site reacts with a different antigen.

[0080] Bispecific antibodies are antibodies that can simultaneously bind to two targets with different structures. Bispecific antibodies (bsAbs) and bispecific antibody fragments (bsFabs) have at least one arm that specifically binds to a first target, e.g., LGALS3, and at least one other arm that specifically binds to a second target, e.g., CD3. Various bispecific fusion proteins can be generated using molecular engineering. In one form, the bispecific fusion protein is bivalent, e.g., consisting of (i) an scFv with a single binding site for one antigen and (ii) an antibody or Fab fragment with a single binding site for a second antigen. In another form, the bispecific fusion protein is tetravalent, e.g., consisting of an IgG with two binding sites for one antigen and two identical scFvs for the second antigen. See, e.g., WO 2011 / 1160119, incorporated herein by reference in its entirety.

[0081] Recent methods for generating bispecific monoclonal antibodies include the use of engineered recombinant monoclonal antibodies with additional cysteine ​​residues to cross-link more strongly than more common immunoglobulin isotypes. See, e.g., FitzGerald et al., Protein Eng. 10(10):1221-1225, 1997. Another approach is to engineer recombinant fusion proteins linking two or more different single-chain antibody or antibody fragment segments with the required bispecificities. See, e.g., Coloma et al., Nature Biotech. 15:159-163, 1997. A variety of bispecific fusion proteins can be generated using molecular engineering.

[0082] Bispecific fusion proteins linking two or more different single-chain antibodies or antibody fragments can be produced in a similar manner. A variety of fusion proteins can be produced using recombinant methods. In certain embodiments, a flexible linker connects an scFv (e.g., an scFv targeting CD3) to the constant region of the light chain of a monoclonal antibody (e.g., the anti-LGALS3 antibody described herein). The appropriate linker sequence required for the in-frame connection of the heavy chain Fc to the scFv can be generated by PCR reaction using the V L The DNA fragment encoding the scFv is then ligated into a staging vector containing a DNA sequence encoding the CHI domain. The resulting construct is excised and the V kappa domain of the antibody (e.g., anti-LGALS3 antibody) is inserted. H The resulting vector is ligated into a vector containing DNA sequences encoding the region, and the resulting vector can be used to transfect suitable host cells, such as mammalian cells, for expression of the bispecific fusion protein.

[0083] In certain embodiments, the anti-LGALS3 antibodies and fragments thereof described herein can also be used to prepare functional bispecific single-chain antibodies (bscAbs), also called diabodies, which can be produced in mammalian cells using recombinant methods. See, e.g., Mack et al., Proc. Natl. Acad. Sci. USA, 92:7021-7025, 1995, incorporated herein by reference. For example, bscAbs can be produced using recombinant methods by connecting two single-chain Fv fragments via a glycine-serine linker. The V of the two antibodies of interest can be linked to each other using recombinant methods. L and V H The domains are isolated using standard PCR methods known in the art. Bispecific single chain antibodies and bispecific fusion proteins are included within the scope of the present technology.

[0084] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein refer to scFvs. scFv is an art-recognized term. scFvs comprise a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin, and the fusion protein retains the same antigen specificity as the whole immunoglobulin. H V via a peptide linker L In certain embodiments, the peptide linker is 5 to 25, 5 to 15, 10 to 20, 10 to 15, or 15 to 25 amino acid residues in length. In certain embodiments, the scFv peptide linker exhibits one or more characteristics suitable for peptide linkers known to those of skill in the art. In certain embodiments, the scFv peptide linker comprises amino acids that allow for solubility of the scFv peptide linker, e.g., serine and threonine. In certain embodiments, the scFv peptide linker comprises amino acids that allow for flexibility of the scFv peptide linker, e.g., glycine. In certain embodiments, the scFv peptide linker comprises a V H N-terminus of V L In certain embodiments, the scFv peptide linker is connected to the C-terminus of V H The C-terminus of V L may be attached to the N-terminus of

[0085] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein refer to chimeric antigen receptors (CARs). CAR is an art-recognized term. CARs can target tumor-associated antigens (e.g., LGALS3). CARs provided herein are typically composed of scFvs derived from LGALS3 glycosylation.

[0086] The CAR comprises an antibody, a transmembrane domain (which in some embodiments is a transmembrane domain derived from a T cell costimulatory molecule (e.g., a transmembrane domain derived from CD28, CD8, CD38, OX-40, or 4-1BB)), and a primary signaling domain, e.g., a T cell receptor (TCR) zeta (ζ) chain cytoplasmic signaling domain. In some embodiments, the CAR further comprises one or more additional regions or domains, such as one or more spacers or linkers, including extracellular spacers, e.g., derived from an antibody or other cell surface molecule, e.g., a spacer containing one or more of an antibody CH2, CH3, and / or hinge region, or a spacer derived from a CD28 molecule or a CD8 molecule, or other spacers. Also provided herein are cells engineered to express such CARs, e.g., T cells, e.g., those recombinantly expressing such CARs. The CAR-expressing T cells, upon recognition of a tumor expressing LGALS3, preferably induce T cell activation, proliferation, and / or lysis of cells of such tumor.

[0087] Anti-LGALS3 antibodies can be of any type (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) of immunoglobulin molecule. In certain embodiments, the antibodies described herein are IgG antibodies, or a class or subclass thereof. In certain embodiments, the antibodies described herein are IgG1 antibodies. In certain embodiments, the antibodies described herein are IgG2 antibodies. In certain embodiments, the antibodies described herein are IgG2a antibodies. In certain embodiments, the antibodies described herein are IgG2b antibodies. In certain embodiments, the antibodies described herein are IgG3 antibodies. In certain embodiments, the antibodies described herein are IgG4 antibodies. In certain embodiments, the antibodies described herein are a mixture of antibody types or subclasses. In certain embodiments, the antibodies described herein are a mixture of IgG2a and IgG2b antibodies. In a specific embodiment, the antibody is a humanized form of a rodent monoclonal antibody.

[0088] An antigen-binding fragment of an anti-LGALS3 antibody can be a Fab fragment, a F(ab')2 fragment, or a portion of an anti-LGALS3 antibody that contains amino acid residues that confer its specificity for the antigen to the anti-LGALS3 antibody (e.g., complementarity-determining regions (CDRs)). Anti-LGALS3 antibodies can be derived from any animal species, including rodents (e.g., mice, rats, or hamsters) and humans.

[0089] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of either the light or heavy chain, typically approximately the amino-terminal 110-120 amino acids in a mature heavy chain and approximately the amino-terminal 90-100 amino acids in a mature light chain, which differs significantly in sequence among antibodies and is responsible for the binding and specificity of a particular antibody for its particular antigen. Sequence variability is concentrated in these regions, called complementarity-determining regions (CDRs), while the more highly conserved regions in the variable domain are called framework regions (FRs). The CDRs are adjacent to the FRs. Generally, the spatial orientation of the CDRs and FRs, from N-terminus to C-terminus, is as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with its antigen. 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 comprises rodent (e.g., mouse or rat) CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or mouse CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0090] CDRs are defined in various ways in the art, including Kabat, Chothia, and IMGT, as well as exemplary definitions. The Kabat definition is based on sequence variability (Kabat, Elvin A. et al., Sequences of Proteins of Immunological Interest. Bethesda: National Institutes of Health, 1983). With respect to the Kabat numbering system, (i) V HCDR1 is typically located at amino acid positions 31 to 35 of the heavy chain, and may optionally include one or two additional amino acids after amino acid position 35 (designated 35A and 35B in the Kabat numbering scheme); (ii) V H CDR2 is typically located at amino acid positions 50-65 of the heavy chain, and (iii) V H CDR2 is typically located at amino acid positions 95-102 of the heavy chain (Kabat, Elvin A. et al., Sequences of Proteins of Immunological Interest. Bethesda: National Institutes of Health, 1983). With respect to the Kabat numbering system, (i) V L CDR1 is typically located at amino acid positions 24-34 of the light chain, and (ii) V L CDR2 is typically located at amino acid positions 50-56 of the light chain, and (iii) V L 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 the Kabat numbering system, the actual linear amino acid sequence of an antibody variable domain may contain fewer or additional amino acids due to shortening or elongation of FRs and / or CDRs, and therefore the Kabat number of an amino acid is not necessarily the same as the number of that linear amino acid.

[0091] The Chothia definition is based on the location of structural loop regions (Chothia et al. (1987) J. Mol. Biol. 196:901-917, and U.S. Pat. No. 7,709,226). The term "Chothia CDR" and similar terms are art-recognized and refer to antibody CDR sequences as determined according to the method of Chothia and Lesk (1987) J. Mol. Biol. 196:901-917, and are referred to herein as "Chothia CDRs" (see also, e.g., U.S. Pat. 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)). With respect to the Chothia numbering system, V H Using the Kabat numbering system to number the amino acid residues in a region, (i) V H CDR1 is typically located at amino acid positions 26-32 of the heavy chain, and (ii) V H CDR2 is typically located at amino acid positions 53-55 of the heavy chain, and (iii) V H CDR3 is typically located at amino acid positions 96 to 101 of the heavy chain. In a specific embodiment, with respect to the Chothia numbering system, V H Using the Kabat numbering system to number the amino acid residues in a region, (i) V H CDR1 is typically located at amino acid positions 26 to 32 or 34 of the heavy chain; and (ii) V H CDR2 is typically located at amino acid positions 52-56 of the heavy chain (in one embodiment, CDR2 is located at positions 52A-56, with 52A being after position 52), and (iii) V H CDR3 is typically located at amino acid positions 95-102 of the heavy chain (in one embodiment, there are no amino acids at positions numbered 96-100). LUsing the Kabat numbering system to number the amino acid residues in a region, (i) V L CDR1 is typically located at amino acid positions 26-33 of the light chain, and (ii) V L CDR2 is typically located at amino acid positions 50-52 of the light chain, and (iii) V L CDR3 is typically located at amino acid positions 91-96 of the light chain. In a specific embodiment, with respect to the Chothia numbering system, V L Using the Kabat numbering system to number the amino acid residues in a region, (i) V L CDR1 is typically located at amino acid positions 24-34 of the light chain, and (ii) V L CDR2 is typically located at amino acid positions 50-56 of the light chain, and (iii) V L CDR3 is typically located at amino acid positions 89-97 of the light chain (in one embodiment, there are no amino acids at positions numbered 96-100). These Chothia CDR positions may vary depending on the antibody and can be determined according to methods known in the art.

[0092] The definition of IMGT is from EVIGT ("IMGT®, the international ImMunoGeneTics Information System® website imgt.org, founder and administrator: Marie-Paule Lefranc, Montpellier, France; see e.g., Lefranc, M.-P., 1999, The Immunologist, 7:132-136, and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212, both of which are incorporated by reference in their entirety). With respect to the IMGT numbering system, (i) V H CDR1 is typically located at amino acid positions 25-35 of the heavy chain, and (ii) V H CDR2 is typically located at amino acid positions 51-57 of the heavy chain, and (iii) V H CDR2 is typically located at amino acid positions 93-102 of the heavy chain.L CDR1 is typically located at amino acid positions 27-32 of the light chain, and (ii) V L CDR2 is typically located at amino acid positions 50-52 of the light chain, and (iii) V L CDR3 is typically located at amino acid positions 89-97 of the light chain.

[0093] Sequence and structure of LGALS3 antibody In certain embodiments, the V of any of the anti-LGALS3 antibodies provided herein, e.g., as shown in Table 6, is provided herein. H Anti-LGALS3 antibodies or antigen-binding fragments thereof are provided, comprising CDRs. In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein comprise the V CDRs of the anti-LGALS3 antibodies shown in Table 6. H In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein comprise CDR1 of the anti-LGALS3 antibodies shown in Table 6. H In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein comprise CDR2 of the anti-LGALS3 antibodies shown in Table 6. H In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein comprise the V CDR3 of the anti-LGALS3 antibodies shown in Table 6. H It may contain one, two, or all three of the CDRs.

[0094] In certain embodiments, the V of any of the anti-LGALS3 antibodies provided herein, e.g., as shown in Table 6, is provided herein. L Anti-LGALS3 antibodies or antigen-binding fragments thereof are provided, comprising CDRs. In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein comprise the V CDRs of the anti-LGALS3 antibodies shown in Table 6. L In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein comprise CDR1 of the anti-LGALS3 antibodies shown in Table 6. LIn certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein comprise CDR2 of the anti-LGALS3 antibodies shown in Table 6. L In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein comprise CDR3 of the anti-LGALS3 antibodies in Table 6. L It may contain one, two, or all three of the CDRs.

[0095] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof, comprising a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ), including (a) V H is selected from the group consisting of SEQ ID NOs: 7, 19, 29, 39, and 49 H - a CDR1 sequence and a V selected from the group consisting of SEQ ID NOs: 8, 20, 30, 40, and 50 H - a CDR2 sequence and a V selected from the group consisting of SEQ ID NOs: 9, 21, 31, 41, and 51 H and / or (b) V L is selected from the group consisting of SEQ ID NOs: 2, 14, 24, 34, and 44 L CDR1 sequence and a V selected from the group consisting of SEQ ID NOs: 3, 15, 25, 35, and 45 L CDR2 sequence and a V selected from the group consisting of SEQ ID NOs: 4, 16, 26, 36, and 46 L and a CDR3 sequence of the antibody or antigen-binding fragment thereof.

[0096] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to galectin-3 (LGALS3), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), including (a) V H V containing SEQ ID NO: 7 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 8 H CDR2 sequence and V comprising SEQ ID NO: 9 H CDR3 sequence, and / or V LV containing SEQ ID NO: 2 L CDR1 sequence and V comprising SEQ ID NO: 3 L CDR2 sequence and V comprising SEQ ID NO: 4 L and (b) a V H V comprising SEQ ID NO: 19 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 20 H CDR2 sequence and V comprising SEQ ID NO: 21 H CDR3 sequence, and / or V L V comprising SEQ ID NO: 14 L CDR1 sequence and V comprising SEQ ID NO: 15 L CDR2 sequence and V comprising SEQ ID NO: 16 L and (c) V H V comprising SEQ ID NO: 29 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 30 H CDR2 sequence and V comprising SEQ ID NO: 31 H CDR3 sequence, and / or V L V comprising SEQ ID NO: 24 L CDR1 sequence and V comprising SEQ ID NO: 25 L CDR2 sequence and V comprising SEQ ID NO: 26 L and (d) V H V comprising SEQ ID NO: 39 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 40 H CDR2 sequence and V comprising SEQ ID NO: 41 H CDR3 sequence, and / or V L V comprising SEQ ID NO: 34 L CDR1 sequence and V comprising SEQ ID NO: 35 L CDR2 sequence and V comprising SEQ ID NO: 36 L CDR3 sequence; or (e) V H V comprising SEQ ID NO: 49 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 50 H CDR2 sequence and V comprising SEQ ID NO: 51 H CDR3 sequence, and / or V L V comprising SEQ ID NO: 44 LCDR1 sequence and V comprising SEQ ID NO: 45 L CDR2 sequence and V comprising SEQ ID NO: 46 L and a CDR3 sequence of the antibody or antigen-binding fragment thereof.

[0097] In certain embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein comprises a V selected from the group consisting of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:38, and SEQ ID NO:48. H Includes the area.

[0098] In certain embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein comprises a V selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 33, and SEQ ID NO: 43. L Includes the area.

[0099] In some embodiments, V H comprises an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:38, and SEQ ID NO:48, and / or V L comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 33, and SEQ ID NO: 43. Additionally or alternatively, in some embodiments, the antigen-binding fragment is a Fab, F(ab')2, Fab', Fv, or scFv.

[0100] Additionally or alternatively, in certain embodiments, the antibody or antigen-binding fragment is selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:1, SEQ ID NO:18 and SEQ ID NO:13, SEQ ID NO:28 and SEQ ID NO:23, SEQ ID NO:38 and SEQ ID NO:33, and SEQ ID NO:48 and SEQ ID NO:43, respectively. H Amino acid sequence and V L Contains the amino acid sequence.

[0101] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are selected from the group consisting of the V and VIII antibodies of the anti-LGALS3 antibodies shown in Table 6. HIn certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein comprise the V region of the anti-LGALS3 antibodies shown in Table 6. L Includes the area.

[0102] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein have the V H Domain only or its V L By domain alone or its three Vs H By CDR alone or its three V L The CDRs may be described by themselves. For example, see Rader C et al. (1998) PNAS 95:8910-8915, which describes the humanization of a murine anti-avP3 antibody, where identifying complementary light or heavy chains from a human light or heavy chain library, respectively, results in humanized antibody variants with affinities as high as or higher than those of the original antibody. Also, certain V H Domain (or V L See Clackson T et al., (1991) Nature 352:624-628, which is incorporated herein by reference in its entirety, which describes a method for generating antibodies that bind to a specific antigen by screening libraries for complementary variable domains using a specific V H domains and complementary V L Domain libraries (e.g., human V L library), and then the selected V L domains to generate additional complementary (e.g., human) V H See Kim and Hong (2007) J Microbiol 45:572-577, which is incorporated by reference in its entirety, describing methods for generating antibodies that bind to specific antigens that can guide domain selection.

[0103] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein can be humanized antibodies, e.g., humanized forms of rodent antibodies. Humanized antibodies can be humanized using techniques such as, but not limited to, CDR-grafting (EP 239,400, WO 91 / 09967, and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), chain shuffling (U.S. Pat. No. 5,565,332), veneering, or resurfacing (EP 592,106 and 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) PNAS91:969-973), as well as, for example, U.S. Pat. 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. al. (1997) J. Biol. Chem. 272(16):10678-84, Couto et al. (1995) Cancer Res. 55(23Supp):5973s-5977s, Caldas et al. (2000) Protein Eng. 13(5):353-60, Morea et al. (2000) Methods 20(3):267-79, and Tan et al. (2002) J. Immunol. 169:1119-25. See also U.S. Patent Publication No. 2005 / 0042664 A1 (February 24, 2005), each of which is incorporated herein by reference in its entirety.

[0104] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are composite human antibodies. Composite human antibodies can be generated, for example, by engineering variable region sequences 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, e.g., 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 antibodies can comprise human constant region sequences, e.g., human light and / or heavy chain constant regions.

[0105] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein may be deimmunized antibodies. Deimmunized antibodies are antibodies from which T cell epitopes have been removed. Methods for producing deimmunized antibodies have been 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.

[0106] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein comprise any of the three V of the antibodies disclosed in Table 6. H CDR and three V L CDR (i.e., V H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2 and V Land humanized immunoglobulins comprising a humanized CDR3, a human-derived framework region, and a human-derived constant region. Non-limiting examples of human framework regions are described in the art, see, e.g., Kabat et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242. In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein comprise framework regions (e.g., V, VB, VC, VD, VE, VF, VH, VF ... L Domain and / or V H In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein comprise framework regions (e.g., framework regions of the V domain) that are primate (e.g., non-human primate) framework regions or are derived from primate (e.g., non-human primate) framework regions. L Domain and / or V H For example, CDRs from an antigen-specific non-human antibody, typically from a rodent (e.g., a mouse or rat), are grafted onto a homologous human or non-human primate (e.g., Old World apes, e.g., Pan troglodytes, Pan paniscus, or Gorilla, Pan troglodytes, Old World monkeys, e.g., monkeys from the genus Macaca, or cynomolgus monkeys, Macaca cynomolgus). Non-human primate framework sequences are described in U.S. Patent Application Publication No. 2005 / 0208625.

[0107] In specific embodiments, the V H V in the region H CDR1, V H CDR2 and / or V H CDR3 position and / or V L V in the regionL CDR1, V L CDR2 and / or V L The position of the CDR3 can be varied by 1, 2, 3, 4, 5, 6, or more amino acid positions, as long as specific binding to LGALS3 or a portion thereof containing the carbohydrate-binding domain is maintained (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, up to at least 95%).

[0108] In another embodiment, the V H V in the region H CDR1, V H CDR2 and / or V H CDR3 length and / or V L V in the region L CDR1, V L CDR2 and / or V L The length of the CDR3 can vary by 1, 2, 3, 4, 5, 6, or more amino acids (e.g., be longer or shorter), as long as specific binding to LGALS3 or a portion thereof containing the carbohydrate-binding domain is maintained (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, up to at least 95%).

[0109] In another embodiment, the V H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2 and / or V LThe amino and / or carboxy termini of CDR3 may be extended or shortened by 1, 2, 3, 4, 5, 6, or more amino acids compared to one or more of the CDRs described herein, as long as specific binding to LGALS3 or a portion thereof containing the carbohydrate-binding domain is maintained (e.g., by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).

[0110] Any method known in the art can be used to determine whether specific binding to LGALS3 is maintained (eg, ELISA binding assay, SPR analysis, or FACS analysis).

[0111] In specific aspects, provided herein are anti-LGALS3 antibodies or antigen-binding fragments thereof that include the heavy and / or light chains of the antibodies, e.g., the heavy chain only, the light chain only, or both the heavy and light chains.

[0112] In some embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain, wherein the amino acid sequence of the variable region of the heavy chain is selected from the group consisting of the V and VL domains of any one of the antibodies listed in Table 6. H CDR1, V H CDR2 and V H The heavy chain constant region comprises a CDR3, and the heavy chain constant region is a human heavy chain constant region. In certain embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein comprises a heavy chain, and the heavy chain variable region has the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively; SEQ ID NOs: 19, 20, and 21, respectively; SEQ ID NOs: 29, 30, and 31, respectively; SEQ ID NOs: 39, 40, and 41, respectively; or SEQ ID NOs: 49, 50, and 51, respectively. H CDR1, V H CDR2 and V H The heavy chain constant region, including CDR3, is a human heavy chain constant region.

[0113] In specific embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof 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: 6, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38, or SEQ ID NO: 48, and the constant region of the heavy chain is a human heavy chain constant region. Non-limiting examples of human constant region sequences are described in the art, see, e.g., Kabat EA et al. (1991), supra.

[0114] In some embodiments, the heavy chain of the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein can 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 antibodies or antigen-binding fragments thereof described herein can comprise a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain, wherein the amino acid sequence of the variable region of the heavy chain is the V of any one of the antibodies listed in Table 6. H CDR1, V H CDR2 and V H The heavy chain constant region, including CDR3, is an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain.

[0115] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain, wherein the amino acid sequences of the variable regions of the heavy chain are V having the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively; SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively; SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, respectively; SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41, respectively; or SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51, respectively. H CDR1, V H CDR2 and V Hand CDR3, and the heavy chain constant region is a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In a specific embodiment, an anti-LGALS3 antibody or antigen-binding fragment thereof described herein comprises a heavy chain, and the amino acid sequence of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:38, or SEQ ID NO:48, and the heavy chain constant region is a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain.

[0116] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein comprise a light chain, wherein the amino acid sequence of the variable region of the light chain is selected from the group consisting of the V and VL domains of any one of the antibodies listed in Table 6. L CDR1, V L CDR2 and V L The light chain constant region comprises a CDR3, and the light chain constant region is a human light chain constant region. In certain embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein comprises a light chain, and the light chain variable region has the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively; SEQ ID NOs: 14, 15, and 16, respectively; SEQ ID NOs: 24, 25, and 26, respectively; SEQ ID NOs: 34, 35, and 36, respectively; or SEQ ID NOs: 44, 45, and 46, respectively. L CDR1, V L CDR2 and V L The antibody or antigen-binding fragment thereof described herein comprises a light chain, the variable region of which comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 33, or SEQ ID NO: 43, and the constant region of the light chain is a human light chain constant region.

[0117] In some embodiments, the light chain of the anti-LGALS3 antibody or antigen-binding fragment thereof described herein is a kappa light chain. In another specific embodiment, the light chain of the anti-LGALS3 antibody or antigen-binding fragment thereof described herein is a lambda light chain. In yet another specific embodiment, the light chain of the anti-LGALS3 antibody or antigen-binding fragment thereof described herein is a human kappa light chain or a human lambda light chain. In certain embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein comprises a light chain, and the amino acid sequence of the variable region of the light chain is the V of any one of the antibodies listed in Table 6. L CDR1, V L CDR2 and V L The light chain constant region comprises a CDR3, and the light chain constant region is a kappa (κ) or lambda (λ) light chain constant region. In certain embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein comprises a light chain, and the light chain variable region has the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively; SEQ ID NOs: 14, 15, and 16, respectively; SEQ ID NOs: 24, 25, and 26, respectively; SEQ ID NOs: 34, 35, and 36, respectively; or SEQ ID NOs: 44, 45, and 46, respectively. L CDR1, V L CDR2 and V L The antibody or antigen-binding fragment thereof described herein comprises a light chain, the amino acid sequence of the variable region of the light chain comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 33, or SEQ ID NO: 43, and the light chain constant region is a kappa or lambda light chain constant region.

[0118] In specific embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain variable region (V H ) and the light chain variable region (V L), wherein the constant region is of a type found in an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In another specific embodiment, the anti-LGALS3 antibody or antigen-binding fragment thereof described herein comprises a V H and V L wherein the constant region is of the type found in IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecules, of any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b). In certain embodiments, the constant region is of the type found in human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecules, of any class of human immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2).

[0119] In certain embodiments, the epitope of an antibody can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography, ELISA assays, 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(Pt4):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 studied using well-known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, ed. Wyckoff HW et al., U.S. Patent Application No. 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 studies can be accomplished using any method known to those skilled in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis techniques, 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 conventional techniques such as immunoassays, for example, by demonstrating the ability of one antibody to block the binding of another antibody to a target antigen (e.g., competitive binding assays). Competitive binding assays can also be used to determine whether two antibodies have similar binding specificities for an epitope. Competitive binding can be determined in an assay in which the immunoglobulin under test inhibits the specific binding of a reference antibody to a common antigen. Many types of competitive binding assays are available, such as solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see Stahli C et al. (1983) Methods Enzymol 9:242-253), solid-phase direct biotin-avidin EIA (see Kirkland TN et al. (1986) J Immunol 137:3614-9), solid-phase direct labeled assays, solid-phase direct labeled sandwich assays (see Harlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press), solid-phase direct labeled RIA using 1-125 label (Morel GA et al. (1988) Mol Immunol 25(1):7-15), solid-phase direct biotin-avidin EIA (Cheung RC et al. (1990) Virology 176:546-52), and direct labeled RIA are known (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 containing either an unlabeled test immunoglobulin and a 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 the test immunoglobulin. Typically, the test immunoglobulin is present in excess.Typically, when present in excess, a competing antibody will inhibit specific binding of a reference antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more. Competitive binding assays can be configured in a number of different formats, using either labeled antigen or labeled antibody. In a typical version of this assay, the antigen is immobilized on a 96-well plate. The ability of unlabeled antibodies to block binding of the labeled antibody to the antigen is then measured using radioactive or enzyme labels. For further details, see, e.g., 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 supra, 386-389.

[0120] In certain aspects, competitive binding assays can be used to determine whether an antibody is competitively blocked (e.g., in a dose-dependent manner) by another antibody when the two antibodies recognize the same or sterically overlapping epitopes in a competitive binding assay, such as a competitive ELISA assay, e.g., whether the antibody essentially binds to the same epitope as a reference antibody or an overlapping epitope; this assay can be configured in any number of different formats, using either labeled antigen or labeled antibody. In certain embodiments, antibodies can be tested in competitive binding assays with the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein.

[0121] In another aspect, provided herein are antibodies that do not compete with the binding of an anti-LGALS3 antibody or antigen-binding fragment thereof described herein to LGALS3 (e.g., in a dose-dependent manner), as determined using an assay known to one of skill in the art or as determined using an assay described herein (e.g., ELISA). In another aspect, provided herein are antibodies that do not competitively inhibit the anti-LGALS3 antibody or antigen-binding fragment thereof described herein from binding to LGALS3 (e.g., in a dose-dependent manner), as determined using an assay known to one of skill in the art or as determined using an assay described herein (e.g., ELISA).

[0122] In certain embodiments, provided herein are antibodies that compete with the antibodies described herein for binding to LGALS3 to the same extent that the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein self-compete for binding to LGALS3. In certain embodiments, provided herein are first antibodies that compete with the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein for binding to LGALS3, where the competition is demonstrated by a reduction in binding of the first antibody to the epitope by greater than 80% (e.g., 85%, 90%, 95%, or 98%, or 80%-85%, 80%-90%, 85%-90%, or 85%-95%). In a specific aspect, provided herein are V antibodies comprising the amino acid sequences set forth in Table 6. H CDR1, V H CDR2 and / or V H V containing CDR3 H and / or V comprising an amino acid sequence listed in Table 6 L CDR1, V L CDR2 and / or V L V containing CDR3 L In a specific aspect, provided herein is an anti-LGALS3 antibody or antigen-binding fragment thereof that competes for specific binding to LGALS3 (e.g., in a dose-dependent manner) with an anti-LGALS3 antibody or antigen-binding fragment thereof comprising: H Domains and / or V listed in Table 6L and an anti-LGALS3 antibody or antigen-binding fragment thereof that competes (e.g., in a dose-dependent manner) for specific binding to LGALS3 with an anti-LGALS3 antibody or antigen-binding fragment thereof comprising:

[0123] In a specific embodiment, provided herein are V antigens comprising the amino acid sequences set forth in Table 6. H CDR1, V H CDR2 and / or V H V containing CDR3 H and / or V comprising an amino acid sequence listed in Table 6 L CDR1, V L CDR2 and / or V L V containing CDR3 L In a specific aspect, provided herein are anti-LGALS3 antibodies or antigen-binding fragments thereof that bind to the same epitope as or an overlapping epitope of an antibody comprising: H Domains and / or V listed in Table 6 L The present invention provides an anti-LGALS3 antibody or antigen-binding fragment thereof that binds to the same epitope or an overlapping epitope of an antibody comprising:

[0124] Assays known to those of skill in the art or described herein (e.g., X-ray crystallography, ELISA assays, surface plasmon resonance (SPR) assays) can be used to determine whether two antibodies bind to the same epitope. Affinity can be measured by, but is not limited to, the equilibrium dissociation constant (K D ), and equilibrium association constant (KA). D can be determined by techniques known to those skilled in the art, such as biolayer interferometry.

[0125] In certain embodiments, the epitope bound by an anti-LGALS3 antibody or antigen-binding fragment thereof described herein is used as an immunogen to generate antibodies, which in some embodiments include all or a portion of the carbohydrate-binding domain of LGALS3.

[0126] Functional characteristics of anti-LGALS3 antibodies In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are administered in an amount of about 0.5×10 -3 / sec, 1×10 -3 / sec, 1.5×10 -3 / sec, 2×10 -3 / sec, 2.5×10 -3 / sec, 3×10 -3 / sec, 4×10 -3 / sec, 5×10 -3 / sec, 6×10 -3 / sec, 7×10 -3 / sec, 8×10 -3 / sec, 9×10 -3 / sec, l×10 -4 / sec, 2×10 -4 / sec, 3×10 -4 / sec, 4×10 -4 / sec, 5×10 -4 / sec, 6×10 -4 / sec, 7×10 -4 / sec, or 8 x 10 -4 k / sec or less d In some embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein bind to LGALS3 at about 0.5 x 10 -3 / sec~8×10 -4 Binds to LGALS3 with a kd of 1 / sec.

[0127] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein have a concentration of at least or about 2.5 x 10 4 / sec, 3×10 4 / sec, 3.5×10 4 / sec, 4×10 4 / sec, 4.5×10 4 / sec, 5×10 4 / sec, 5.5×10 4 / sec, 6×10 4 / sec, 6.5×10 4 / sec, 7×10 4 / sec, 7.5×10 4 / sec, 8×10 4 / sec, 9x10 4 / sec, or 9 x 105 In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein bind to LGALS3 with a binding affinity of at least or about 2.5 x 10 4 / sec~9×10 5 Binds to LGALS3 at ka / sec.

[0128] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein have a K 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. D In some embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein bind to LGALS3 with a K of about 1 nM to about 1000 nM. D In some embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein bind to LGALS3 with a K of about 1 nM to about 100 nM. D In some embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein bind to LGALS3 with a K of about 5 nM to about 50 nM. D In some embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein bind to LGALS3 with a K of about 5 nM to about 20 nM. D binds to LGALS3.

[0129] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein bind to LGALS3 at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 250, 500, or 1000 times more strongly than an isotype control antibody binds to LGALS3. An isotype control antibody is an art-recognized term that refers to an antibody that lacks specificity for the target but matches the class and type of the primary antibody (e.g., an anti-LGALS3 antibody or antigen-binding fragment thereof provided herein). An isotype control is used as a negative control to help distinguish nonspecific background signal from specific antibody signal.

[0130] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein bind to LGALS3 with a 100- to 1,000-fold increased affinity compared to native ligand binding.

[0131] Assays for determining anti-LGAL S3 antibody or antigen-binding fragment-mediated inhibition of Gal-3-PE binding to cancer cells are known to those skilled in the art. For example, OVCAR3 cells can be purchased from ATCC, Manassas, VA. PE-labeled Gal-3 (Gal-3-PE) can be purchased from Abcam, Cambridge, MA. Assays for inhibition of Gal-3-PE binding to OVCAR3 cells can be performed as follows. Briefly, cultured SKOV3 cells were trypsinized and washed with culture medium. 500, 1000, or 2000 cells were plated in a 24-well plate and grown at 37°C in a 5% CO2 humidified incubator. After incubation, the cells were incubated with various concentrations of Gal-3-PE and the test antibody. Gal-3-PE binding to OVCAR3 cells was detected. The degree of inhibition was calculated using a standard curve such as that in Figure 3A.

[0132] In specific embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein inhibit Gal-3-PE binding to cancer cells 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 mock-treated mice, as assessed by methods described herein or known to those of skill in the art. In specific embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein inhibit Gal-3-PE binding to cancer cells by at least about 25% or 35%, optionally by about 75%, compared to mock-treated mice, as assessed by methods described herein or known to those of skill in the art. In specific embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein inhibit Gal-3-PE binding to cancer cells by at least about 1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold compared to mock-treated cancer cells as assessed by methods described herein or known to one of skill in the art. Mock-treated cancer cells can be treated with, for example, phosphate-buffered saline or a control (e.g., an anti-IgG antibody).

[0133] antibody conjugates In some embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein are conjugated to another molecule, eg, an organic moiety, a detectable label, and / or an isotope.

[0134] In certain embodiments, provided herein are conjugates of anti-LGALS3 antibodies or antigen-binding fragments thereof, wherein the anti-LGALS3 antibodies or antigen-binding fragments thereof are conjugated to one or more agents, such as imaging agents or cytotoxic agents. Also provided herein are bispecific antibody conjugates, wherein the bispecific antibodies are conjugated to one or more agents, such as imaging agents or cytotoxic agents. Also provided herein are antibody heavy chain conjugates, wherein the antibody heavy chains are conjugated to one or more agents, such as imaging agents or cytotoxic agents. Also provided herein are antibody light chain conjugates, wherein the antibody light chains are conjugated to one or more agents, such as imaging agents or cytotoxic agents. Also provided herein are fusion protein conjugates, wherein the fusion proteins are conjugated to agents, such as imaging agents or cytotoxic agents. In certain embodiments, the agents are conjugated covalently or non-covalently. In certain embodiments, the imaging agent is a detectable label, such as a chromogenic, enzymatic, radioisotope, isotopic, fluorescent, toxic, chemiluminescent, nuclear magnetic resonance imaging agent, or other label.

[0135] Non-limiting examples of suitable chromogenic labels include diaminobenzidine and 4-hydroxyazo-benzene-2-carboxylic acid.

[0136] Non-limiting examples of suitable enzyme labels 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.

[0137] Non-limiting examples of suitable radioisotope labels 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 In certain embodiments, Pd is 111 In the liver 125 I or 131 To avoid the problem of dehalogenation of the I-labeled anti-LGALS3 antibody or its antigen-binding fragment, it is used for in vivo imaging. 111 In has a more favorable gamma emission energy for imaging (Perkins et al. (1985) Eur. J. Nucl. Med. 70:296-301, Carasquillo et al. (1987) J. Nucl. Med. 25:281-287). For example, In is linked to a monoclonal antibody with l-(P-isothiocyanatobenzyl)-DPTA. 111 In is poorly taken up by non-tumor tissues, especially the liver, thus enhancing the specificity of tumor localization (Esteban et al., (1987) J. Nucl. Med. 28:861-870).

[0138] Non-limiting examples of suitable non-radioisotopic labels include: 157 Gd, 55 Mn, 162 Dy, 52 Tr, and 56 Fe is one example.

[0139] Non-limiting examples of suitable fluorescent labels include: 152 These include Eu labels, fluorescein labels, isothiocyanate labels, rhodamine labels, phycoerythrin labels, phycocyanin labels, allophycocyanin labels, green fluorescent protein (GFP) labels, o-phthaldehyde labels, and fluorescamine labels.

[0140] 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.

[0141] Non-limiting examples of nuclear magnetic resonance imaging agents include heavy metal nuclei such as Gd, Mn, and iron.

[0142] Techniques known to those skilled in the art for conjugating the above-mentioned labels to the above-mentioned anti-LGALS3 antibodies or antigen-binding fragments thereof, bispecific antibodies, antibody heavy chains, antibody light chains, and fusion proteins are described, for example, in Kennedy et al. (1976) Clin. Chm. Acta 70:1-31 and Schurs et al. (1977) Clin. Chm. Acta 81:1-40. The coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, and the m-maleimidobenzyl-N-hydroxy-succinimide ester method, all of which are incorporated herein by reference.

[0143] Non-limiting examples of cytotoxic agents include cytostatic or cytocidal agents, radioactive metal ions such as alpha emitters, and toxins such as Pseudomonas exotoxin, abrin, cholera toxin, ricin A, and diphtheria toxin.

[0144] In certain embodiments, the agent is a diagnostic agent. A diagnostic agent is an agent useful for diagnosing or detecting disease by locating cells containing an antigen. Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as those containing biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancing agents for magnetic resonance imaging (MRI) (e.g., paramagnetic ions). U.S. Patent No. 6,331,175 describes MRI techniques and the preparation of antibodies conjugated to MRI enhancing agents and is incorporated by reference in its entirety. In some embodiments, the diagnostic agent is selected from the group consisting of radioisotopes, enhancing agents for use in magnetic resonance imaging, and fluorescent compounds. To incorporate a radiometal or paramagnetic ion into an anti-LGALS3 antibody or antigen-binding fragment thereof, it may be necessary to react it with a reagent having a long tail to which multiple chelating groups are attached for binding to the ion. Such tails can be polymers such as polylysine, polysaccharides, or other derivatized or derivatizable chains having pendant groups that can be attached to chelating groups known to be useful for this purpose, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and the like. Chelators are linked to antibodies using standard chemistries. Chelators are typically linked to antibodies by groups that allow for the formation of bonds to the molecule with minimal loss of immunoreactivity, minimal aggregation and / or other internal cross-linking; more unusual methods and reagents for conjugating chelators to antibodies are disclosed in U.S. Patent No. 4,824,659, issued April 25, 1989, to Hawthorne, entitled "Antibody Conjugates," the disclosure of which is incorporated herein by reference in its entirety. Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, which are used in conjunction with diagnostic isotopes for radioimaging.The same chelators are useful for MRI when used with the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein when complexed with non-radioactive metals such as manganese, iron, and gadolinium. Macrocyclic chelators such as NOTA, DOTA, and TETA are useful when used with a variety of metals and radiometals, most particularly gallium, yttrium, and copper radionuclides, respectively. Such metal-chelator complexes can be made highly stable by adjusting the ring size to the metal of interest. Other ring-type chelators, such as macrocyclic polyethers, are intended for stably binding nuclides, such as 223Ra for RAIT.

[0145] In certain embodiments, the drug is an organic drug. Such organic drugs can produce conjugates with improved pharmacokinetic properties (e.g., increased in vivo serum half-life). The organic moiety can be a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" encompasses monocarboxylic and dicarboxylic acids. As used herein, "hydrophilic polymer group" refers to an organic polymer that is more soluble in water than in octane, such as polylysine. Hydrophilic polymers suitable for modifying the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein can 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 polyaspartate), polyalkane oxides (e.g., polyethylene oxide and polypropylene oxide), and polyvinylpyrrolidone. In certain embodiments, the hydrophilic polymer modifying an anti-LGALS3 antibody or antigen-binding fragment thereof, bispecific antibody, antibody heavy chain, antibody light chain, or fusion protein provided herein has a molecular weight of about 800 to about 150,000 daltons as a separate molecular entity, e.g., PEG. 5000 and PEG20,000 (The subscript is the average molecular weight of the polymer in Daltons) can be used. The hydrophilic polymer group can be substituted with 1 to about 6 alkyl, fatty acid, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups can be prepared by using appropriate methods. For example, a polymer containing an amine group can be linked to the carboxylate of a fatty acid or fatty acid ester, and an activated carboxylate on the fatty acid or fatty acid ester (e.g., activated with N,N-carbonyldiimidazole) can be linked to a hydroxyl group on the polymer.

[0146] Fatty acids and fatty acid esters suitable for modifying the anti-LGALS3 antibodies or antigen-binding fragments thereof, bispecific antibodies, antibody heavy chains, antibody light chains, or fusion proteins provided herein can be saturated or contain one or more unsaturated units. Fatty acids suitable for modifying the anti-LGALS3 antibodies or antigen-binding fragments thereof, bispecific antibodies, antibody heavy chains, antibody light chains, or fusion proteins provided herein include, for example, n-dodecanoate, n-tetradecanoate, n-octadecanoate, n-eicosanoate, n-docosanoate, n-triacontanoate, n-tetracontanoate, cis-delta-9-octadecanoate, all-cis-delta-5,8,11,14-eicosatetranoate, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like. Suitable fatty acid esters include monoesters of dicarboxylic acids containing straight-chain or branched-chain lower alkyl groups. A lower alkyl group can contain 1 to about 12, preferably 1 to about 6, carbon atoms.

[0147] The conjugates provided herein can be prepared using suitable methods, for example, by reaction with one or more modifying 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 modifying agent and the second chemical group. For example, amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl ester (NHS), etc. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), etc. Aldehyde functional groups can be linked to amine- or hydrazine-containing molecules, and azide groups can react with trivalent phosphorus groups to form phosphoramidate or phosphorimide bonds. Suitable methods for introducing activating groups into molecules are known in the art (see, e.g., Hernanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)). Activating groups can be attached directly to organic groups (e.g., hydrophilic polymers, fatty acids, fatty acid esters) or to linker moieties, e.g., divalent C-C 12The linker moiety may be linked via a group, and one or more carbon atoms may be replaced with a heteroatom such as oxygen, nitrogen, or sulfur. Suitable linker moieties include, for example, tetraethylene glycol, (CH2)3, and NH. A modifying agent containing a linker moiety 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 l-ethyl-3-(3-dimethylaminopropyl)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 (TFA) to expose a primary amine, which can be coupled with another carboxylate as shown, or reacted with maleic anhydride, and the resulting product can be cyclized to produce an activated maleimide derivative of the fatty acid. (See, for example, Thompson, et al., WO92 / 16221, the teachings of which are incorporated herein by reference in their entirety.)

[0148] A "modifying agent" can refer to a suitable organic group (e.g., hydrophilic polymers, fatty acids, and fatty acid esters) containing an activating group. For example, an organic moiety can be attached to an anti-LGALS3 antibody or antigen-binding fragment thereof in a non-site-specific manner by using an amine-reactive modifying agent, such as an N-hydroxysuccinimide ester of PEG. Modified anti-LGALS3 antibodies or antigen-binding fragments thereof can also be prepared by reducing disulfide bonds (e.g., intrachain disulfide bonds) of an anti-LGALS3 antibody or antigen-binding fragment thereof, bispecific antibody, antibody heavy chain, antibody light chain, or fusion protein. The reduced anti-LGALS3 antibody or antigen-binding fragment thereof, bispecific antibody, antibody heavy chain, antibody light chain, or fusion protein can then be reacted with a thiol-reactive modifying agent to produce a conjugate provided herein. Conjugates comprising an organic moiety that binds to a specific site on the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein can be prepared using suitable methods, such as reverse proteolysis (Fisch et al. (1992) Bioconjugate Chem., 3:147-153; Werlen et al. (1994) Bioconjugate Chem., 5:41 1-417; 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 the methods described in Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996).

[0149] antibody generation Antibody generation and screening In another aspect, provided herein are methods for producing anti-LGALS3 antibodies or antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof described herein can be produced by any method known in the art for the synthesis of antibodies, for example, by chemical synthesis or by recombinant expression techniques. The methods described herein employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the art. These techniques are described, for example, in the references cited herein and are fully explained therein.See, for example, 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); Current Protocols in Immunology, John Wiley & Sons (1987 and annually updated); 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. (ed.) (1999) Genome See Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0150] In specific embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are antibodies (e.g., recombinant antibodies) that are prepared, expressed, generated, or isolated by any means involving, for example, synthesis, creation through genetic manipulation of DNA sequences. In certain embodiments, such antibodies comprise a sequence encoded by a DNA sequence that does not naturally occur in vivo within the antibody germline repertoire of an animal or mammal (e.g., a human). In specific embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are made by a method that includes using the carbohydrate-binding domain of LGALS3 or a portion thereof. For example, see Examples 1-2 for a detailed description of exemplary methods of how to generate the antibodies described herein.

[0151] In certain embodiments, the anti-LGALS3 antibodies provided herein are generated using immunogenic peptides comprising: ADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPYGAPAGPLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYTMI (SEQ ID NO: 53).

[0152] In certain embodiments, the anti-LGALS3 antibodies provided herein are generated using an immunogenic peptide comprising an Fc fusion protein containing the Fc region (CH2 and CH3 domains) of a human IgG1 heavy chain and amino acids 117-224 of LGALS3 linked to a hinge region. Amino acids 117-224 of LGALS3 are represented by the following amino acid sequence: PYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTS (SEQ ID NO: 54).

[0153] In some embodiments, amino acids 117-224 of LGALS3 can be encoded by the following exemplary nucleotide sequence: CCTTATAACCTGCCTTTGCCTGGGGGAGTGGTGCCTCGCATGCTGATAACAATTCTGGGCACGGTGAAGCCCAATGCAAACAGAATTGCTTTAGATTTCCAAAGAGGGAATGATGTTGCCTTCCACTTTAACCCACGCTTCAATGAGAACAACAGGAGAGTCATTGTTTGCAATACAAAGCTGGATAATAACTGGG GAAGGGAAGAAAGACAGTCGGTTTTCCCATTTGAAAGTGGGAAACCATTCAAAATACAAGTACTGGTTGAACCTGACCACTTCAAGGTTGCAGTGAATGATGCTCACTTGTTGCAGTACAATCATCGGGTTAAAAAAACTCAATGAAATCAGCAAACTGGGAATTTCTGGTGACATAGACCTCACCAGT (SEQ ID NO: 55).

[0154] In certain embodiments, immunogenic peptides are conjugated to immunogenic carrier proteins. In most cases, small antigens (e.g., short peptides or small haptens) are not complex enough to induce antibody production. Due to their large size and complex structure, immunogenic carrier proteins may confer immunogenicity to the conjugated small antigen, resulting in antibodies being generated against epitopes on the small antigen and immunogenic carrier protein. Therefore, small antigens are always chemically conjugated to immunogenic carrier proteins to enhance the immune response for successful antibody production. 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 a specific embodiment, immunogenic peptides are conjugated to 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 keyhole limpets (Megathura crenulata). Due to its evolutionary distance from mammals, high molecular weight, complex structure, and large surface containing hundreds of lysine groups that provide primary amines as targets for conjugation, KLH is a highly immunogenic and effective carrier protein in mammals.

[0155] In certain embodiments, the immunogenic peptide is 10 to 60 amino acid residues in length. In some embodiments, the immunogenic peptide is 10 to 30 amino acid residues in length. In some embodiments, the immunogenic peptide is 15 to 25 amino acid residues in length. In some embodiments, the immunogenic peptide is 15 to 20 amino acid residues in length. In specific embodiments, the immunogenic peptide is 15 to 18 amino acid residues in length.

[0156] In certain embodiments, the immunogenic peptide comprises at least a 10 amino acid portion of the LGALS3CBD domain. In certain embodiments, the immunogenic peptide comprises at least a 10 amino acid portion of the amino acid sequence of SEQ ID NO: 53. In certain other embodiments, the immunogenic peptide comprises at least a 15, 20, 25, or 30 amino acid portion of the amino acid sequence of SEQ ID NO: 53. In specific embodiments, the immunogenic peptide consists of 15 to 30 consecutive amino acid residues of SEQ ID NO: 53.

[0157] In another aspect, provided herein is a method for generating an antibody or antigen-binding fragment thereof that specifically binds to LGALS3, the method comprising immunizing a subject with the immunogenic peptide described above. A subject immunized according to the methods described herein can be, but is not limited to, a goat, sheep, donkey, chicken, guinea pig, rat, rabbit, or mouse. In some embodiments, a subject immunized according to the methods described herein is a rat, rabbit, or mouse. In some embodiments, a subject immunized according to the methods described herein is the AlivaMab® Mouse platform (Ablexis). In a specific embodiment, a subject immunized according to the methods described herein is a mouse. In some embodiments, a subject immunized according to the methods described herein is a transgenic mouse expressing chimeric forms of various immunoglobulin chains. In some embodiments, the transgenic mouse expresses a human variable region linked to a mouse constant region. (See, e.g., U.S. Patent Nos. 850,2018, 9580,491, U.S. Patent Publication Nos. 2017 / 0218090, 2016 / 0222093, and 2013 / 0167256, each of which is incorporated by reference in its entirety. See also the AlivaMab® Mouse platform (Ablexis).)

[0158] Immunization of a subject can be carried out by any method known in the art, for example, by administering to the subject an immunogenic peptide and an adjuvant.

[0159] In another aspect, the present specification also provides methods for preparing the immunogenic peptides described herein. In certain embodiments, the method for preparing the immunogenic peptides comprises 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.

[0160] Methods for producing the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are known to those of skill in the art, e.g., by chemical synthesis, by purification from biological sources, or by recombinant expression techniques, including, e.g., from mammalian cells or transgenic preparations. The methods described herein employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the art. These techniques are described, for example, in the references cited herein and are explained more fully in the literature.See, for example, Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annually updated); Current Protocols in Immunology, John Wiley & Sons (1987 and annually updated); Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren et al. (ed.) (1999) Genome Analysis: A Laboratory See Manual, Cold Spring Harbor Laboratory Press.

[0161] Various methods exist in the art for producing the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein. For example, anti-LGALS3 antibodies or antigen-binding fragments thereof can be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. One or more DNAs encoding the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies, or such chains from human, humanized, or other sources). Once isolated, the DNA can be placed into an expression vector and then transformed into host cells that do not otherwise produce immunoglobulin proteins, such as NSO cells, monkey COS cells, Chinese hamster ovary (CHO) cells, yeast cells, algae cells, eggs, or myeloma cells, to obtain the synthesis of the anti-LGALS3 antibody or antigen-binding fragment thereof in the recombinant host cells. The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains of the desired species in place of the homologous human sequences (U.S. Pat. No. 4,816,567, Morrison et al., supra), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide can be substituted for the constant domain of an anti-LGALS3 antibody or antigen-binding fragment thereof provided herein. In certain embodiments, DNA encoding the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein can also be prepared using a polynucleotide encoding at least one anti-LGALS3 antibody or antigen-binding fragment thereof to provide a transgenic animal or mammal, such as a goat, cow, horse, sheep, etc., that produces such an antibody in its milk. Such animals can be provided using known methods.See, for example, but not by way of limitation, U.S. Patent Nos. 5,827,690, 5,849,992, 4,873,316, 5,849,992, 5,994,616, 5,565,362, and 5,304,489, each of which is incorporated herein by reference in its entirety.

[0162] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein can be additionally prepared using a polynucleotide encoding at least one anti-LGALS3 antibody or antigen-binding fragment thereof provided herein to provide transgenic plants and cultured plant cells (e.g., but not limited to, tobacco and corn), plant parts, or specific portions or variants in cells cultured therefrom that produce such antibodies. As a non-limiting example, transgenic tobacco leaves expressing recombinant proteins have been successfully used to provide large amounts of recombinant protein, for example, using an inducible promoter. See, e.g., Cramer et al. (1999) Curr. Top. Microbol. Immunol. 240:95-118 (1999) and references cited therein. Transgenic corn has also been used to express mammalian proteins at commercial production levels with biological activity equivalent to that produced in other recombinant systems or purified from natural sources. See, e.g., Hood et al. (1999) Adv. Exp. Med. Biol. 464:127-147, and references cited therein. Antibodies have also been produced in large quantities from transgenic plant seeds containing antibody fragments, e.g., scFvs, including tobacco seeds and potato tubers. See, e.g., Conrad et al. (1998) Plant Mol. Biol. 38:101-109, and references cited therein. Thus, transgenic plants can also be used to produce anti-LGALS3 antibodies or antigen-binding fragments thereof, according to known methods. See also, e.g., 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 references cited therein.Each of the above references is incorporated herein by reference in its entirety.

[0163] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein can be prepared using a polynucleotide encoding at least one anti-LGALS3 antibody or antigen-binding fragment thereof provided herein, and bacteria producing such anti-LGALS3 antibodies or antigen-binding fragments can be provided. As a non-limiting example, recombinant protein-expressing E. coli has been successfully used to provide large amounts of recombinant protein. See, e.g., Verma et al. (1998) 216(1-2):165-181 and references cited therein.

[0164] Methods for making multispecific (e.g., bispecific) antibodies have been described, see, e.g., 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.

[0165] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein are used to generate bispecific antibodies. Bispecific antibodies can be produced by fusing two hybridomas to create a hybrid immunoglobulin molecule with two binding sites. Bispecific antibodies not only restrict tumor activity to T cells, but also crosslink CD3 on T cells, initiating an activation cascade. In this way, T cell receptor-based cytotoxicity is redirected to the desired tumor target, bypassing MHC restriction. Arming polyclonal activated T cells (ATCs) with anti-CD3-anti-LGALS3 bispecific binding molecules combines the targeting specificity of anti-LGALS3 antibodies with the non-MHC-restricted perforin / granzyme-mediated cytotoxicity of T cells. The bispecific binding molecules, BsAb or BiTE, can arm activated T cells expanded ex vivo before infusion into patients. This strategy converts any ATC into a specific CTL (Thakur and Lum (2010) Curr Opin Mol Ther 12, 340-349; Grabert et al. (2006) Clin Cancer Res 12, 569-576).

[0166] The bispecific binding molecule can be composed of an anti-LGALS3 antibody, where the anti-LGALS3 antibody is an immunoglobulin and each light chain of the immunoglobulin is a fusion protein, where the fusion protein is an immunoglobulin light chain linked via a peptide linker to an scFv that targets CD3, and where the N297A mutation in the CH2 domain results in glycosylation that does not result in FcR or Clq binding.

[0167] The anti-LGALS3 antibodies or antigen-binding fragments thereof provided herein can be used to generate CARs. CARs are most commonly composed of a single-chain variable fragment length antibody (scFv), such as one derived from a monoclonal antibody targeting a given tumor-associated antigen and / or variants thereof, a transmembrane domain (e.g., a transmembrane domain derived from a T cell surface molecule such as a costimulatory molecule, such as CD8, CD28, OX-40, and 4-1BB), a signaling portion of the TCR complex, such as the intracellular domain of the TCR zeta (ζ) chain, and / or additional portions, such as its cytoplasmic signaling domain.

[0168] In a specific embodiment, the heavy and light chain variable regions of the monoclonal anti-LGALS3 antibodies 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. H and V L The chain variable regions are cloned by standard PCR using primers specific for such variable regions. H and V L The fragment is subcloned into a shuttle vector, such as the TopoTA PCR 2.1 cloning vector (Invitrogen), and sequenced. H and V LThe fragment was then ligated to a (Gly4Ser)3 spacer domain (SEQ ID NO: 56) to generate the anti-LGALS3 antibody scFv, which was fused to the human CD8 leader peptide (CD8L) by overlap PCR (CD8L-anti-LGALS3 antibody scFv) (see, e.g., Maher et al. (2002) Nat Biotechnol 20(1):70-5 and Gong et al. (1999) Neoplasia 1(2):123-7). The coding region for the CD8L-anti-LGALS3 antibody scFv is fused to the human CD8 hinge and transmembrane domain, or alternatively, to the CD28 transmembrane and cytoplasmic signaling domains and to the T cell receptor 0)3-zeta signaling domain (see, e.g., Maher et al. (2002) Nat Biotechnol 20(1):70-5, Brentjens et al. (2003) Nat Med 9(3):279-86, and Brentjens et al. (2007) Clin Cancer Res 13(18Pt 1):5426-35).

[0169] Also provided herein are T cells expressing the CARs described herein. Methods for generating T cells expressing CARs are known in the art. For example, a CAR construct can be subcloned into a 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-based vector. VSV-G pseudotyped retroviral supernatant from transduced gpg29 fibroblasts can be used to construct a cell line producing stable PG13 gibbon ape leukemia virus (GaLV) envelope pseudotyped retrovirus (see, e.g., Gong et al. (1999) Neoplasia 1(2):123-7). Isolated peripheral blood mononuclear cells (PBMCs) from healthy donors can be activated with 2 μg / ml phytohemagglutinin (PHA) (Sigma, St. Louis, MO) and retrovirally transduced on retronectin-coated non-tissue culture plates (Quintas-Cardama A, et al. (2007) Hum Gene Ther 18(12):1253-60) to generate T cells recombinantly expressing the CAR. Gene transfer of the CAR into T cells can be assessed by FACS.

[0170] Single domain antibodies, e.g., antibodies lacking light chains, can be generated by methods well known in the art (see, e.g., 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 WO 94 / 04678, WO 94 / 25591, and WO 01 / 44301, each of which is incorporated by reference in its entirety.

[0171] In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein bind to the same epitope or an overlapping epitope as the anti-LGALS3 antibodies described herein and are human anti-LGALS3 antibodies or antigen-binding fragments thereof. In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein competitively block any of the antibodies described herein (e.g., in a dose-dependent manner) from binding to LGALS3 and are human anti-LGALS3 antibodies or antigen-binding fragments thereof.

[0172] Human antibodies can be produced using any method known in the art. For example, transgenic mice incapable of expressing functional endogenous immunoglobulins but capable of expressing human immunoglobulin genes can be used. In particular, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells in addition to human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered nonfunctional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to generate homozygous offspring that express human antibodies. The transgenic mice are immunized in the usual manner with a selected antigen, e.g., all or a portion of the antigen. Monoclonal antibodies directed against an antigen can be obtained from immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such technology, it is possible to generate therapeutically useful IgG, IgA, IgM, and IgE antibodies. For an overview of this technology for producing human antibodies, see, e.g., Lonberg and Huszar (1995) Int Rev Immunol 13:65-93. For a detailed description of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735, and U.S. Pat. Nos. 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 the Xenomouse™ (Abgenix, Inc., U.S. Pat. Nos. 6,075,181 and 6,150,184), HuAb-Mouse™ (Mederex, Inc. / Gen Pharm, U.S. Pat. Nos. 5,545,806 and 5,569,825), Trans Chromo Mouse™ (Kirin), and KM Mouse™ (Medarex / Kirin).

[0173] Human antibodies that specifically bind to LGALS3 can be produced by various methods known in the art, including the phage display method described above, using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887, 4,716,111, and 5,885,793, and International Publication Nos. WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741.

[0174] In some embodiments, human antibodies can be produced using mouse-human hybridomas. For example, Epstein-Barr virus (EBV)-transformed human peripheral blood lymphocytes can be fused with mouse myeloma cells to generate mouse-human hybridomas that secrete human monoclonal antibodies, and these mouse-human hybridomas can be screened to determine those that secrete human monoclonal antibodies that specifically bind to the target antigen. 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.

[0175] Exemplary methods for generating antibodies or antigen-binding fragments thereof that specifically bind to LGALS3, as well as methods for screening and selecting antibodies or antigen-binding fragments thereof that specifically bind to LGALS3, are described in Example 2. In exemplary embodiments, monoclonal antibodies secreted by hybridomas are screened for binding to the LGALS3 carbohydrate-binding domain peptide (CBD), the full-length LGALS3 protein. In some embodiments, the LGALS3 N-terminus, which contains the polymerization domain, is used as a control. In some embodiments, monoclonal antibodies are further screened against human Gal3 (or other members of the galectin family, e.g., LGAL1, LGAL7, LGAL8, and LGAL9). In some embodiments, monoclonal antibodies are further screened using a functional assay for inhibition of binding of human Gal-3 to suitable cancer cells.

[0176] Once the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are produced, they can be purified by any method known in the art for the purification of immunoglobulin molecules, such as, for example, chromatography (e.g., by ion exchange, affinity, particularly for specific antigens followed by Protein A and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for the purification of proteins. Additionally, to facilitate purification, the antibodies described herein can be fused to heterologous polypeptide sequences described herein or known in the art.

[0177] In specific embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are isolated or purified. Generally, an isolated antibody is one that is substantially free of other antibodies having antigenic specificities different from that of the isolated antibody. For example, in certain embodiments, preparations of anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are substantially free of cellular material and / or chemical precursors. The language "substantially free of cellular material" includes preparations of anti-LGALS3 antibodies or antigen-binding fragments thereof in which the antibodies are separated from cellular components of the cells from which they are isolated or recombinantly produced. Thus, an anti-LGALS3 antibody or antigen-binding fragment thereof that is substantially free of cellular material includes preparations of antibodies that have less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins") and / or variants of the anti-LGALS3 antibody or antigen-binding fragment thereof, e.g., differently post-translationally modified forms of the anti-LGALS3 antibody or antigen-binding fragment thereof, or other different aspects of the anti-LGALS3 antibody or antigen-binding fragment thereof (e.g., antibody fragments). When the antibody is recombinantly produced, it also generally is substantially free of culture medium, i.e., culture medium represents 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 generally is substantially free of chemical precursors or other chemicals, i.e., separated from chemical precursors or other chemicals involved in the synthesis of the protein. Accordingly, such preparations of antibodies have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In specific embodiments, the antibodies described herein are isolated or purified.

[0178] Polynucleotides In certain embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-LGALS3 antibody or antigen-binding fragment thereof. Also provided herein are vectors comprising such polynucleotides. Also provided herein are polynucleotides encoding antigens of the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein. Also provided herein are polynucleotides that hybridize to polynucleotides encoding the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein under stringent or less stringent hybridization conditions.

[0179] The language "purified" includes preparations of polynucleotides or nucleic acid molecules that have less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (particularly less than 10%) of other materials, e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals. In specific embodiments, nucleic acid molecules encoding anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are isolated or purified.

[0180] The nucleic acid molecules provided herein may be in the form of RNA, such as mRNA, hnRNA, tRNA, or any other form, or in the form of DNA, including but not limited to cDNA and genomic DNA obtained by cloning or produced synthetically, or any combination thereof. The DNA may be triple-stranded, double-stranded, or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA may be the coding strand, also known as the sense strand, or the non-coding strand, also referred to as the antisense strand.

[0181] In certain embodiments, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-LGALS3 antibody or antigen-binding fragment thereof described herein. Also provided herein in certain aspects are polynucleotides comprising a nucleotide sequence encoding an anti-LGALS3 antibody or antigen-binding fragment thereof, which specifically binds to LGALS3 and comprises an amino acid sequence described herein, as well as an antibody that competes with such an anti-LGALS3 antibody or antigen-binding fragment thereof for binding to LGALS3 or binds to the same epitope as such an antibody.

[0182] The polynucleotides provided herein can be obtained by any method known in the art. For example, if the nucleotide sequence encoding an anti-LGALS3 antibody or antigen-binding fragment thereof described herein is known, a polynucleotide encoding the anti-LGALS3 antibody or antigen-binding fragment thereof can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al. (1994) BioTechniques 17:242), which briefly involves synthesizing overlapping oligonucleotides containing portions of the antibody-encoding sequence, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0183] Alternatively, polynucleotides encoding anti-LGALS3 antibodies or antigen-binding fragments thereof can be generated from nucleic acid from a suitable source. If a clone containing nucleic acid encoding a particular anti-LGALS3 antibody or antigen-binding fragment thereof is not available but the sequence of the anti-LGALS3 antibody or antigen-binding fragment thereof is known, nucleic acid encoding the anti-LGALS3 antibody or antigen-binding fragment thereof can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated therefrom, or nucleic acid, preferably polyA+ RNA isolated therefrom, any tissue or cell that expresses an antibody, e.g., hybridoma cells selected to express the anti-LGALS3 antibody or antigen-binding fragment thereof provided herein) by PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for a particular gene sequence to identify cDNA clones from a cDNA library encoding the antibody. Amplified nucleic acids generated by PCR can then be cloned into a replicable cloning vector using any method known in the art. In such embodiments, polynucleotides encoding such anti-LGALS3 antibodies or antigen-binding fragments thereof can be manipulated using methods well known in the art for the manipulation of nucleotide sequences, such as recombinant DNA technology, site-directed mutagenesis, PCR, etc. (e.g., the techniques described in Sambrook et al. (19900 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 which are incorporated by reference in their entireties) to generate anti-LGALS3 antibodies or antigen-binding fragments thereof having different amino acid sequences, e.g., to create amino acid substitutions, deletions, and / or insertions.For example, such manipulations can be performed to deglycosylate the encoded amino acids, or to destroy the ability of the antibody to bind to Clq, Fc receptors, or to activate the complement system.

[0184] The isolated nucleic acid molecules provided herein may include nucleic acid molecules comprising an open reading frame (ORF), optionally with one or more introns, including, but not limited to, at least one identified portion of at least one complementarity determining region (CDR), such as CDR1, CDR2 and / or CDR3 of at least one heavy or light chain; nucleic acid molecules comprising a coding sequence or variable region for an anti-LGALS3 antibody; and nucleic acid molecules that are substantially different from those described above but that, due to the degeneracy of the genetic code, still comprise a nucleotide sequence encoding at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein.

[0185] Also provided herein are isolated nucleic acids that hybridize under selective hybridization conditions to the polynucleotides described herein. Thus, the polynucleotides of this embodiment can be used to isolate, detect, and / or quantify nucleic acids that contain such polynucleotides. For example, the polynucleotides provided herein can be used to identify, isolate, or amplify partial-length or full-length clones in a deposited library. In some embodiments, the polynucleotides are genomic or cDNA sequences isolated from a human or mammalian nucleic acid library or otherwise complementary to cDNAs from a human or mammalian nucleic acid library.

[0186] The nucleic acids may advantageously contain sequences in addition to the polynucleotides 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 polynucleotides. In addition, a translatable sequence can be inserted to aid in the isolation of the translated polynucleotides provided herein. For example, a hexa-histidine marker sequence (SEQ ID NO: 57) provides a convenient means for purifying the polypeptides provided herein. The nucleic acids provided herein (excluding coding sequences) are optionally vectors, adapters, or linkers for cloning and / or expression of the polynucleotides provided herein.

[0187] Additional sequences can also be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to aid in the isolation of polynucleotides, or to improve the introduction of polynucleotides into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra, or Sambrook, supra.)

[0188] In specific embodiments, one or more of the CDRs of an anti-LGALS3 antibody or antigen-binding fragment thereof described herein can be inserted into a known framework region using conventional recombinant DNA techniques. The framework region can be a naturally occurring or consensus framework region, preferably a human framework region (e.g., see Chothia et al., (1998) J. Mol. Biol. 278:457-479 for a list of human framework regions). In some embodiments, the polynucleotide generated by combining the framework regions and CDRs encodes an anti-LGALS3 antibody or antigen-binding fragment thereof that specifically binds to LGALS3. One or more amino acid substitutions may be made within the framework region, and preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods can be used to make amino acid substitutions or deletions of one or more variable region cysteine ​​residues involved in intrachain disulfide bonds to generate antibody molecules lacking one or more intrachain disulfide bonds. Other modifications to the polynucleotide are provided herein and within the skill of the art.

[0189] In certain embodiments, the isolated or purified nucleic acid molecule, or a fragment thereof, when linked to another nucleic acid molecule, can encode a fusion protein. The generation of fusion proteins is within the skill of one in the art and may involve the use of restriction enzymes or recombinant cloning techniques (see, e.g., Gateway™ (Invitrogen)). See also U.S. Patent No. 5,314,995.

[0190] In certain embodiments, the polynucleotides provided herein are in the form of a vector (e.g., an expression vector). In certain aspects, provided herein are polynucleotides comprising an anti-LGALS3 antibody or antigen-binding fragment thereof described herein, or an antigen-binding fragment thereof that specifically binds to LGALS3 (e.g., a variable light chain region and / or a variable heavy chain region), as well as vectors, e.g., vectors comprising such polynucleotides, for their efficient expression in host cells (e.g., E. coli and mammalian cells). In some embodiments, the polynucleotides are isolated or purified.

[0191] In certain aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding an anti-LGALS3 antibody or antigen-binding fragment thereof, including an amino acid sequence described herein, and an antibody that competes with such an antibody (e.g., in a dose-dependent manner) for binding to LGALS3 or binds to the same or overlapping epitope as such an antibody.

[0192] In certain aspects, provided herein are polynucleotides comprising a nucleic acid sequence encoding the light chain or heavy chain of an anti-LGALS3 antibody or antigen-binding fragment thereof described herein. The polynucleotides may be any of the V and V sequences described herein. H The polynucleotide may comprise a nucleotide sequence encoding a heavy chain comprising the CDRs (see, e.g., Table 6). L It may include a nucleotide sequence encoding a light chain comprising the CDRs (see, for example, Table 6).

[0193] In certain embodiments, provided herein are polynucleotides comprising three V H CDRs, e.g., V as set forth in Table 6 H CDR1, V H CDR2 and V HPolynucleotides are provided that include a nucleotide sequence encoding an anti-LGALS3 antibody containing CDR3, wherein the antibody specifically binds to LGALS3. In specific embodiments, the polynucleotides described herein include the V and V sequences of the 46H02 antibody (i.e., SEQ ID NOs: 7, 8, and 9, respectively), the 12H07 antibody (i.e., SEQ ID NOs: 19, 20, and 21, respectively), the 20F08 antibody (i.e., SEQ ID NOs: 29, 30, and 31, respectively), the 38E05 antibody (i.e., SEQ ID NOs: 39, 40, and 41, respectively), or the 39F02 antibody (i.e., SEQ ID NOs: 49, 50, and 51, respectively). H CDR1, V H CDR2 and V H In certain embodiments, the polynucleotides described herein comprise a nucleotide sequence encoding an anti-LGALS3 antibody described herein, comprising a heavy chain variable region comprising an amino acid sequence described herein (e.g., SEQ ID NO: 6, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38, or SEQ ID NO: 48), wherein the antibody specifically binds to LGALS3.

[0194] In certain embodiments, provided herein are polynucleotides comprising three V L CDRs, e.g., V as set forth in Table 6 L CDR1, V L CDR2 and V LPolynucleotides are provided that include a nucleotide sequence encoding an anti-LGALS3 antibody containing CDR3, wherein the antibody specifically binds to LGALS3. In specific embodiments, the polynucleotides described herein include the V and V sequences of the 46H02 antibody (i.e., SEQ ID NOs: 2, 3, and 4, respectively), the 12H07 antibody (i.e., SEQ ID NOs: 14, 15, and 16, respectively), the 20F08 antibody (i.e., SEQ ID NOs: 24, 25, and 26, respectively), the 38E05 antibody (i.e., SEQ ID NOs: 34, 35, and 36, respectively), or the 39F02 antibody (i.e., SEQ ID NOs: 44, 45, and 46, respectively). L CDR1, V L CDR2 and V L In certain embodiments, the polynucleotides described herein comprise a nucleotide sequence encoding an anti-LGALS3 antibody described herein, comprising a light chain variable region comprising an amino acid sequence described herein (e.g., SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 33, or SEQ ID NO: 43), wherein the antibody specifically binds to LGALS3.

[0195] In certain embodiments, the polynucleotides described herein comprise a nucleotide sequence encoding an anti-LGALS3 antibody described herein comprising a heavy chain variable region comprising an amino acid sequence described herein (e.g., SEQ ID NO: 6, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38, or SEQ ID NO: 48) and a light chain variable region comprising an amino acid sequence described herein (e.g., SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 33, or SEQ ID NO: 43), wherein the antibody specifically binds to LGALS3.

[0196] In specific aspects, provided herein are polynucleotides comprising nucleotide sequences encoding an anti-LGALS3 antibody or antigen-binding fragment thereof comprising a light chain and a heavy chain, e.g., a separate light chain and heavy chain. With respect to the heavy chain, in specific embodiments, the polynucleotides provided herein comprise 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 polynucleotides provided herein comprise a nucleotide sequence encoding a kappa (κ) or lambda (λ) light chain.

[0197] Cells and Vectors In certain embodiments, provided herein are cells (e.g., isolated cells or ex vivo cells) that (e.g., recombinantly) express one or more anti-LGALS3 antibodies or antigen-binding fragments thereof. Also provided herein are vectors (e.g., expression vectors) that include nucleotide sequences encoding the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein for recombinant expression in a host cell, preferably a mammalian cell. Also provided herein are cells (e.g., isolated cells or ex vivo cells) that include such vectors or nucleotide sequences for recombinantly expressing the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein. Also provided herein are methods for producing the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein, comprising expressing such anti-LGALS3 antibodies or antigen-binding fragments thereof from cells (e.g., isolated cells or ex vivo cells).

[0198] A vector (e.g., an expression vector) is a DNA molecule that contains a gene to be expressed in a cell (e.g., an ex vivo cell). Typically, gene expression is placed under the control of specific regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. Such a gene is said to be "operably linked" to a regulatory element, e.g., a promoter. A recombinant host can be any prokaryotic or eukaryotic cell that contains either a cloning vector or an expression vector. The term also includes prokaryotic or eukaryotic cells and transgenic animals that have been genetically engineered to contain a cloned gene in the chromosome or genome of the host cell or in the cells of the host cell (e.g., an ex vivo cell). In one embodiment, the promoter is a CMV promoter.

[0199] In certain embodiments, provided herein are vectors comprising one or more polynucleotides described herein. In certain embodiments, the polynucleotides described herein can be cloned into a suitable vector and used to transform or transfect a suitable host. Vectors and methods for constructing such vectors are known to those of skill in the art and are described in general technical reference works (see generally, "Recombinant DNA Part D," Methods in Enzymology, Vol. 153, Wu and Grossman, eds., Academic Press (1987)). In certain embodiments, the vector contains regulatory sequences, e.g., transcription and translation initiation and termination codons, that are specific to the type of host into which the vector will be introduced (e.g., bacteria, fungi, plants, insects, or mammals), taking into account whether the vector is DNA or RNA, as appropriate. In certain embodiments, the vector contains regulatory sequences that are specific to the genus of the host. In certain embodiments, the vector contains regulatory sequences that are specific to the species of the host. In certain embodiments, the vector contains one or more marker genes that allow for the selection of transformed or transfected hosts. Non-limiting examples of marker genes include biocide resistance, resistance to, e.g., antibiotics, heavy metals, etc., complementation in auxotrophic hosts to provide prototrophy, etc. In certain embodiments, the vector contains ampicillin and hygromycin selectable markers.

[0200] In certain embodiments, the expression vector may comprise a native promoter or a canonical promoter operably linked to the polynucleotide described herein. For example, the selection of strong, weak, inducible, tissue-specific, and development-specific promoters is within the skill of one in the art. Similarly, as described above, combining a nucleic acid molecule or a fragment thereof with a promoter is also within the skill of the art.

[0201] Non-limiting examples of suitable vectors include those designed for propagation or propagation, or for 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-GT11, lambda-ZapII (Stratagene), lambda-EMBL4, and lambda-NM1149, can also be used. Non-limiting examples of plant expression vectors include pBI110, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Non-limiting examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The TOPO cloning system (Invitrogen, Carlsbad, Calif.) can also be used according to the manufacturer's recommendations.

[0202] In certain embodiments, the vector is a mammalian vector. In certain embodiments, the mammalian vector contains at least one promoter element that mediates the initiation of transcription of the sequence encoding the mRNA, anti-LGALS3 antibody, or antigen-binding fragment thereof, and signals necessary for the termination of transcription and polyadenylation of the transcript. In certain embodiments, the mammalian vector contains additional elements, such as an enhancer flanking donor and acceptor sites for RNA splicing, a Kozak sequence, and an intervening sequence. In certain embodiments, highly efficient transcription can be achieved using, for example, the early and late promoters from SV40, the long terminal repeats (LTRS) from retroviruses such as RSV, HTLV-1, and HIV-1, and the early promoter of cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter). Non-limiting examples of mammalian expression vectors include vectors such as 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, Cos1, Cos7, and CV1, quail QC1-3 cells, mouse L cells, and Chinese hamster ovary (CHO) cells.

[0203] In certain embodiments, the vector is a viral vector, such as a retroviral vector, a parvovirus-based vector, such as an adeno-associated virus (AAV)-based vector, an AAV-adenovirus chimeric vector, or an adenovirus-based vector, or a lentiviral vector, such as a herpes simplex (HSV)-based vector. In certain embodiments, the viral vector is engineered to be defective in viral replication. In certain embodiments, the viral vector is engineered to eliminate toxicity to the host. These viral vectors can be prepared using standard recombinant DNA techniques, as described, for example, in Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd 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).

[0204] In certain embodiments, the vectors or polynucleotides described herein can be transferred 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 antibodies or antigen-binding fragments thereof described herein. Accordingly, provided herein are cells comprising a polynucleotide encoding an anti-LGALS3 antibody or antigen-binding fragment thereof, its heavy chain or light chain, or its light chain fusion polypeptide operably linked to a promoter for expression of such sequences in a host cell. In certain embodiments, a vector encoding a heavy chain operably linked to a promoter and a vector encoding a light chain operably linked to a promoter can be co-expressed in a cell for expression of the entire anti-LGALS3 antibody. In certain embodiments, a vector encoding a heavy chain operably linked to a promoter and a vector encoding a light chain fusion polypeptide operably linked to a promoter can be co-expressed in a cell for expression of the entire bispecific binding molecule. In certain embodiments, a cell comprises a vector comprising a polynucleotide encoding both the heavy and light chain polypeptides of an anti-LGALS3 antibody described herein operably linked to a promoter. In certain embodiments, the cell contains a vector comprising a polynucleotide encoding both the heavy chain and the light chain fusion polypeptide of a bispecific binding molecule described herein operably linked to a promoter. In certain embodiments, the cell contains two different vectors: a first vector comprising a polynucleotide encoding the heavy chain operably linked to a promoter, and a second vector comprising a polynucleotide encoding the light chain polypeptide operably linked to a promoter. In certain embodiments, the cell contains two different vectors: a first vector comprising a polynucleotide encoding the heavy chain operably linked to a promoter, and a second vector comprising a polynucleotide encoding the light chain fusion polypeptide operably linked to a promoter.In certain embodiments, a first cell comprises a first vector comprising a polynucleotide encoding the heavy chain of an anti-LGALS3 antibody described herein, and a second cell comprises a second vector comprising a polynucleotide encoding the light chain polypeptide of an anti-LGALS3 antibody described herein. In certain embodiments, provided herein is a mixture of cells comprising such a first cell and such a second cell. In certain embodiments, the first cell comprises a first vector comprising a polynucleotide encoding the heavy chain of a bispecific binding molecule described herein, and the second cell comprises a second vector comprising a polynucleotide encoding the light chain fusion polypeptide of a bispecific binding molecule described herein. In certain embodiments, provided herein is a mixture of cells comprising such a first cell and such a second cell. In certain embodiments, the cell expresses the vector or vectors such that the polynucleotides are efficiently transcribed and translated by the cell.

[0205] In some embodiments, the cell expresses the vector, such that the polynucleotide, or a fragment thereof, is both transcribed and efficiently translated by the cell.

[0206] In certain embodiments, the cell is present in a host, which may be an animal, e.g., a mammal. Exemplary cells include, but are not limited to, human cells, human cell lines, E. coli (e.g., E. coli TB-1, TG-2, DH5a, XL-Blue MRF (Stratagene), SA2821, and Y1090), B. subtilis, P. aeruginosa, S. cerevisiae, N. crassa, insect cells (e.g., Sf9, Ea4), and others described below. In certain embodiments, the cell is a CHO cell. In another specific embodiment, the cell is a CHO-S cell.

[0207] In certain embodiments, the polynucleotides described herein can be expressed in stable cell lines containing the polynucleotide integrated into a chromosome by introducing the polynucleotide into cells. In certain embodiments, the polynucleotide is introduced into cells, for example, by electroporation. In certain embodiments, the polynucleotide is introduced into cells, for example, by transfecting a vector containing the polynucleotide into the cells. In certain embodiments, the vector is co-transfected with a selectable marker, such as DHFR, GPT, neomycin, or hygromycin, to allow for identification and isolation of transfected cells. In certain embodiments, the transfected polynucleotide can be amplified to express large amounts of the encoded anti-LGALS3 antibody or antigen-binding fragment thereof. For example, a DHFR (dihydrofolate reductase) marker can be used to develop cell lines carrying hundreds or even thousands of copies of a polynucleotide of interest. Another example of a selectable 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 selective medium and the cells with the highest resistance are selected. These cell lines contain an amplified gene integrated into the chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for antibody production.

[0208] In some embodiments, the vector comprises (i) a first polynucleotide sequence encoding an immunoglobulin light chain that binds to LGALS3 operably linked to a first promoter, and (ii) a second polynucleotide sequence encoding an immunoglobulin heavy chain that binds to LGALS3 operably linked to a second promoter. In certain embodiments, the vector is a viral vector.

[0209] In some embodiments, the vector comprises (i) a first polynucleotide sequence encoding a light chain fusion polypeptide comprising an immunoglobulin light chain fused to an scFv via a peptide linker, wherein the light chain binds to LGALS3 and the scFv binds CD3, operably linked to a first promoter, and (ii) a second polynucleotide encoding an immunoglobulin heavy chain that binds LGALS3, operably linked to a second promoter. In certain embodiments, the vector is a viral vector.

[0210] Pharmaceutical Compositions In certain embodiments, provided herein are compositions (e.g., pharmaceutical compositions) and kits comprising a pharmaceutically effective amount of one or more anti-LGALS3 antibodies or antigen-binding fragments thereof. In certain embodiments, the pharmaceutical compositions comprise immune cells, e.g., T cells, recombinantly expressing an antibody, antigen-binding fragment thereof, and / or CAR described herein. The compositions can be used to prepare individual single-unit dosage forms. The compositions provided herein can be formulated for parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intrachondral, intracavitary, intracavity, intracerebral, intraventricular, intraocular, intravitreal, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, intrathecal, intraventricular, intraparenchymal, or transdermal administration.

[0211] In certain embodiments, provided herein are compositions comprising one or more polynucleotides comprising a nucleotide sequence encoding an anti-LGALS3 antibody or antigen-binding fragment thereof described herein. In certain embodiments, provided herein are compositions comprising cells, wherein the cells comprise one or more polynucleotides comprising a nucleotide sequence encoding an anti-LGALS3 antibody or antigen-binding fragment thereof described herein. In certain embodiments, provided herein are compositions comprising vectors, wherein the vectors comprise one or more polynucleotides comprising a nucleotide sequence encoding an anti-LGALS3 antibody or antigen-binding fragment thereof described herein. In certain embodiments, provided herein are compositions comprising cells, wherein the cells comprise vectors, wherein the vectors comprise one or more polynucleotides comprising a nucleotide sequence encoding an anti-LGALS3 antibody or antigen-binding fragment thereof described herein.

[0212] In certain embodiments, the compositions described herein are stable or preserved formulations. In certain embodiments, stable formulations include saline or a phosphate buffer with a selected salt. In certain embodiments, the compositions described are versatile, preserved formulations suitable for pharmaceutical or veterinary use. In certain embodiments, the compositions described herein include a preservative. 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, phenylmercuric nitrate, 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 an aqueous diluent. Any suitable concentration or mixture, for example, 0.001-5%, or any range or value therein, for example, but not limited to, 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, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21, 2.22, 2.23, 2.24, 2.25, 2.26, 2.27, 2.28, 2.29, 2.30, 2.31, 2.32, 2.33, 2.34, 2.35, 2.36, 2.37, 2.38, 2.39, 2.40, 2.41, 2.42, 2.43, 2.44, 2.45, 2.46, 2.47, 2.48, 2.49, 2.50, 2.51, 2.52, 2.53, 2.54, 2.5 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 therein, can be used as known in the art.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%), 0.001-2.0% phenol (e.g., Examples include 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005 to 1.0% alkylparabens (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.

[0213] It may be desirable to deliver the compositions provided herein to a subject over an extended period of time, e.g., from one week to a year or more following a single administration. A variety of sustained-release, depot, or implant dosage forms are available. For example, the dosage form may contain a pharmaceutically acceptable, non-toxic salt of a compound having low solubility in body fluids, such as (a) an acid addition salt with a polybasic acid, such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or disulfonic acid, or polygalacturonic acid; (b) a salt with a polyvalent metal ion, such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, or cadmium, or an organic cation, such as formed from N,N'-dibenzyl-ethylenediamine or ethylenediamine; or (c) a combination of (a) and (b), e.g., zinc tannate. In addition, the compositions provided herein, preferably the relatively insoluble salts described immediately above, can be formulated in gels suitable for injection, such as sesame oil-containing gels, such as aluminum monostearate gels. Particularly specific salts include zinc salts, zinc tannate salts, and pamoate salts. Another type of sustained-release depot formulation for injection can contain the compound or salt dispersed for encapsulation in a slowly degrading, nontoxic, and nonantigenic polymer, such as polylactic acid / polyglycolic acid polymer, as described, for example, in U.S. Pat. No. 3,773,919. The compound, or preferably the relatively insoluble salts described above, can also be formulated in cholesterol matrix silastic pellets, particularly for use in animals. Additional sustained-release, depot, or implant compositions, such as gas or liquid liposomes, are known in the literature (U.S. Pat. No. 5,770,222 and "Sustained and Controlled Release Drug Delivery Systems," J.R. Robinson ed., Marcel Dekker, Inc., NY, 1978).

[0214] Compositions of at least one anti-LGALS3 antibody or antigen-binding fragment thereof provided herein range from about 1.0 micrograms / ml to about 1000 mg / ml when in a wet / dry system, but lower and higher concentrations are also operable and depend on the intended delivery vehicle; for example, a solution formulation will differ from a transdermal patch, pulmonary, transmucosal, or osmotic or micropump method.

[0215] In certain embodiments, the compositions provided herein comprise any suitable auxiliary substance, such as, but not limited to, at least one of a diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, etc. In certain embodiments, the pharmaceutically acceptable auxiliary substance is specified. Non-limiting examples of such sterile solutions and methods for preparing them are well known in the art, such as, but not limited to, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990.

[0216] Pharmaceutically acceptable carriers suitable for the mode of administration, solubility, and / or stability of the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein can be routinely selected.

[0217] In certain embodiments, the compositions provided herein contain one or more pharmaceutical excipients and / or additives. Non-limiting examples of pharmaceutical excipients and additives include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars, and polysaccharides or sugar polymers), which may be present alone or in combination and comprise 1 to 99.99% by weight or volume, alone or in combination. Non-limiting examples of protein excipients include serum albumins, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Non-limiting examples of amino acids / antibody components that may also function in a 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 such as fructose, maltose, galactose, glucose, D-mannose, and sorbose; disaccharides such as lactose, sucrose, trehalose, and cellobiose; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, and starch; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), and myo-inositol. In certain embodiments, the carbohydrate excipient is mannitol, trehalose, or raffinose.

[0218] In certain embodiments, the compositions provided herein include one or more buffering agents or pH adjusting agents. Typically, the buffering agent is a salt prepared from an organic acid or base. Non-limiting examples of buffering agents include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris; tromethamine hydrochloride; or phosphate buffer. In certain embodiments, the buffering agent is an organic acid salt such as citrate. Other excipients, such as isotonicity agents, buffering agents, antioxidants, and preservative enhancers, may optionally, but preferably, be added to the diluent. Isotonicity agents such as glycerin are generally used at known concentrations. The addition of a physiologically tolerable buffering agent is preferred to provide improved pH control. The compositions can cover a wide range of pH, for example, from about pH 4 to about pH 10, with a specific range of from about pH 5 to about pH 9, and a most specific range of from about pH 6.0 to about pH 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, particularly phosphate buffered saline (PBS).

[0219] In certain embodiments, the compositions provided herein comprise one or more polymeric excipients / additives, such as polyvinylpyrrolidone, Ficoll (a polymeric sugar), dextrates (e.g., cyclodextrins, e.g., 2-hydroxypropyl-beta-cyclodextrin), polyethylene glycol, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "TWEEN 20" and "TWEEN 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and / or chelating agents (e.g., EDTA).

[0220] Other additives, 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, pharmaceutically acceptable solubilizers, and chelating agents such as EDTA and EGTA, can optionally be added to the composition to reduce aggregation. These additives are particularly useful when a pump or plastic container is used to administer the composition. The presence of a pharmaceutically acceptable surfactant mitigates the tendency of proteins to aggregate.

[0221] Additional pharmaceutical excipients and / or additives suitable for use in the compositions provided herein are known to those skilled in the art and are referred to, for example, in "Remington: The Science & Practice of Pharmacy," 19th ed., Williams & Williams, (1995), and "Physician's Desk Reference," 52nd ed., Medical Economics, Montvale, NJ (1998), which are incorporated herein by reference in their entireties. In certain embodiments, the carrier or excipient material is a carbohydrate (e.g., sugar and alditol) and a buffer (e.g., citrate) or a polymeric agent.

[0222] In some embodiments, the aqueous diluent optionally further comprises a pharmaceutically acceptable preservative. Exemplary preservatives include those selected from the group consisting of 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 provide an antibacterial effect. Such a concentration depends on the preservative selected and can be easily determined by one skilled in the art.

[0223] The compositions provided herein can be prepared by a process comprising mixing at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein and a preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl parabens (e.g., methyl, ethyl, propyl, butyl), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or a mixture thereof, in an aqueous diluent. Mixing at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein and a preservative in an aqueous diluent is carried out using conventional dissolution and mixing procedures. To prepare a suitable composition, for example, a measured amount of at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein in a buffer solution is combined with the desired preservative in a buffer solution in an amount sufficient to provide the anti-LGALS3 antibody or antigen-binding fragment thereof described herein and the preservative at the desired concentration. The compositions provided herein can be prepared by a process comprising mixing at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein and a selected buffer, preferably a phosphate buffer containing a salt or a selected salt. Mixing at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein and a buffer in an aqueous diluent can be carried out using conventional dissolution and mixing procedures. To prepare a suitable composition, for example, a measured amount of at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein in water or buffer is combined with the desired buffer in water in an amount sufficient to provide the protein and buffer at the desired concentration. Variations on these processes will be recognized by those skilled in the art. For example, the order in which components are added, whether additional additives are used, and the temperature and pH at which the composition is prepared are all factors that can be optimized for the concentration and means of administration used.

[0224] Parenteral formulations 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 can be formulated as a solution, suspension, emulsion, or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle, or can be provided separately. Non-limiting examples of such vehicles include water, saline, Ringer's solution, dextrose solution, glycerol, ethanol, and 1-10% human serum albumin. Non-aqueous vehicles such as liposomes and fixed 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 and preservatives). The formulation is sterilized by known or suitable techniques.

[0225] Suitable pharmaceutical carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field.

[0226] Preparations for parenteral administration may contain, as common excipients, sterile water or physiological saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalenes, etc. Aqueous or oily suspensions for injection can be prepared according to known methods by using appropriate emulsifiers or wetting agents and suspending agents. Injectable preparations can be non-toxic parenterally administrable diluents, such as aqueous solutions, or sterile injection solutions or suspensions in solvents. Acceptable vehicles or solvents include water, Ringer's solution, isotonic saline, etc. Sterile fixed oils can be used as common solvents or suspension media. For these purposes, any type of fixed oil and fatty acid can be used, including natural, synthetic, or semi-synthetic aliphatic oils or fatty acids, and natural, synthetic, or semi-synthetic monoglycerides, diglycerides, or triglycerides. Parental administration is known in the art and includes, but is not limited to, conventional injection means, gas pressurized needleless injection devices such as those described in U.S. Pat. No. 5,851,198, and laser perforation devices such as those described in U.S. Pat. No. 5,839,446, which is incorporated herein by reference in its entirety.

[0227] Pulmonary preparations In certain embodiments, compositions comprising the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are formulated for pulmonary administration, where the compositions are delivered in a particle size effective to reach the lower airways or sinuses of the lung.

[0228] Compositions for pulmonary administration can be delivered by any of a variety of inhalation or nasal devices known in the art for administering therapeutic agents by inhalation. These devices, which can deposit an aerosolized formulation into a patient's sinuses or alveoli, include metered-dose inhalers, nebulizers, dry powder generators, sprayers, and the like. Other devices suitable for pulmonary or nasal administration of the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein are also known in the art. All such devices use formulations suitable for administration of the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein for aerosol dispensing. Such aerosols may be composed of either solutions (both aqueous and non-aqueous) or solid particles. Metered-dose inhalers, such as the Ventolin® metered-dose inhaler, typically use a propellant gas and require actuation during inhalation (see, e.g., WO 94 / 16970, WO 98 / 35888). Dry powder inhalers, such as Turbuhaler™ (Astra), Rotahaler® (Glaxo), Diskus® (Glaxo), and devices marketed by Inhale Therapeutics, to name a few, use breath actuation of a mixed powder (U.S. Pat. No. 4,668,218 Astra, EP 237507 Astra, WO 97 / 25086 Glaxo, WO 94 / 08552 Dura, U.S. Pat. No. 5,458,135 Inhale, WO 94 / 06498 Fisons, which are incorporated herein by reference in their entirety).

[0229] Nebulizers such as the Ultravent® nebulizer (Mallinckrodt) and the Acorn II® nebulizer (Marquest Medical Products) (U.S. Pat. No. 5,404,871 Aradigm, WO 97 / 22376) (the above references are incorporated herein by reference in their entirety) generate aerosols from solutions, while metered dose inhalers, dry powder inhalers, and the like generate aerosols of small particles. These examples of commercially available inhalation devices are non-limiting examples and are not intended to limit the scope.

[0230] In certain embodiments, a spray comprising an anti-LGALS3 antibody or antigen-binding fragment thereof described herein can be generated by forcing a suspension or solution of at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein through a nozzle under pressure. The nozzle size and configuration, applied pressure, and liquid feed rate can be selected to achieve a desired output and particle size. Electrosprays can be produced, for example, by an electric field in combination with a capillary or nozzle feed. Advantageously, particles of a composition comprising at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein delivered by a sprayer have a particle size of less than about 10 μm, e.g., in the range of about 1 μm to about 5 μm, e.g., about 2 μm to about 3 μm.

[0231] Formulations of compositions comprising at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein suitable for use with a nebulizer typically comprise at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein in aqueous solution at a concentration of about 0.1 mg to about 100 mg per ml of solution, or mg / gm, or any range or value therein, for example, but not limited to, 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. The formulation may contain agents such as excipients, buffers, isotonicity agents, preservatives, surfactants, and the like, preferably zinc. The formulation may also contain excipients or agents for stabilization of the anti-LGALS3 antibody or antigen-binding fragment thereof composition, such as buffers, reducing agents, bulk proteins, or carbohydrates. Bulk proteins useful in formulating such compositions include albumin and protamine. Typical carbohydrates useful in formulating antibody composition proteins include sucrose, mannitol, lactose, trehalose, glucose, and the like. The composition may also contain a surfactant to reduce or prevent surface-induced aggregation of the composition caused by atomization of the solution during aerosol formation. Various conventional surfactants can be used, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. The amount generally ranges from 0.001% to 14% by weight of the formulation. Preferred surfactants include polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, and the like.

[0232] In certain embodiments, the composition is administered via a nebulizer, such as a jet nebulizer or ultrasonic nebulizer. Typically, jet nebulizers use a compressed air source to generate a high-velocity air jet through an orifice. As the gas expands beyond the nozzle, a low-pressure region is created, drawing the antibody composition protein solution through a capillary tube connected to a liquid container. The liquid flow from the capillary tube is sheared into unstable filaments and droplets as it exits the tube, generating an aerosol. Various configurations, flow rates, and baffle types can be used to achieve desired performance characteristics from a given jet nebulizer. Ultrasonic nebulizers use high-frequency electrical energy, typically a piezoelectric transducer, to generate vibrational mechanical energy. This energy is transferred to the antibody composition protein formulation, either directly or via a fluidic interface, to generate an aerosol containing the antibody composition protein. Advantageously, the antibody composition protein particles delivered by the nebulizer have a particle size of less than about 10 μm, preferably in the range of about 1 μm to about 5 μm, and most preferably in the range of about 2 μm to about 3 μm.

[0233] In certain embodiments, the compositions are administered via a metered dose inhaler (MDI), in which a propellant, at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein, and any excipients or other additives are contained in a canister as a mixture with a liquefied compressed gas.

[0234] Actuation of the metering valve releases the dye mixture as an aerosol, which preferably contains particles in the size range of less than about 10 um, preferably about 1 um to about 5 um, and most preferably about 2 um to about 3 um. The desired aerosol particle size can be achieved by using formulations of the antibody composition protein produced by a variety of methods known to those skilled in the art, including jet milling, spray drying, critical point condensation, etc. Preferred metered dose inhalers include those manufactured by 3M or Glaxo, which use hydrofluorocarbon propellants.

[0235] Formulations of anti-LGALS3 antibodies or antigen-binding fragments thereof described herein for use with metered-dose inhalation devices generally comprise a finely divided powder containing at least one anti-IL-6 antibody as a suspension in a non-aqueous medium, e.g., suspended in a propellant with the aid of a surfactant. The propellant can be any conventional material used for this purpose, such as a chlorofluorocarbon, hydrochlorofluorocarbon, hydrofluorocarbon, or hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, HFA-134a (hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227), and the like. In some embodiments, the propellant is a hydrofluorocarbon. A surfactant can be selected to stabilize at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein as a suspension in the propellant, such as to protect the active agent from chemical degradation. Suitable surfactants include sorbitan trioleate, soybean lecithin, oleic acid, and the like. In some cases, the solution aerosol uses a solvent, such as ethanol, among others.

[0236] Additional agents known in the art for formulation of proteins can also be included in the formulation.

[0237] Oral formulation 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 antigen-binding fragment thereof described herein for oral administration rely on the co-administration of adjuvants, such as resorcinol and non-ionic surfactants, such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether, to artificially increase the permeability of the intestinal wall, and enzyme inhibitors, such as pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DFF) and trasylol, to inhibit enzymatic degradation. The active ingredient compound of a solid dosage form for oral administration can be mixed with at least one additive, including sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, and glycerides. These dosage forms may also contain other types of additives, such as, for example, inert diluents, lubricants such as magnesium stearate, preservatives such as parabens, sorbic acid, ascorbic acid, alpha-tocopherol, antioxidants such as cysteine, disintegrating agents, binders, thickeners, buffers, sweetening agents, flavoring agents, and perfuming agents.

[0238] In certain embodiments, tablets and pills for oral administration can be further processed into enteric-coated preparations. In certain embodiments, liquid preparations for oral administration include, for example, emulsions, syrups, elixirs, suspensions, and solution preparations that are acceptable for medical use. These preparations may contain inert diluents commonly used in the art, such as water. Liposomal preparations can be used for oral administration preparations, for example, as described for insulin and heparin (U.S. Pat. No. 4,239,754). In addition, microparticles of artificial polymers of mixed amino acids (proteinoids) can be used for oral administration of pharmaceuticals, for example, as described in U.S. Pat. No. 4,925,673. Furthermore, carrier compounds, such as those described in U.S. Pat. Nos. 5,879,681 and 5,871,753, can be used for oral administration of biologically active agents.

[0239] Mucosal preparations In certain embodiments, the compositions provided herein are formulated for absorption through mucosal surfaces. In certain embodiments, compositions and methods for administering at least one anti-LGALS3 antibody or antigen-binding fragment thereof described herein for absorption through mucosal surfaces include emulsions comprising a plurality of submicron particles, a mucoadhesive polymer, a bioactive peptide, and an aqueous continuous phase, achieving mucoadhesion of the emulsion particles and thereby promoting absorption through mucosal surfaces (U.S. Patent No. 5,514,670). Mucous surfaces suitable for application of the emulsions provided herein may include, for example, corneal, conjunctival, oral, sublingual, nasal, vaginal, pulmonary, gastric, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, such as suppositories, may contain excipients such as polyalkylene glycols, petrolatum, cocoa butter, etc. Formulations for intranasal administration may be solid, may contain excipients such as lactose, or may be aqueous or oily solutions for nasal sprays. For buccal administration, excipients include, for example, sugars, calcium stearate, magnesium stearate, pregelatinized starch, and the like (US Pat. No. 5,849,695).

[0240] Transdermal preparations 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 antigen-binding fragment thereof described herein encapsulated in a delivery device such as, for example, a liposome or polymer nanoparticle, a microparticle, a microcapsule, or a microparticle (collectively referred to as a microparticle unless otherwise specified). For transdermal administration, several suitable devices are known, including microparticles made from synthetic polymers such as polyhydroxy acids (e.g., polylactic acid, polyglycolic acid), and their copolymers, polyorthoesters, polyanhydrides, and polyphosphazenes, as well as natural polymers (e.g., collagen, polyamino acids, albumin, and other proteins, alginate, and other polysaccharides), and combinations thereof (U.S. Patent No. 5,814,599).

[0241] kit Also disclosed herein are kits for the detection and / or treatment of galectin-3-associated diseases or conditions (e.g., galectin-3-associated cancers, cardiovascular diseases, fibrosis, rheumatic diseases, and inflammation), comprising at least one immunoglobulin-related composition of the present technology (e.g., any of the antibodies, antigen-binding fragments, antibody conjugates, bispecific antibodies or antibody conjugates, scFvs, scFv conjugates, CARs, T cells, recombinant nucleic acid sequences, vectors, or isolated cells described herein) and instructions for use. In certain embodiments, the immunoglobulin-related composition is linked to one or more detectable labels. In one embodiment, the one or more detectable labels comprise a radioactive label, a fluorescent label, or a chromogenic label. Additionally or alternatively, in some embodiments, the kit further comprises a secondary antibody that specifically binds to the immunoglobulin-related composition described herein. In some embodiments, the secondary antibody is linked to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, or a chromogenic label.

[0242] In specific embodiments, provided herein are pharmaceutical packs or kits comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, e.g., one or more antibodies or antigen-binding fragments thereof described herein. In some embodiments, the kit contains a pharmaceutical composition described herein and a prophylactic or therapeutic agent.

[0243] Optionally, associated with such a container may be a notice, dosage form, and / or instructions for its use in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products. In certain embodiments, the instructions included in the kit provide guidance regarding dosages and / or dosing regimens for administration of the pharmaceutical composition.

[0244] Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, packets, sachets, tubes, inhalers, pumps, bags, vials, containers, syringes, and any packaging material suitable for the selected pharmaceutical composition and intended mode of administration and treatment.

[0245] The kits provided herein may further comprise a device used to administer the active ingredient. Examples of such devices include, but are not limited to, syringes, needleless syringes, drip bags, patches, and inhalers.

[0246] The kits provided herein may further include a pharmaceutically acceptable vehicle that can be used to administer the components. For example, if the components are provided in a solid form that must be reconstituted for parenteral administration, the kit may include a sealed container of a suitable vehicle in which the components can be dissolved to form a particulate-free sterile solution suitable for parenteral administration, or reconstituted as a suspension for oral administration. Examples of pharmaceutically acceptable vehicles include, but are not limited to, aqueous vehicles (including, but not limited to, Water for Injection USP, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and the like).

[0247] Injection, and 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).

[0248] Use and Method Therapeutic Uses and Methods In certain embodiments, provided herein are methods for treating an LGALS3-associated disease or condition, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-LGALS3 antibody or antigen-binding fragment thereof. Exemplary LGALS3-associated diseases or conditions include, but are not limited to, cancer, tumor metastasis, tumor angiogenesis, heart failure, pulmonary fibrosis, renal glomerular disease, inflammatory disease, and rheumatic disease.

[0249] In specific embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof is administered in combination with one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents inhibit or treat one or more symptoms of an LGALS3-associated disease or condition.

[0250] In certain embodiments, provided herein are methods for treating cancer in a subject, particularly an LGALS3-positive cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-LGALS3 antibody or antigen-binding fragment thereof. In specific embodiments, the LGALS3-positive cancer is ovarian cancer, lung cancer, pancreatic cancer, breast cancer, fallopian tube cancer, uterine (e.g., endometrial) cancer, primary peritoneal cancer, or cancer of any other tissue that expresses LGALS3.

[0251] For use of an anti-LGALS3 antibody or fragment thereof in a subject of a particular species, an anti-LGALS3 antibody or fragment thereof that binds to LGALS3 of that particular species is used. For example, to treat humans, an anti-LGALS3 antibody or antigen-binding fragment thereof that binds to human LGALS3 is used. In a specific embodiment, the anti-LGALS3 antibody or antigen-binding fragment thereof is an immunoglobulin.

[0252] Additionally, for use of an anti-LGALS3 antibody or fragment thereof in a subject of a particular species, the constant region of the anti-LGALS3 antibody, and in particular embodiments, the anti-LGALS3 antibody or antigen-binding fragment thereof, is derived from that particular species. For example, to treat humans, the anti-LGALS3 antibody or fragment thereof may include an anti-LGALS3 antibody or antigen-binding fragment thereof that is an immunoglobulin, and the immunoglobulin comprises a human constant region. In a specific embodiment, the subject is human.

[0253] In specific embodiments, treatment can be achieving beneficial or desired clinical results, whether detectable or undetectable, including, but not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delaying or slowing disease progression, improvement or palliation of the disease state, and remission (whether partial or complete). In specific embodiments, "treatment" can also prolong survival as compared to expected survival in the absence of treatment. In specific embodiments, administration of an anti-LGALS3 antibody or antigen-binding fragment thereof described herein, or a pharmaceutical composition described herein, to a subject with 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 that expresses LGALS3) achieves at least one, two, three, four, or more of the following effects:(i) a reduction or amelioration of the severity of one or more symptoms of cancer, (ii) a reduction in the duration of one or more symptoms associated with cancer, (iii) prevention of the recurrence of symptoms associated with cancer, (iv) a reduction in the hospitalization of a subject, (v) a reduction in the length of hospitalization, (vi) an increase in the survival rate of a subject, (vii) an enhancement or improvement in the therapeutic efficacy of another therapy, (viii) inhibition of the onset or occurrence of one or more symptoms associated with cancer, (ix) a reduction in the number of symptoms associated with cancer, (x) an improvement in quality of life as assessed by methods well known in the art, (x) inhibition of tumor recurrence, (xi) reversal of the tumor and / or one or more symptoms associated therewith, (xii) inhibition of the progression of the tumor and / or one or more symptoms associated therewith, (xiii) a reduction in tumor growth, (xiv) a decrease in tumor size (e.g., (xv) reduction in the formation of newly formed tumors, (xvi) prevention, eradication, removal, or control of primary, regional, and / or metastatic tumors, (xvii) reduction in the number or size of metastases, (xviii) reduction in mortality, (xix) increase in recurrence-free survival, (xx) maintenance of tumor size, no increase in tumor size, or an increase in tumor size that is less than that seen after standard therapy, as measured by conventional methods available to those skilled in the art, such as magnetic resonance imaging (MRI), dynamic contrast-enhanced MRI (DCE-MRI), X-ray, and computed tomography (CT) scan, or positron emission tomography (PET) scan, and / or (xxi) increase in the length of remission in a patient. Treatment can be the achievement of one or more of the foregoing.

[0254] Diagnostic Use In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof described herein can be used for diagnostic purposes to detect, diagnose, or monitor a condition described herein (e.g., a condition involving LGALS3-positive cancer cells). In certain embodiments, the anti-LGALS3 antibodies or antigen-binding fragments thereof for use in diagnostic purposes are labeled.

[0255] In certain embodiments, provided herein are methods for detecting a condition described herein, comprising: (a) assaying expression of LGALS3 or a fragment thereof in a cell or tissue sample from a subject using one or more anti-LGALS3 antibodies or antigen-binding fragments thereof described herein; and (b) comparing the level of LGALS3 or a fragment thereof expression to a control level, e.g., by comparing it to the level in a normal tissue sample (e.g., from a subject not having a condition described herein or from the same patient prior to the onset of the condition), wherein an increase or decrease in the assayed level of LGALS3 or a fragment thereof expression is indicative of a condition described herein.

[0256] The antibodies described herein can be used to assay levels of LGALS3 or fragments thereof in biological samples using classical immunohistological methods described herein or known to those skilled in the art (see, e.g., 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, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are known in the art and include enzyme labels, e.g., glucose oxidase; radioisotopes, e.g., iodine ( 125 I, 121I), 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.

[0257] In certain embodiments, monitoring of a condition described herein (eg, LGALS3-positive cancer) is performed by repeating the method for diagnosis over a period of time after initial diagnosis.

[0258] The presence of the labeled molecule can be detected in a subject using methods known in the art for in vivo scanning. Those skilled in the art will be able to determine the appropriate method for detecting a particular label. Methods and devices that can be used in the diagnostic methods of the present disclosure include, but are not limited to, whole-body scans such as computed tomography (CT), position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.

[0259] Dosage and Regimen The anti-LGALS3 antibodies or antigen-binding fragments thereof, or compositions, or cells expressing the antibodies or antigen-binding fragments thereof, described herein, can be delivered to a subject by various routes. These routes include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival, and subcutaneous routes. Pulmonary administration can also be used, for example, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent for use as a spray. In one embodiment, the anti-LGALS3 antibodies or antigen-binding fragments thereof, or compositions described herein are parenterally administered to a subject. In a specific embodiment, the parenteral administration is intravenous, intramuscular, or subcutaneous.

[0260] The amount of the anti-LGALS3 antibody or antigen-binding fragment thereof, or composition that will be effective in the treatment and / or prevention of a condition will depend on the nature of the disease, and can be determined by standard clinical techniques.

[0261] The exact dosage used in the composition also depends on the route of administration and the type of cancer, and should be determined according to the judgment of the physician and the circumstances of each patient.For example, the effective dosage may also vary depending on the means of administration, the target site, the physiological condition of the patient (including age, weight, and health), whether the patient is human or animal, other medications administered, or whether the treatment is prophylactic or therapeutic.The treatment dosage is optimally titrated to optimize safety and effectiveness.

[0262] In certain embodiments, in vitro assays are used to help identify optimal dosage ranges, and effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0263] For anti-LGALS3 antibodies or antigen-binding fragments thereof, dosages can range from about 0.0001 to 100 mg / kg, more usually 0.01 to 15 mg / kg, of patient body weight. For example, for a 70 kg patient, dosages can be 1 mg / kg body weight, 10 mg / kg body weight, or in the range of 1 to 10 mg / kg, or in other words, 70 mg or 700 mg, or in the range of 70 to 700 mg, respectively. Generally, human antibodies have a longer half-life in the human body than antibodies from other species due to the immune response to foreign polypeptides. Therefore, lower dosages of human antibodies and less frequent administration are often possible.

[0264] In certain embodiments, e.g., upon administration of an antibody or antigen-binding fragment thereof, or an engineered cell expressing a CAR, a subject is administered a range of about 1 million to about 100 billion cells, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the above values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, In some embodiments, the total cell dose and / or the dose of individual subpopulations of cells is administered at a dose of 10, 20, 30, 40, 50, 60, 70, 80, 90, 10, 25, 30, 45, 50, 75 ... 4 Or about 10 4 ~10 9 Or about 10 9 cells / kilogram (kg) body weight, e.g., 10 5 ~10 6 in the range of cells / kg body weight, e.g., 1 x 10 5 or about 1 x 10 5 cells / kg, 1.5 x 10 5 or about 1.5 x 10 5 cells / kg, 2 x 10 5 or about 2 x 10 5 cells / kg, or 1 x 10 6 or about 1 x 10 6 cells / kg, 2 x 10 6 or about 2 x 10 6 5 x 10 cells / kg 6 Or about 5 x 106 cells / kg, or 10 x 10 6 Or about 10 x 10 6 For example, in some embodiments, the cells are 10 4 Or about 10 4 ~10 9 Or about 10 9 T cells / kilogram (kg) body weight, e.g., 10 5 ~10 7 T cells / kg body weight or within their specified tolerance range.

[0265] The anti-LGALS3 antibody or antigen-binding fragment thereof can be administered multiple times, with the interval between doses being, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, or 2 years.

[0266] Combination therapy In specific embodiments, the methods provided herein for treating cancer in a subject (e.g., ovarian cancer, pancreatic cancer, lung cancer, breast cancer, fallopian tube cancer, uterine (e.g., endometrial) cancer, or primary peritoneal cancer) comprise administering to a subject in need thereof a pharmaceutical composition comprising an anti-LGALS3 antibody or antigen-binding fragment thereof described herein, and further comprise administering one or more additional therapeutic agents to the subject. In specific embodiments, the additional therapeutic agent is for treating cancer (e.g., ovarian cancer, pancreatic cancer, lung cancer, breast cancer, fallopian tube cancer, uterine (e.g., endometrial) cancer, and primary peritoneal cancer) in the subject. In specific embodiments, the additional therapeutic agent is for treating any side effects of treatment with the anti-LGALS3 antibody or antigen-binding fragment thereof described herein.

[0267] In a specific embodiment, the additional agent is an agent used to treat ovarian cancer. In a specific embodiment, the additional agent is an agent used to treat pancreatic cancer. In a specific embodiment, the additional agent is an agent used to treat lung cancer. In a specific embodiment, the additional agent is an agent used to treat breast cancer. In a specific embodiment, the additional agent is an agent used to treat fallopian tube cancer. In a specific embodiment, the additional agent is an agent used to treat uterine (e.g., endometrial) cancer. In a specific embodiment, the additional agent is an agent used to treat primary peritoneal cancer.

[0268] The anti-LGALS3 antibodies or antigen-binding fragments thereof described herein may be administered simultaneously with or sequentially (before and / or after) an additional therapeutic agent. The antibodies or antigen-binding fragments thereof and the additional therapeutic agent may be administered in the same or different compositions, and may be administered by the same or different routes of administration. A first therapy (which is an anti-LGALS3 antibody or antigen-binding fragment thereof described herein, or an additional therapeutic agent) may be administered (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 30 minutes, 45 minutes, 1 hour, 2 hours, 5 minutes, 6 hours, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 14 days, 15 days, 16 days, 18 days, 20 days, 22 days, 24 days, 26 days, 28 days, 30 days, 45 minutes, 1 hour, 28 days, 30 days, 55 days, 30 days, 45 minutes, 1 hour, 28 days, 28 days, 29 days, 30 days, 35 days, 45 days, 50 days, 55 days, 60 days, 70 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 140 days, 150 days, 160 days, 180 days, 180 days, 190 days, 210 days, 220 days, 230 days, 240 days, 250 days, 260 days, 270 days, 280 days, 290 days, 300 days, 350 days, 400 days, 450 days, 500 days, 600 days, 700 days, 800 days, 900 days, 1 The additional therapeutic agent may be administered at any time (e.g., 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, 8 weeks, or 12 weeks before), simultaneously with, or after (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, 8 weeks, or 12 weeks after) the administration of the anti-LGALS3 antibody or antigen-binding fragment thereof described herein. In certain embodiments, additional therapeutic agents administered to a subject in combination with an anti-LGALS3 antibody or antigen-binding fragment thereof described herein are administered in the same composition (pharmaceutical composition). In other embodiments, additional therapeutic agents administered to a subject in combination with an anti-LGALS3 antibody or antigen-binding fragment thereof described herein are administered to a subject in a composition that is different from the anti-LGALS3 antibody or antigen-binding fragment thereof described herein (e.g., two or more pharmaceutical compositions are used).

[0269] Patient population The subject treated according to the methods provided herein can be any mammal, such as a rodent, cat, dog, horse, cow, pig, monkey, primate, or human. In certain embodiments, the subject is a human. In another specific embodiment, the subject is a dog. As used herein, the terms "subject" and "patient" are used interchangeably.

[0270] In certain embodiments, the subject treated in accordance with the methods provided herein has been diagnosed with an LGALS3-positive cancer, including, but not limited to, cancer of the ovary, lung, pancreas, breast, uterus, fallopian tube, or primary peritoneum, or any other tissue that expresses LGALS3.

[0271] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0272] Example 1. Generation of anti-galectin-3 hybridomas using AlivaMab® Mouse The amino acid sequence of the Gal-3 immunogen used to generate antibodies in Ablexis AlivaMab® Mouse is: ADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPYGAPAGPLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYTMI (SEQ ID NO: 53)

[0273] To elicit anti-Gal-3 antibodies in AlivaMab® Mouse, multiple tolerance-breaking strategies were used to enhance antigen presentation or stimulate T helper cells. Once the titers in the animals' serum reached high levels, splenocytes from several mice were used in fusions to generate hybridomas.

[0274] Example 2. Hybridoma screening As a primary screen, ELISA using human Gal-3 was used, and 10 enriched clones specific for Gal-3 were obtained.

[0275] An assay for Gal-3-PE binding to cancer cells was developed as a secondary screen. huGal-3 (Abcam ab89487) was labeled with PE (Innova Biosciences) according to the manufacturer's instructions. Assays for Gal-3-PE binding to OVCAR3 cells were performed as follows: Cultured SKOV3 cells were trypsinized and washed with culture medium. 500, 1000, or 2000 cells were incubated with various concentrations of Gal-3-PE. The indicated number of OVCAR3 cells was pipetted into a 96-well plate with the indicated amount of Gal-3-PE and incubated on the bench in the dark for 2 hours before reading with a Mirrorball® device (TTP Labtech). Binding of Gal-3-PE to OVCAR3 cells was detected using a Mirrorball® flow cytometer. As shown in Figure 3A, OVCAR3 cells exhibited dose-dependent binding to Gal-3-PE. Similarly, MOLT-4 also showed dose-dependent binding to Gal-3-PE, as shown in Figure 3B.

[0276] The binding assay described above was converted to an assay for inhibition of Gal-3-PE binding to OVCAR3 cells as follows. Binding reactions to measure Gal-3-PE binding to OVCAR3 cells were set up in the presence of PBS (negative control) or the indicated antibodies obtained by enrichment (crude purification) from clones selected in the primary binding screen. The extent of binding was measured by flow cytometry and plotted. As shown in Figure 4, five antibodies (12H7, 20F08, 38E05, 39F02, and 46H02) blocked Gal-3-PE binding to OVCAR3 cells. Clone 14D11 (also known as 14D11.2D3) did not block Gal-3-PE binding to OVCAR3 cells in this assay and therefore serves as a negative control.

[0277] Example 3. Antibody Characterization To determine the species cross-reactivity of the obtained antibodies, binding to human Gal-3 or mouse Gal-3 was detected using ELISA. As shown in Figures 5A-5B, clones 39F02, 38E05, and 46H02 bound to mouse Gal-3, but clones 20F08 and 12H07 did not.

[0278] The clones were then subjected to a dose-response determination of Gal-3-PE binding blockade to OVCAR3 cells. Assays for inhibition of Gal-3-PE binding to OVCAR3 cells were performed in the presence of increasing concentrations of antibody. Binding reactions to measure Gal-3-PE binding to OVCAR3 cells were set up in the presence of PBS (negative control) or increasing amounts of purified antibody. As shown in Figure 6A, clones 20F08 and 12H07 exhibited very similar dose responses for blocking Gal-3-PE binding to OVCAR3 cells. Interestingly, 20F08 and 12H07 were found to have identical sequences. As shown in Figure 6B, clone 46H02 exhibited a unique dose response for blocking Gal-3-PE binding to OVCAR3 cells. Clones 38E05 and 39F02, which also have identical sequences, showed very similar dose responses for blocking Gal-3-PE binding to OVCAR3 cells, as shown in Figure 6C. Clone 14D11 did not block Gal-3-PE binding to OVCAR3 cells in this assay (Figure 6D).

[0279] Next, binding parameters were determined for clones 46H02 and 20F08 using surface plasmon resonance (SPR) in the presence of increasing concentrations of these antibodies. As shown in Figures 7A-7B, monoclonal antibodies (mAbs) 46H02 (Figure 7A) and 20F08 (Figure 7B) exhibited characteristic binding kinetics for human Gal-3. These data demonstrate that mAbs 46H02 and 20F08 exhibit K values ​​of 10 nM and 44 nM, respectively. D This shows that the antibody bound to human Gal-3.

[0280] Because the first-generation AlivaMab® Mouse (Ablexis) used here produces a mixture of antibodies with human heavy and light chains or hybrid antibodies with human heavy and mouse light chains, clones 12H07, 20F08, 38E05, 39F02, and 46H02 were subjected to ELISA light chain species determination using anti-mouse kappa, anti-mouse lambda, anti-human kappa, and anti-human lambda antibodies. As shown in Figure 8, 46H02 has a fully human F(ab)2, while the other hits have a human HC and a mouse LC.

[0281] To analyze whether 46H02 and 14D11.2D3 bind to overlapping epitopes on Gal-3, we performed an epitope binning assay using a premixed epitope binning assay strategy (Figure 9A). In this strategy, the test antibody (analyte) is immobilized on a capture surface, the ligand is flowed over the analyte, and binding is detected by SPR. The ligand is either the antigen alone or a premixed solution of the antigen complexed with a second antibody. As shown, the antigen alone generates a distinctive SPR profile indicating analyte binding to the antigen (left panel). When the ligand is a premixed solution of the antigen complexed with a second antibody, an SPR profile indicating analyte binding to the antigen is obtained if the second antibody binds to a distinct epitope on the antigen (center panel). If the second antibody blocks analyte binding, the SPR profile shows no binding, indicating that the second antibody blocks analyte binding (right panel). The test antibody (analyte) itself was used as a control to mimic a second antibody blocking analyte binding. As shown in Figure 9B, when mAb 2D3 was used as the analyte, the Gal-3 + 14D11.2D3 complex completely inhibited binding. In contrast, Gal-3 or Gal-3 + 46H02 complexes did not inhibit binding (Figure 9B). Similarly, as shown in Figure 9C, when mAb 46H02 was used as the analyte, the Gal-3 + 46H02 complex completely inhibited binding. In contrast, Gal-3 or Gal-3 + 14D11.2D3 complexes did not inhibit binding (Figure 9C). Thus, mAb 46H02 and 14D11 do not compete with each other and therefore bind to distinct epitopes. [Table 1] TIFF0007825633000002.tif197142 TIFF0007825633000003.tif166139 TIFF0007825633000004.tif161139 TIFF0007825633000005.tif161139 TIFF0007825633000006.tif182140 TIFF0007825633000007.tif166140 TIFF0007825633000008.tif151139

[0282] All publications, patents, and patent applications cited in this specification are incorporated by reference herein as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Although the foregoing technology of the present invention has been described in some detail by way of illustration and example for clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the technology that certain changes and modifications can be made without departing from the spirit or scope of the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to galectin-3 (LGALS3), The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), (a) the V H V includes SEQ ID NO: 7 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 8 H CDR2 sequence and V comprising SEQ ID NO: 9 H CDR3 sequence, and L V includes SEQ ID NO: 2 L CDR1 sequence and V comprising SEQ ID NO:3 L CDR2 sequence and V comprising SEQ ID NO:4 L and a CDR3 sequence, or (b) the V H V comprising SEQ ID NO: 19 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 20 H CDR2 sequence and V comprising SEQ ID NO: 21 H CDR3 sequence, and L V comprising SEQ ID NO: 14 L CDR1 sequence and V comprising SEQ ID NO: 15 L CDR2 sequence and V comprising SEQ ID NO: 16 L and a CDR3 sequence, or (c) Said V H V comprising SEQ ID NO: 29 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 30 H CDR2 sequence and V comprising SEQ ID NO: 31 H CDR3 sequence, and L V comprising SEQ ID NO: 24 L CDR1 sequence and V comprising SEQ ID NO: 25 L CDR2 sequence and V comprising SEQ ID NO: 26 L and a CDR3 sequence, or (d) the V H V comprising SEQ ID NO: 39 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 40 H CDR2 sequence and V comprising SEQ ID NO: 41 H CDR3 sequence, and L V comprising SEQ ID NO: 34 L CDR1 sequence and V comprising SEQ ID NO: 35 L CDR2 sequence and V comprising SEQ ID NO: 36 L and a CDR3 sequence; or (e) the V H V comprising SEQ ID NO: 49 H Complementarity determining region (CDR) 1 sequence and V comprising SEQ ID NO: 50 H CDR2 sequence and V comprising SEQ ID NO: 51 H CDR3 sequence, and L V comprising SEQ ID NO: 44 L CDR1 sequence and V comprising SEQ ID NO: 45 L CDR2 sequence and V comprising SEQ ID NO: 46 L and a CDR3 sequence, the antibody or antigen-binding fragment thereof inhibits Gal-3-phycoerythrin (PE) binding to ovarian tumor cells, and the ovarian tumor cells are OVCAR3 cells. The antibody or antigen-binding fragment thereof.

2. The V H comprises an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:38, and SEQ ID NO:48; or The V L 2. The antibody or antigen-binding fragment of claim 1, wherein said antibody or antigen-binding fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 33, and SEQ ID NO:

43.

3. V selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:1, SEQ ID NO:18 and SEQ ID NO:13, SEQ ID NO:28 and SEQ ID NO:23, SEQ ID NO:38 and SEQ ID NO:33, and SEQ ID NO:48 and SEQ ID NO:43, respectively. H Amino acid sequence and V L 3. The antibody or antigen-binding fragment of claim 1 or 2, comprising the amino acid sequence:

4. The antibody comprises a heavy chain constant region and a light chain constant region of human origin, or the antibody is an immunoglobulin comprising two identical heavy chains and two identical light chains, or the antibody or antigen-binding fragment thereof inhibits binding of LGALS3 to a glycosylated cell surface receptor, or the antibody or antigen-binding fragment thereof inhibits binding of LGALS3 to a glycosylated growth factor receptor, or the antibody or antigen-binding fragment inhibits binding of glycosylated mucin-1 (MUC1), mucin-4 (MUC4), or mucin-5 (MUC5). 4), inhibits binding of LGALS3 to one or more of mucin-16 (MUC16), disialoganglioside, GD2, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), insulin-like growth factor receptor (IGFR), integrin, or CTLA4; or the antibody or antigen-binding fragment inhibits growth of tumors expressing a glycosylated form of MUC16; or the antibody is a monoclonal antibody; or the antigen-binding fragment is a Fab, F(ab') 2 , Fab′, Fv, or scFv; The antibody or antigen-binding fragment of any one of claims 1 to 3.

5. The antibody or antigen-binding fragment of claim 4, wherein the heavy chain constant region has an isotype selected from the group consisting of gamma 1, gamma 2, gamma 3, and gamma 4, or the light chain constant region has an isotype selected from the group consisting of kappa and lambda, or the immunoglobulin is IgG.

6. An antibody conjugate comprising the antibody or antigen-binding fragment of any one of claims 1 to 5 conjugated to a drug.

7. The antibody conjugate of claim 6, wherein the drug is an imaging agent or a cytotoxic agent.

8. An antibody or antigen-binding fragment described in any one of claims 1 to 5, which is a bispecific antibody.

9. The antibody or antigen-binding fragment of claim 8, wherein the bispecific antibody specifically binds to CD3, or the bispecific antibody comprises an immunoglobulin that specifically binds to LGALS3, the light chain of the immunoglobulin being conjugated via a peptide linker to a single-chain variable fragment (scFv) that specifically binds to CD3.

10. A bispecific antibody conjugate comprising the bispecific antibody of claim 8 or 9 conjugated to a drug.

11. The bispecific antibody conjugate of claim 10, wherein the agent is an imaging agent or a cytotoxic agent.

12. A chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment of any one of claims 1 to 5.

13. A T cell recombinantly expressing the CAR of claim 12.

14. A polynucleotide comprising a nucleic acid sequence encoding the antibody or antigen-binding fragment according to any one of claims 1 to 5 or 8 to 9, or a nucleic acid sequence encoding the CAR according to claim 12.

15. A polynucleotide described in claim 14, comprising any one of the polynucleotides of SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:47, SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:42, or SEQ ID NO:

52.

16. A vector or host cell comprising the polynucleotide of claim 14 or 15.

17. A vector or host cell as described in claim 16, operably linked to a promoter.

18. A cell comprising the vector of claim 16 or 17.

19. 19. A pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 5, the antibody conjugate of claim 6 or 7, the bispecific antibody of claim 8 or 9, the bispecific antibody conjugate of claim 10 or 11, the CAR of claim 12, the T cell of claim 13, the polynucleotide of claim 14 or 15, the vector of claim 16 or 17, or the cell of any one of claims 16 to 18.

20. 20. The pharmaceutical composition of claim 19 for treating cancer in a patient.

21. 21. The pharmaceutical composition of claim 20, wherein the cancer is metastatic cancer or cancer of the ovary, lung, pancreas, breast, uterus, fallopian tube, or primary peritoneum.

22. 22. The pharmaceutical composition of claim 20 or 21, which inhibits metastasis in a patient.

23. The pharmaceutical composition of any one of claims 20 to 22, wherein the patient is a human patient.

24. A pharmaceutical composition according to any one of claims 20 to 23, for administration in combination with a therapeutically effective amount of an additional therapeutic agent.

Images