Anti-bag2 antibodies and methods of treating cancer

Abstract

To provide anti-BAG2 antibodies and methods of treating cancer.SOLUTION: An antibody or antigen binding fragment thereof that specifically binds to a BAG2 polypeptide, and a method for treating cancer therewith.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] 2. Background of the Invention 1. Field of the invention The present disclosure relates to antibodies or antigen-binding fragments thereof that specifically bind to a BAG2 polypeptide or a fragment thereof. The present application also relates to anti-cancer therapeutic agents using the compositions of the present invention. [Background technology]

[0002] 2. General Background and State of the Art The co-chaperone Bcl-2-associated athanogene (BAG) protein family mediates various physiological processes, including intracellular protein folding, stress response, neuronal differentiation, apoptosis, and cell proliferation, and functionally binds to various cooperating proteins. BAG2, a member of the BAG domain family with anti-apoptotic activity, is a negative regulator of the C-terminus of Hsc70-interacting protein (CHIP), a chaperone-associated ubiquitin ligase. The primary role of BAG2 in protein regulation through inhibition of CHIP activity is associated with neurodegenerative and autosomal recessive disorders through stabilization of chaperone-related proteins such as PINK1 and CFTR. It has been reported that BAG2 has pro-apoptotic activity, such that expression of BAG2 increases proteasome inhibitor-induced apoptosis, and BAG2 knockdown partially inhibits apoptosis when thyroid cancer cells are exposed to the proteasome inhibitor MG132. Apart from the formation of the BAG2-Hsp70 complex, BAG protein functionally interacts with various binding partners and regulates various cellular processes such as stress signaling, cell division, apoptosis and cell differentiation. It has also been reported that overexpression of BAG2 promotes the stabilization of STK33 protein, a potent oncogene, in various mutant K-Ras-induced tumors, thereby promoting tumor development. In addition, it has been suggested that the expression and location of BAG2 protein may vary depending on the histopathological and molecular genetic pathology of cancer cells during breast cancer progression or metastasis. It has been found that BAG2 protein is secreted from cells during tumor formation by interacting with cathepsin B, a proteolytic enzyme, and it has been confirmed that the loss of BAG2 protein completely inhibits tumor formation and metastasis to the lung in animal models of breast cancer. As a result, the development of BAG2 protein inhibitors will contribute to the treatment and survival of cancer patients. Summary of the Invention [Problem to be solved by the invention]

[0003] However, despite these findings, the role of BAG2 in cancer progression and metastasis is not clearly known. In addition, detailed testing has not been performed on BAG2 monoclonal antibodies. Therefore, there is a need to develop antibodies or antigen-binding fragments thereof that specifically bind to BAG2 polypeptide or fragments thereof. [Means for solving the problem]

[0004] Summary of the Invention These and other objects of the present invention will be more fully understood from the following description of the invention, the accompanying reference drawings and the appended claims.

[0005] One embodiment provides an antibody or antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or a fragment thereof.

[0006] Another aspect provides a polynucleotide encoding the antibody or antigen-binding fragment thereof.

[0007] Another aspect provides a host cell comprising the polynucleotide.

[0008] Another aspect provides a method of producing the antibody or antigen-binding fragment thereof.

[0009] Additional aspects will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the embodiments presented in this disclosure.

[0010] One embodiment provides an antibody or antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or a fragment thereof.

[0011] The BAG2 polypeptide may be derived from a mammal. The mammal may be a human (Homo sapiens), mouse (Mus musculus), monkey, cow, or horse. The BAG2 may comprise the amino acid sequence of SEQ ID NO:69. The amino acid sequence of SEQ ID NO:69 corresponds to NCBI reference SEQ ID NO:NM_004282.4. The BAG2 protein includes variants having biologically equivalent activity to the amino acid sequence of SEQ ID NO:69, but whose amino acid sequences may not match the amino acid sequence of SEQ ID NO:69. The BAG2 polypeptide may comprise an amino acid sequence having at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO:69. A BAG2 protein may be a polypeptide having the same sequence as SEQ ID NO:69 except for at least one amino acid residue, at least two amino acid residues, at least three amino acid residues, at least four amino acid residues, at least five amino acid residues, at least six amino acid residues, or at least seven amino acid residues. As used herein, "polypeptide" may be used interchangeably with "protein."

[0012] Antibody refers to a specific immunoglobulin against an antigenic site. Antibody refers to a polypeptide or combination of polypeptides that specifically binds to a BAG2 polypeptide or a fragment thereof. Antibody may include polyclonal antibodies, monoclonal antibodies, or recombinant antibodies, such as ScFv fragments, diabodies, single chain antibodies, and the like, and may include all immunoglobulin antibodies. Antibody may include the complete form of an antibody with two full-length light chains and two full-length heavy chains, and may include functional fragments of an antibody molecule that retain antigen binding function by including a specific antigen binding site, i.e., a binding domain, despite the absence of the structure of a complete intact antibody with two light chains and two heavy chains.

[0013] An antigen-binding fragment refers to a portion of a polypeptide that is a fragment of the entire structure of an immunoglobulin and contains the portion to which an antigen can bind. For example, the antigen-binding fragment may be an scFv, (scFv)2, Fv, Fab, Fab', FvF(ab')2, or a combination thereof.

[0014] There are five types of heavy chains, gamma, delta, alpha, mu and epsilon, and the heavy chain may determine the type of antibody. Alpha and gamma each contain 450 amino acids, mu and epsilon each contain 550 amino acids. The heavy chain has two regions, the variable region and the constant region.

[0015] There are two types of light chains, kappa and lambda, which may contain from about 211 amino acids to about 217 amino acids. The light chain may have a constant region and a variable region.

[0016] The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity determining region (VH-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 33, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 45, and a light chain variable region comprising a complementarity determining region (VL-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 51, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 57, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 63; a heavy chain variable region comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 34, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 40, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 52, and a light chain variable region comprising a complementarity determining region (VL-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 51, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 57, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 63, and a light chain variable region comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 34, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 40, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 52, and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 52, and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence a light chain variable region comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 35, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 41, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 47, and a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 53, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 59, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 65; a heavy chain variable region comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 36, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 42, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 48, and a a light chain variable region comprising a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO:54, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO:60, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO:66;a heavy chain variable region comprising VH-CDR1 having the amino acid sequence of SEQ ID NO: 37, VH-CDR2 having the amino acid sequence of SEQ ID NO: 43, and VH-CDR3 having the amino acid sequence of SEQ ID NO: 49, and a light chain variable region comprising VL-CDR1 having the amino acid sequence of SEQ ID NO: 55, VL-CDR2 having the amino acid sequence of SEQ ID NO: 61, and VL-CDR3 having the amino acid sequence of SEQ ID NO: 67; a heavy chain variable region comprising VH-CDR1 having the amino acid sequence of SEQ ID NO: 38, VH-CDR2 having the amino acid sequence of SEQ ID NO: 44, and VH-CDR3 having the amino acid sequence of SEQ ID NO: 50, and a VL-CDR1 having the amino acid sequence of SEQ ID NO: 56, VL-CDR2 having the amino acid sequence of SEQ ID NO: 62, and a light chain variable region comprising VL-CDR1 having the amino acid sequence of SEQ ID NO: 55, VL-CDR2 having the amino acid sequence of SEQ ID NO: 61, and VL-CDR3 having the amino acid sequence of SEQ ID NO: 67, and a light chain variable region comprising VH-CDR1 having the amino acid sequence of SEQ ID NO: 38, VH-CDR2 having the amino acid sequence of SEQ ID NO: 44, and VH-CDR3 having the amino acid sequence of SEQ ID NO: 50, and a light chain variable region comprising VL-CDR1 having the amino acid sequence of SEQ ID NO: 56, VL-CDR2 having the amino acid sequence of SEQ ID NO: 62, and a light chain variable region comprising VL-CDR1 having the amino acid sequence of SEQ ID NO: 55, VL-CDR2 having the amino acid sequence of SEQ ID NO: 61, and VL-CDR3 having the amino acid sequence of SEQ ID a light chain variable region comprising a VL-CDR3 consisting of the amino acid sequence of NO:68; or a combination thereof;

[0017] The sixth and seventh Xaa in SEQ ID NO:39 may be glycine (Gly) or alanine (Ala). The second Xaa in SEQ ID NO:35 may be tyrosine (Tyr) or histidine (His). The eighth Xaa in SEQ ID NO:41 may be serine (Ser) or threonine (Thr). The twelfth Xaa in SEQ ID NO:47 may be tyrosine (Tyr) or histidine (His). The third Xaa in SEQ ID NO:53 may be methionine (Met) or isoleucine (Lie). The second Xaa in SEQ ID NO:59 may be Ala or Ser.

[0018] The antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising any one of amino acid sequences selected from SEQ ID NOs:21 to 26; a light chain variable region comprising any one of amino acid sequences selected from SEQ ID NOs:27 to 32; or the heavy chain variable region and the light chain variable region.

[0019] The antibody or antigen-binding fragment thereof may comprise the heavy chain variable region of SEQ ID NO:21 and the light chain variable region of SEQ ID NO:27; the heavy chain variable region of SEQ ID NO:22 and the light chain variable region of SEQ ID NO:28; the heavy chain variable region of SEQ ID NO:23 and the light chain variable region of SEQ ID NO:29; the heavy chain variable region of SEQ ID NO:24 and the light chain variable region of SEQ ID NO:30; the heavy chain variable region of SEQ ID NO:25 and the light chain variable region of SEQ ID NO:31; or the heavy chain variable region of SEQ ID NO:26 and the light chain variable region of SEQ ID NO:32; or a combination thereof.

[0020] Xaa at positions 56 and 57 in SEQ ID NO:21 may be Gly or Ala, respectively. In SEQ ID NO:23, Xaa at position 1 may be glutamine (Gln) or glutamic acid (Glu), Xaa at position 7 may be Ser or proline (Pro), Xaa at position 12 may be valine (Val) or alanine (Ala), Xaa at position 27 may be Tyr or His, Xaa at position 58 may be Ser or Thr, Xaa at position 61 may be asparagine (Asn) or Ser, Xaa at position 74 may be arginine (Arg) or lysine (Lys), Xaa at position 83 may be phenylalanine (Phe) or leucine (Leu), Xaa at position 92 may be Gly or Ala, and Xaa at position 108 may be His or Tyr. In SEQ ID NO:27, Xaa at position 53 can be He or Phe. In SEQ ID NO:29, Xaa at position 29 can be Met or He, Xaa at position 51 can be Ala or Ser, Xaa at position 79 can be Glu or aspartic acid (Asp), and Xaa at position 106 can be Met or He.

[0021] The antibody or antigen-binding fragment thereof includes a plurality of antibodies or antigen-binding fragments thereof and may be a combination of antibodies selected from one of Set No. 1 and one of Set No. 2; one of Set No. 1 and one of Set No. 3; and one of Set No. 2 and one of Set No. 3. Set No. 1 may be antibodies or antigen-binding fragments thereof that bind to the central region of BAG2 protein, including VH of SEQ ID NO: 21 and VL of SEQ ID NO: 27, and VH of SEQ ID NO: 22 and VL of SEQ ID NO: 28. Set No. 2 may be antibodies or antigen-binding fragments thereof that bind to the N-terminal region of BAG2 protein, including VH of SEQ ID NO: 23 and VL of SEQ ID NO: 29, and VH of SEQ ID NO: 24 and VL of SEQ ID NO: 30. Set No. 3 may be an antibody or antigen-binding fragment thereof that binds to the C-terminal region of BAG2 protein, including VH of SEQ ID NO:25 and VL of SEQ ID NO:31, and VH of SEQ ID NO:26 and VL of SEQ ID NO:32.

[0022] The antibody or antigen-binding fragment thereof may be a monoclonal antibody.

[0023] The antibody or antigen-binding fragment thereof may be marked with a detectable label or a label capable of emitting a detectable signal. A label refers to a detectable compound or composition that is directly or indirectly conjugated to the antibody to produce a labeled antibody or antigen-binding fragment thereof. The label may itself be detectable or may catalyze the chemical modification of a substrate compound or composition that is detectable.

[0024] The label may be an immunofluorescent label, a chemiluminescent label, a phosphorescent label, a radioactive label, an epitope tag, avidin / biotin, a colloidal gold particle, a colored particle, a magnetic particle, a chromophore label, an ECL label, an enzyme, or the like.

[0025] The antibody or antigen-binding fragment thereof may be produced by a hybridoma cell selected from the hybridoma cells deposited under accession numbers KCTC 137378P, KCTC 137388P, KCTC 137398P, KCTC 137408P, KCTC 137418P, KCTC 137428P, KCTC 137438P, KCTC 137448P, KCTC 137458P and KCTC 137468P.

[0026] Hybridoma cells refer to hybrid cells that have tumorigenic potential due to artificial fusion of two types of cells, and can generally be used to continuously produce antibodies by fusing B cells with plasmacytoma cells isolated from immunized subjects. In this specification, hybridoma cells may also be referred to as hybrid cells or fusion cells.

[0027] The present inventors have identified anti-BAG2 antibodies, including VH of SEQ ID NO:21 and VL of SEQ ID NO:27 (2A11, 4C2, 8C4); VH of SEQ ID NO:22 and VL of SEQ ID NO:28 (3B5); VH of SEQ ID NO:23 and VL of SEQ ID NO:29 (9B3, 9B12, 3B10); VH of SEQ ID NO:24 and VL of SEQ ID NO:30 (10H7); VH of SEQ ID NO:25 and VL of SEQ ID NO:31 (3G8); and VH of SEQ ID NO:26 and VL of SEQ ID NO:32 (3F12), and hybridoma cells producing the same. It has been confirmed that the identified anti-BAG2 antibodies show antigen-antibody reaction in breast cancer cells.

[0028] Another aspect provides polynucleotides comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof.

[0029] The polynucleotide may be any one of nucleotide sequences selected from the group consisting of SEQ ID NOs:1 to 10 encoding a heavy chain variable region, and any one of nucleotide sequences selected from the group consisting of SEQ ID NOs:11 to 20 encoding a light chain variable region.

[0030] The polynucleotide may be a vector. The vector may be obtained by replicating and / or expressing the polynucleotide in a cell. The cell may be a eukaryotic or prokaryotic cell. The eukaryotic cell may be a mammalian cell, a plant cell, a yeast cell, or an insect cell. The mammal may be a human, a monkey, a rabbit, a rat, a hamster, or a mouse. The prokaryotic cell may be a bacterial cell. The bacterium may be E. coli. The vector may be an expression vector. In an expression vector, the polynucleotide is operably linked to an appropriate regulatory region so that the polynucleotide is expressed in the host cell. The regulatory region may be a promoter, an enhancer, or a terminator. The vector may also include a selection marker. The vector may be a phage, a plasmid, a cosmid, a minichromosome, a virus, or a retroviral vector. The vector may include a polynucleotide encoding the heavy chain variable region or the light chain variable region of an antibody, or may include both a polynucleotide encoding the heavy chain variable region and a polynucleotide encoding the light chain variable region.

[0031] Polynucleotide may be conjugated with detectable label or label capable of emitting detectable signal.Label may be fluorescent dye, phosphorescent dye, radioisotope, chromophore, quantum dot, quencher, magnetic bead nanoparticle, gold nanoparticle, nanophosphor, silicon nanoparticle, semiconductor particle with luminescent property, and the like.For example, fluorescent dye may be cyanine (cyanine 2), amidomethylcoumarin, fluorescein, indocarbocyanine (cyanine 3), cyanine 3.5, tetramethylrhodamine, rhodamine red, Texas red, indiancarbocyanine (cyanine 5), cyanine 5.5, cyanine 7, Oyster, and the like. For example, the semiconductor particles may be cadmium selenium (CdSe), cadmium tellurium (CdTe), indium gallium phosphide (InGaP), silver indium zinc sulfide (AgInZnS), or the like. The method of conjugation with the label may be a method of attaching a label to the 3' end of the polynucleotide, a method of attaching a label to the 5' end, or a method of including a nucleotide to which a label is attached in the polynucleotide. The conjugation of the label to the 3' end or 5' end may be performed by an enzymatic reaction, and the enzyme may be T4 RNA ligase, terminal transferase, polyA polymerase, or the like. The label may be a fluorescence microscope, a scanning electron microscope, a transmission electron microscope, a computed tomography, a magnetic resonance imaging, or the like.

[0032] Another aspect provides a host cell comprising the polynucleotide.

[0033] The polynucleotide is the same as described above.

[0034] The host cell may be a bacterial cell, a yeast cell, a fungal cell, an insect cell, an animal cell or a plant cell, each of which contains the polynucleotide. The bacterial cell may be Escherichia coli (E. coli), Streptomyces, or Salmonella typhimurium. The yeast cell may be Pichia pastoris. The insect cell may be a Drosophila or Spodoptera Sf9 cell. The animal cell may be a Chinese hamster ovary cell (CHO), mouse myeloma (SP2 / 0), human lymphoblastoid, COS, mouse myeloma (NSO), 293T, bovine (bow) melanoma cell, HT-1080, baby hamster kidney cell (BHK), human embryonic kidney cell (HEK), or PERC.6 (human retinal cell).

[0035] The polynucleotide may be introduced into the host cell. Introduction refers to the method of delivering the vector containing the polynucleotide encoding the antibody into the host cell. Such introduction may be carried out by various methods known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polybrene mediated transfection, electric shock, microinjection, liposome fusion, Lipofectamine and protoplast fusion. In addition, transduction refers to the delivery of the target product into the cell by infection with a viral particle. In addition, the vector may be introduced into the host cell by gene bombardment or the like. Introduction may also be referred to as transformation.

[0036] Another aspect provides a method of producing an antibody or antigen-binding fragment thereof.

[0037] The method may include culturing the host cells; and isolating the antibody or antigen-binding fragment thereof from the resulting culture.

[0038] The host cells are the same as those described above.

[0039] The culture may be carried out according to suitable media and culture conditions known in the art. Those skilled in the art can easily control the media and culture conditions according to the selected microorganism. The culture method may include, for example, batch, continuous and fed-batch culture.

[0040] The medium may contain various carbon sources, nitrogen sources, and trace element components.

[0041] The carbon source may be a carbohydrate, such as glucose, sucrose, lactose, fructose, maltose, starch, or cellulose; a fat, such as soybean oil, sunflower oil, castor oil, or coconut oil; a fatty acid, such as palmitic acid, stearic acid, and linoleic acid, glycerol; an alcohol, such as ethanol; an organic acid, such as acetic acid; or a combination thereof. The nitrogen source may include inorganic nitrogen sources, such as peptone, yeast extract, meat extract, malt extract, corn steep liquor (CSL), and soybean wheat, organic nitrogen sources, such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrogen, or a combination thereof. The medium as a source of phosphorus may contain, for example, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the corresponding sodium-containing salts, or metal salts, such as magnesium antioxidants or iron antioxidants. In addition, amino acids, vitamins, suitable precursors, and the like, may be included in the medium. The medium or individual components may be added to the culture in a batch or continuous manner.

[0042] In addition, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid may be added to the bacterial culture in an appropriate manner to adjust the pH of the culture during the period in which the host cells are cultured. In addition, antifoaming agents such as fatty acid polyglycol esters may be used to suppress foam formation during the culture of the host cells.

[0043] The cells may be cultured under aerobic, microaerobic, or anaerobic conditions. Microaerobic conditions refer to culture conditions in which a level of oxygen lower than atmospheric oxygen levels is dissolved in the medium. The low level of oxygen may be, for example, about 0.1% to about 10%, about 1% to about 9%, about 2% to about 8%, about 3% to about 7%, or about 4% to about 6%. In addition, microaerobic conditions may include conditions in which the concentration of oxygen dissolved in the medium ranges from about 0.9 ppm to about 3.6 ppm. The culture temperature may be, for example, about 20°C to about 45°C, or about 25°C to about 40°C. Incubation may be continued until the desired amount of the antibody or antigen-binding fragment thereof reaches the target level.

[0044] Separation may be carried out by methods known in the art for isolating antibodies, and may involve carrying out one or more steps selected from centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution, and chromatography.

[0045] The method may further comprise labeling the isolated antibody or antigen-binding fragment thereof, which may be an immunofluorescent label, a chemiluminescent label, a phosphorescent label, a radioactive label, an epitope tag, avidin / biotin, a colloidal gold particle, a colored particle, a magnetic particle, a chromophore label, an ECL label, an enzyme, or the like.

[0046] In one embodiment, the present invention is directed to a method for treating cancer, either primary or metastatic cancer, in an individual, comprising administering to an individual in need thereof anti-BAG2 or an antigen-binding fragment thereof as discussed above. The method may include co-administering or sequentially administering an existing therapeutic agent. The existing treatment may be a cancer immunotherapy agent, which may include an inhibitor to an immune checkpoint molecule, such as PD-1, PD-L1, or CTLA4. The immunotherapeutic agent may be granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 12 (IL-12), interleukin 15 (IL-15), B7-1 (CD80), B7-2 (CD86), 4-1BB ligand, GITRL, OX-40L, anti-CD3 antibody, anti-CD27 antibody, anti-CTLA4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-GITR antibody, anti-OX-40 antibody, anti-4-1BB antibody, anti-LAG-3 antibody, and anti-TIM-3 antibody. The cancer may be breast cancer, colorectal cancer, head and neck cancer, colon cancer, skin cancer, pancreatic cancer, lung cancer, stomach cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, clear cell sarcoma, melanoma, brain and spinal cord tumors, brain cancer, thymus, mesothelioma, esophageal cancer, bile duct cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, cervical cancer, ovarian cancer, endometrial cancer, lymphoma, myelodysplastic syndrome (MOS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, Hodgkin's disease, endocrine cancer, and sarcoma.

[0047] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent Office upon request and payment of the necessary fee.

[0048] The present invention will become more fully understood from the detailed description given herein below and the accompanying drawings, which are illustrative only and therefore not limiting of the invention. [Brief description of the drawings]

[0049] [Figure 1] 1 shows the results of Western blotting of anti-BAG2 antibodies produced from 10 mouse hybridoma cells. [Figure 2A] 1 shows the results of Western blotting of anti-BAG2 antibodies or fragments thereof for the full-length BAG2 polypeptide. [Figure 2B] 1 shows the results of Western blotting of anti-BAG2 antibodies or fragments thereof for the full-length BAG2 polypeptide. [Diagram 3] The BAG2 domains that react with each anti-BAG2 antibody are shown. [Figure 4A] Figure 1 shows the therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-L1 antibody in a breast cancer model. (a) BALB / c mice were injected with 2x105 EMT6 cells. Starting on day 12 after tumor cell injection, some mice were intraperitoneally (ip) injected with anti-BAG2 antibody 3B10 (200μg / mouse) or isotype control antibody once every 2 days. Starting on day 14, some mice received single injections of anti-PD-L1 antibody (100μg / mouse ip) or isotype control antibody once every 2 days. Experimental design is shown. t-test: all data are expressed as mean±SEM (standard error of the mean). *p<0.05, ***p<0.001 (vs. control mouse IgG2a-treated group). [Figure 4B] The therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-L1 antibody in a breast cancer model is shown. (b and c) Tumor volumes of control mice (black line, n=5), mice treated with anti-BAG2 antibody 3B10 alone (green line, n=5), mice treated with anti-PD-L1 antibody alone (blue line, n=5), and mice treated with both 3B10 and anti-PD-L1 antibody (red line, n=5). Tumor growth was monitored and tumor volumes were measured with electronic calipers on days 12, 14, 16, 18, 20, 22, and 24. Mice were sacrificed on day 25. (b) Tumor volumes of individual mice within groups. t-test: all data are presented as mean ± SEM (standard error of the mean). *p<0.05, ***p<0.001 vs. control mouse IgG2a treatment group. [Figure 4C] The therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-L1 antibody in a breast cancer model is shown. (b and c) Tumor volumes of control mice (black line, n=5), mice treated with anti-BAG2 antibody 3B10 alone (green line, n=5), mice treated with anti-PD-L1 antibody alone (blue line, n=5), and mice treated with both 3B10 and anti-PD-L1 antibody (red line, n=5). Tumor growth was monitored and tumor volumes were measured with electronic calipers on days 12, 14, 16, 18, 20, 22, and 24. Mice were sacrificed on day 25. (c) Mean tumor growth of the four treatment groups. t-test: all data are presented as mean ± SEM (standard error of the mean). *p<0.05, ***p<0.001 vs. control mouse IgG2a treatment group. [Figure 4D] Therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-L1 antibody in a breast cancer model. (d) CD3+ / CD8+ T cell profile in the EMT6 tumor microenvironment (TME) of all groups. Flow cytometry data are the average of two independent experiments. t-test: all data are presented as mean ± SEM (standard error of the mean). *p<0.05, ***p<0.001 vs. control mouse IgG2a treatment group. [Figure 5A] The therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-L1 antibody in a lung cancer model is shown. (a) C57BL / 6 mice were injected with 5x105 LLC cells. Approximately 8 days later, when the average tumor size reached approximately 60-80mm3, mice were sorted into groups (n=5) such that the average tumor size of all groups was similar, and treatment by ip injection was initiated. Starting 8 days after tumor cell injection, some mice were ip injected with anti-BAG2 antibody 3B10 (200μg / mouse), anti-PD-L1 antibody (100μg / mouse), a combination of anti-PD-L1 antibody and 3B10, or an isotype control antibody once every 3 days until the end of the study. Experimental design is shown. t-test: All data are expressed as mean ± SEM (standard error of the mean). *p<0.05, **p<0.01 (vs. control mouse IgG2a-treated group). [Figure 5B]The therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-L1 antibody in a lung cancer model is shown. (b and c) Tumor volumes of control mice (black line, n=5), mice treated with anti-BAG2 antibody 3B10 alone (green line, n=5), mice treated with anti-PD-L1 antibody alone (blue line, n=5), and mice treated with both anti-BAG2 and anti-PD-L1 antibodies (red line, n=5). Tumor growth was monitored and tumor volumes were measured with electronic calipers on days 8, 11, 14, 17, and 20. On day 20, mice were sacrificed. (b) Tumor volumes of individual mice within groups. t-test: all data are presented as mean ± SEM (standard error of the mean). *p<0.05, **p<0.01 vs. control mouse IgG2a treatment group. [Figure 5C] The therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-L1 antibody in a lung cancer model is shown. (b and c) Tumor volumes of control mice (black line, n=5), mice treated with anti-BAG2 antibody 3B10 alone (green line, n=5), mice treated with anti-PD-L1 antibody alone (blue line, n=5), and mice treated with both anti-BAG2 and anti-PD-L1 antibodies (red line, n=5). Tumor growth was monitored and tumor volumes were measured with electronic calipers on days 8, 11, 14, 17, and 20. Mice were sacrificed on day 20. (c) Mean tumor growth of the four treatment groups. t-test: all data are presented as mean ± SEM (standard error of the mean). *p<0.05, **p<0.01 vs. control mouse IgG2a treatment group. [Figure 5D] The therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-L1 antibody in a lung cancer model. (d) CD3+ / CD8+ T cell profile in LLC tumor microenvironment (TME) of all groups. Flow cytometry data are the average of two independent experiments. t-test: All data are presented as mean ± SEM (standard error of the mean). *p<0.05, **p<0.01 vs. control mouse IgG2a treatment group. [Figure 6A]Figure 1 shows the therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-L1 antibody in a melanoma lung metastasis model. (a) C57BL / 6 mice were intravenously injected with 2x105 B16-F10-Luc2 cells. When performing BLI, mice received intraperitoneal (ip) injections of D-luciferin to background their BLI signals. Melanoma lung metastasis tumor growth was monitored by BLI signals on days 14, 20, 26, 32 and 38, and mice were sorted into groups (n=4) such that the average BLI signals of the four groups were similar. Starting from day 15 after tumor cell injection, some mice were intraperitoneally (ip) injected with anti-BAG2 antibody 3F12 (200μg / mouse) or isotype control antibody once every 4 days. From day 23, some mice received a single injection of anti-PD-L1 (100μg / mouse ip) or isotype control antibody every 4 days until the end of the study. Mice were sacrificed on day 39. Experimental design shown. t-test: All data are presented as mean±SEM (standard error of the mean). *p<0.05, **p<0.01, ***p<0.001 compared to control mouse IgG2a treatment group. [Figure 6B] The therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-L1 antibody in a melanoma lung metastasis model is shown. (b) Representative BLI signals of control mice (n=4), mice treated with anti-PD-L1 antibody only (n=4), mice treated with anti-BAG2 antibody 3F12 only (n=4), and mice treated with both anti-BAG2 antibody 3F12 and anti-PD-L1 antibody (n=4) at day 38. t-test: All data are presented as mean ± SEM (standard error of the mean). *p<0.05, **p<0.01, ***p<0.001 compared to control mouse IgG2a treatment group. [Figure 6C] Therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-L1 antibody in a melanoma lung metastasis model. (c) Quantification of BLI signal of tumors in the four treatment groups. t-test: All data are presented as mean ± SEM (standard error of the mean). *p<0.05, **p<0.01, ***p<0.001 vs. control mouse IgG2a treatment group. [Figure 7A]The therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-1 antibody in a colon cancer model is shown. (a) C57BL / 6 mice were injected with 5x105 MC38 cells. After about 8 days, when the average tumor size reached about 30mm3, mice were sorted into groups (n=6) such that the average tumor size of all groups was similar, and treatment by ip injection was started. Starting from day 8 after tumor cell injection, some mice were treated with anti-BAG2 antibody 3B10 (200μg / mouse), anti-PD-1 antibody (50μg / mouse), combination of anti-PD-1 antibody and 3B10, or isotype control antibody once every 4 days until the end of the study. Experimental design is shown. t-test: All data are expressed as mean ± SEM (standard error of the mean). *p<0.05, **p<0.01, ***p<0.001 (vs. control mouse IgG2a-treated group). [Figure 7B] The therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-1 antibody in a colon cancer model is shown. (b and c) Tumor volumes of control mice (black line, n=6), mice treated with anti-BAG2 antibody 3B10 alone (green line, n=6), mice treated with anti-PD-1 antibody alone (blue line, n=6), and mice treated with both anti-BAG2 and anti-PD-1 antibodies (red line, n=6). Tumor growth was monitored and tumor volumes were measured with electronic calipers on days 8, 10, 12, 14, 16, 18, and 20. On day 20, mice were sacrificed. (b) Tumor volumes of individual mice within a group. t-test: all data are presented as mean ± SEM (standard error of the mean). *p<0.05, **p<0.01, ***p<0.001 vs. control mouse IgG2a-treated group. [Figure 7C]The therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-1 antibody in a colon cancer model is shown. (b and c) Tumor volumes of control mice (black line, n=6), mice treated with anti-BAG2 antibody 3B10 alone (green line, n=6), mice treated with anti-PD-1 antibody alone (blue line, n=6), and mice treated with both anti-BAG2 and anti-PD-1 antibodies (red line, n=6). Tumor growth was monitored and tumor volumes were measured with electronic calipers on days 8, 10, 12, 14, 16, 18, and 20. On day 20, mice were sacrificed. (c) Mean tumor growth of the four treatment groups. t-test: all data are presented as mean ± SEM (standard error of the mean). *p<0.05, **p<0.01, ***p<0.001 vs. control mouse IgG2a treatment group. [Figure 7D] The therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-1 antibody in a colon cancer model. (d) CD3+ / CD8+ T cell profile in the MC38 tumor microenvironment (TME) of all groups. Flow cytometry data are the average of two independent experiments. t-test: all data are presented as mean ± SEM (standard error of the mean). *p<0.05, **p<0.01, ***p<0.001 vs. control mouse IgG2a-treated group. [Figure 8A] The therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-L1 antibody in a lung cancer model is shown. (a) BALB / c mice were injected with 3×105 CT26 cells. After about 11 days, when the average tumor size reached about 70 mm3, mice were sorted into groups (n=7) such that the average tumor size of all groups was similar, and treatment by ip injection was started. Starting on day 11 after tumor cell injection, some mice were treated with anti-BAG2 antibody 3B10 (250 μg or 750 μg / mouse), anti-PD-L1 antibody (200 μg / mouse), a combination of anti-PD-L1 antibody and 3B10 (250 μg or 750 μg / mouse), or an isotype control antibody every 2 days until the end of the study. Experimental design is shown. t-test: All data are presented as mean ± SEM (standard error of the mean). *p<0.05, ***p<0.001 (compared to the control mouse IgG2a-administered group). [Figure 8B]The therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-L1 antibody in a lung cancer model is shown. (b and c) Tumor volumes of control mice (black line, n=7), mice treated with 3B10 only (250μg, green line, n=7), mice treated with 3B10 only (750μg, brown line, n=7), mice treated with anti-PD-L1 antibody only (blue line, n=7), mice treated with both 3B10 (250μg) and anti-PD-L1 antibody (red line, n=7), and mice treated with both 3B10 (750μg) and anti-PD-L1 antibody (purple line, n=7). Tumor growth was monitored and tumor volumes were measured with electronic calipers on days 11, 13, 15, 17, 19, 21 and 23. On day 20, mice were sacrificed. (b) Tumor volumes of individual mice within a group. t-test: all data are expressed as mean ± SEM (standard error of the mean). *p<0.05, ***p<0.001 (vs. control mouse IgG2a-treated group). [Figure 8C] The therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-L1 antibody in a lung cancer model is shown. (b and c) Tumor volumes of control mice (black line, n=7), mice treated with 3B10 only (250μg, green line, n=7), mice treated with 3B10 only (750μg, brown line, n=7), mice treated with anti-PD-L1 antibody only (blue line, n=7), mice treated with both 3B10 (250μg) and anti-PD-L1 antibody (red line, n=7), and mice treated with both 3B10 (750μg) and anti-PD-L1 antibody (purple line, n=7). Tumor growth was monitored and tumor volumes were measured with electronic calipers on days 11, 13, 15, 17, 19, 21 and 23. On day 20, mice were sacrificed. (c) Mean tumor growth of the four treatment groups. t-test: all data are expressed as mean ± SEM (standard error of the mean). *p<0.05, ***p<0.001 (vs. control mouse IgG2a-treated group). [Figure 8D]The therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-L1 antibody in a lung cancer model. (d) CD3+ / CD8+ T cell profile in the MC38 tumor microenvironment (TME) of all groups. Flow cytometry data are the average of two independent experiments. t-test: all data are presented as mean ± SEM (standard error of the mean). *p<0.05, ***p<0.001 vs. control mouse IgG2a treatment group. [Figure 9A] The therapeutic efficacy of anti-BAG2 antibodies in combination with anti-PD-1 or anti-CTLA4 antibodies in pancreatic cancer models is shown. (a) C57BL / 6 mice were orthotopically injected with 2x106 PANC02-Luc cells. Approximately 15 days later, when BLI was performed, mice received an intraperitoneal (ip) injection of D-luciferin to background their BLI signals. Primary pancreatic tumor growth was monitored by BLI signals, and mice were sorted into groups (n=6-7) such that the mean BLI signals of the 15 groups were similar. Mice were treated with 200μg (low dose) / mouse (8 times on days 16-32) and 400μg (high dose) / mouse (3 times on days 35-39) of four anti-BAG2 antibodies (3B10, 3F12, 3B5, and 3G8), or an isotype control antibody, 3 times per week until the end of the study. Starting 28 days after implantation, mice were treated with anti-PD-1 (75 μg / mouse) or anti-CTLA4 (200 μg / mouse) or isotype control antibody three times a week until the end of the study. Mice were sacrificed on day 40. Experimental design shown. t-test: All data are presented as mean ± SEM (standard error of the mean). *p<0.05, **p<0.01, ***p<0.001 vs. control mouse IgG2a treatment group. ns (not significant). Tumor-specific lymphocytes (CD3+ / CD8+ T cells and CD45+ / CD3- / CD19+ B cells); tumor-specific NK cells (CD45+ / CD3- / CD49b+); tumor-specific myeloid cells (CD45+ / CD11b+ / Gr1- / F4 / 80+ macrophages, CD45+ / CD11b+ / Gr1+ MDSC); tumor-specific stromal cells (CD45- / CD90.2+). [Figure 9B]Therapeutic efficacy of anti-BAG2 antibodies in combination with anti-PD-1 or anti-CTLA4 antibodies in a pancreatic cancer model. (b) Tumor growth was monitored by IVIS imaging of BLI signals at 15, 27, and 39 days after implantation. BLI signals of mice treated with four anti-BAG2 antibodies alone (3B10, 3F12, 3B5, and 3G8) or an isotype control antibody (white bars, n=7 / group), mice additionally treated with anti-PD-1 antibodies (black bars, n=6 / group), and mice additionally treated with anti-CTLA4 antibodies (grey bars, n=4 / group). t-test: All data are presented as mean±SEM (standard error of the mean). *p<0.05, **p<0.01, ***p<0.001 (vs. control mouse IgG2a treatment group). ns (non-significant). Tumor-specific lymphocytes (CD3+ / CD8+ T cells and CD45+ / CD3- / CD19+ B cells); tumor-specific NK cells (CD45+ / CD3- / CD49b+); tumor-specific myeloid cells (CD45+ / CD11b+ / Gr1- / F4 / 80+ macrophages, CD45+ / CD11b+ / Gr1+ MDSC); tumor-specific stromal cells (CD45- / CD90.2+). [Figure 9C] Therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-1 or anti-CTLA4 antibody in pancreatic cancer model. (c) Primary tumor weight (mg) of individual mice within group at day 40. t-test: All data are expressed as mean ± SEM (standard error of the mean). *p<0.05, **p<0.01, ***p<0.001 (vs. control mouse IgG2a treatment group). ns (non-significant). Tumor-specific lymphocytes (CD3+ / CD8+ T cells and CD45+ / CD3- / CD19+ B cells); tumor-specific NK cells (CD45+ / CD3- / CD49b+); tumor-specific bone marrow cells (CD45+ / CD11b+ / Gr1- / F4 / 80+ macrophages, CD45+ / CD11b+ / Gr1+ MDSC); tumor-specific stromal cells (CD45- / CD90.2+). [Figure 9D]Therapeutic efficacy of anti-BAG2 antibodies in combination with anti-PD-1 or anti-CTLA4 antibodies in a pancreatic cancer model. (d) Mean metastases to liver (left), pleura (middle) and diaphragm (right) in the 15 treatment groups. t-test: All data are presented as mean ± SEM (standard error of the mean). *p<0.05, **p<0.01, ***p<0.001 vs. control mouse IgG2a treatment group. ns (not significant). Tumor-specific lymphocytes (CD3+ / CD8+ T cells and CD45+ / CD3- / CD19+ B cells); tumor-specific NK cells (CD45+ / CD3- / CD49b+); tumor-specific myeloid cells (CD45+ / CD11b+ / Gr1- / F4 / 80+ macrophages, CD45+ / CD11b+ / Gr1+ MDSC); tumor-specific stromal cells (CD45- / CD90.2+). [Figure 9E] The therapeutic efficacy of anti-BAG2 antibody in combination with anti-PD-1 or anti-CTLA4 antibody in pancreatic cancer model. (e) Profile of lymphoid, NK, myeloid and stromal cell populations in the PANC02-Luc tumor microenvironment (TME) of all groups. Tumor-specific CD45+ / CD11b+ / Gr1- / F4 / 80+ macrophages, CD45+ / CD11b+ / Gr1+ MDSCs and CD45- / CD90.2+ stromal cells. Flow cytometry data are the average of two independent experiments. t-test: all data are presented as mean ± SEM (standard error of the mean). *p<0.05, **p<0.01, ***p<0.001 (vs. control mouse IgG2a treatment group). ns (non-significant). Tumor-specific lymphocytes (CD3+ / CD8+ T cells and CD45+ / CD3- / CD19+ B cells); tumor-specific NK cells (CD45+ / CD3- / CD49b+); tumor-specific myeloid cells (CD45+ / CD11b+ / Gr1- / F4 / 80+ macrophages, CD45+ / CD11b+ / Gr1+ MDSC); tumor-specific stromal cells (CD45- / CD90.2+). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] Detailed Description of the Preferred Embodiments definition In this application, "a" and "an" are used to refer to both a singular and plural entity.

[0051] As used herein, "about" or "substantially" generally provides a tolerance from being limited to an exact number. For example, "about" or "substantially" used in the context of the length of a polypeptide sequence indicates that the polypeptide is not limited to the recited number of amino acids. A few amino acids may be included in addition to or subtracted from the N-terminus or C-terminus, as long as functional activity, such as binding activity, exists.

[0052] As used herein, administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0053] As used herein, "hot spots" in reference to sequences in the complementarity determining region refer to the presence of amino acid residues that cause instability to the antigen binding region, and therefore such motifs should be replaced for better binding efficiency. Although there is no list of amino acids that are typically replaced, one way to consider replacing amino acid residues on CDRs is to consider germline conservation and antibody sequence structure, and select amino acids to replace with similar structures. For example, NG can be mutated to NA, since the structures of both amino acids are similar. Such modifications allow greater binding efficiency of CDR to BAG2.

[0054] As used herein, "amino acid" and "amino acids" refer to all naturally occurring L-α-amino acids. This definition is meant to include norleucine, ornithine, and homocysteine.

[0055] The term "amino acid sequence variant" as generally used herein refers to a molecule having some differences in amino acid sequence compared to a reference (e.g., native sequence) polypeptide. The amino acid modifications may be substitutions, insertions, deletions in the native amino acid sequence or any desired combination of such changes.

[0056] Substitution variants are those that have at least one amino acid residue in the native sequence removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.

[0057] Substitutes for an amino acid within a sequence may be selected from other members of the class to which the amino acid belongs. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the present invention are proteins or fragments or derivatives thereof that exhibit the same or similar biological activity, and derivatives that are differentially modified during or after translation, for example, by glycosylation, proteolytic cleavage, linkage to antibody molecules or other cellular ligands, etc.

[0058] Insertional variants are those that have one or more amino acids inserted at a position immediately adjacent to an amino acid at a unique location in the native amino acid sequence, where immediately adjacent to an amino acid means linked to either the α-carboxy or α-amino functionality of the amino acid.

[0059] Deletion variants are those in which one or more amino acids in the native amino acid sequence have been removed. Usually, deletion variants will have one or two amino acids deleted in a unique region of the molecule.

[0060] As used herein, "fragment" or "functional derivative" refers to biologically active amino acid sequences or fragments of the polypeptides of the invention, as well as derivatives obtained by reaction with organic derivatizing agents, post-translational modification, derivatives with nonproteinaceous polymers, and covalent modifications, including immunoadhesins.

[0061] As used herein, "carrier" includes pharma- ceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed at the dosages and concentrations used. In many cases, pharma-ceutically acceptable carriers are aqueous pH buffered solutions. Examples of pharma-ceutically acceptable carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrose; clating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or non-ionic surfactants, such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

[0062] As used herein, "pharmaceutically acceptable carrier and / or diluent" includes any solvent, dispersion medium, coating antibacterial and antifungal agents, isotonic and absorption retarding agents, and the like.The use of such media and agents for pharmaceutically active substances is well known in the art.Except where any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.Auxiliary active ingredients can also be incorporated into the composition.

[0063] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form.Dosage unit form as used herein refers to a physically separate unit suitable as a single dose for the mammalian subject to be treated; each unit contains a predetermined amount of active material calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification for dosage unit form of the present invention is determined by and directly depends on (a) the specific characteristics of active material and the inherent therapeutic effect to be realized, and (b) the limitations inherent in the art of compounding such active material for the treatment of disease in living subjects with disease state that impairs physical health.

[0064] The main active ingredient is combined with a suitable pharma- ceutically acceptable carrier in a dosage unit form for convenient and effective administration in an effective amount. A dosage unit form may contain, for example, the main active compound in an amount ranging from 0.5 μg to about 2000 mg. Expressed in percentages, the active compound is generally present in about 0.5 μg / ml of carrier. In the example of a composition containing a supplementary active ingredient, the dosage is determined by referring to the usual dosage and administration method of the ingredient.

[0065] As used herein, "vector", "polynucleotide vector", "construct" and "polynucleotide construct" are used interchangeably herein. The polynucleotide vector of the present invention may exist in any of several forms, including, but not limited to, RNA, DNA, RNA encapsulated in a retroviral coat, DNA encapsulated in an adenoviral coat, DNA packaged in another virus or virus-like form (such as adenovirus structures such as herpes simplex and polyamidos).

[0066] As used herein, a "host cell" includes an individual cell or cell culture that can be or has been a recipient of a vector of the invention. A host cell includes the progeny of a single host cell, which progeny may not necessarily be completely identical (morphologically or in complementary total DNA) to the original parent cell due to natural, accidental, or deliberate mutation and / or change.

[0067] As used herein, a "subject" is a vertebrate, preferably a mammal, more preferably a human.

[0068] As used herein, for purposes of treatment, "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, etc. Preferably, the mammal is a human.

[0069] As used herein, a therapeutic agent that "prevents" a disorder or disease refers to a compound that reduces the occurrence of a disorder or disease in a treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of a disorder or disease, or reduces the severity of a disorder or disease in a statistical sample relative to an untreated control sample.

[0070] The terms "reduce", "reduced", "reduction" or "inhibit" are all used herein to mean a decrease or lowering of a characteristic, level or other parameter by a statistically significant amount. In some embodiments, "reduce", "reduction" or "reduce" or "inhibit" typically means a decrease of at least 10% compared to a reference level (e.g., in the absence of a given treatment), and can include, for example, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more decreases. As used herein, "reduce" or "inhibit" does not include complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. The decrease can preferably be a drop to a level that is accepted as within the normal range for individuals without a given disorder.

[0071] The terms "increased," "increase," or "enhance" or "activate" are all used herein to mean an increase in a property, level or other parameter, generally in a statistically significant amount, and for the avoidance of any doubt, the terms "increased," "increase," or "enhance" or "activate" mean an increase of at least 10% compared to a reference level, such as an increase of at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or less including 100%, or any increase between 10-100%, or mean an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or at least about 20-fold, or at least about 50-fold, or at least about 100-fold, or more compared to a reference level.

[0072] "Cancer" or "tumor" as used herein refers to the uncontrolled growth of cells that interfere with the normal function of bodily organs and tissues. A subject with cancer or tumor is one who has objectively measurable cancer cells present in the subject's body. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer that migrates from its original location and disseminates to vital organs may ultimately lead to the death of the subject through the functional destruction of the affected organ. Examples of cancer include, but are not limited to, B-cell lymphoma (Hodgkin's lymphoma and / or non-Hodgkin's lymphoma), brain cancer, breast cancer, colon cancer, lung cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, kidney cancer, carcinoma, melanoma, head and neck cancer, brain cancer such as glioblastoma, and prostate cancer, including but not limited to androgen-dependent prostate cancer and androgen-independent prostate cancer.

[0073] The term "effective amount" or "therapeutically effective amount" as used herein refers to an amount of one or more anti-BAG2 antibodies or fragments thereof disclosed herein, or an amount of a pharmaceutical composition comprising one or more anti-BAG2 antibodies or fragments thereof, to reduce at least one or more symptoms of a disease or disorder, and relates to a sufficient amount of a pharmacological composition to provide a desired effect. As used herein, the phrase "therapeutically effective amount" refers to a sufficient amount of a composition to treat a disorder at a reasonable benefit / risk ratio applicable to any medical treatment.

[0074] The term "administering" as used herein refers to the placement of an agent or composition disclosed herein into a subject by a method or route that results in at least partial localization of the agent or composition at a desired site. "Route of administration" may refer to any route of administration known in the art, including, but not limited to, oral, topical, aerosol, nasal, via inhalation, anal, intraanal, perianal, transmucosal, transdermal, parenteral, enteral, or topical. "Parenteral" refers to a route of administration that is generally associated with injection, including intratumoral, intracranial, intraventricular, intrathecal, epidural, intradural, intraorbital, infusion, intravesical, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intravascular, intravenous, intraarterial, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via parenteral route, the agent or composition may be in the form of a solution or suspension for infusion or injection, or as a lyophilized powder. Via the enteral route, the agent or composition may be in the form of a capsule, gel capsule, tablet, dragee, syrup, suspension, solution, powder, granule, emulsion, microsphere or nanosphere, or lipid or polymer vesicle that allows controlled release. Via the external route, the agent or composition may be in the form of an aerosol, lotion, cream, gel, ointment, suspension, solution or emulsion. In one embodiment, the agent or composition may be provided in powder form and mixed with a liquid such as water to form a drink. According to the present invention, "administering" may be self-administration. For example, a subject consuming a composition disclosed herein is considered to be "administering".

[0075] As used herein, "subject" refers to a human or an animal. Typically, an animal is a vertebrate, such as a primate, a rodent, a livestock animal, or a game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, squirrels, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, and canine species, such as dogs, foxes, and wolves. The terms "patient," "individual," and "subject" are used interchangeably herein. In one embodiment, the subject is a mammal. The mammal can be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. In addition, the methods described herein can be used to treat livestock and / or pets. In one embodiment, the subject is a human.

[0076] "Mammal" as used herein refers to any member of the class Mammalia, including, but not limited to, humans and non-human primates, such as chimpanzees and other ape and monkey species; farm animals, such as cattle, sheep, pigs, goats and horses; domestic mammals, such as dogs and cats; laboratory animals, including rodents, such as mice, rats and guinea pigs, and the like. The term does not denote a specific age. Thus, adult and newborn subjects, as well as fetuses, are intended to be included within the scope of this term.

[0077] The subject may be suffering from or previously diagnosed or identified as having a disease (e.g., cancer or an autoimmune disease) that requires treatment, or one or more complications related to the disease, and optionally have already been treated for the disease or one or more complications related to the disease. Alternatively, the subject may not have previously been diagnosed with the disease or one or more complications related to the disease. For example, the subject may be one that exhibits one or more risks for the disease or one or more complications related to the disease, or one that does not exhibit risk factors. For example, the subject may be one that exhibits one or more symptoms for the disease or one or more complications related to the disease, or one that does not exhibit symptoms. A "subject in need" of diagnosis or treatment for a particular disease may be a subject suspected of having the disease, a subject that has been diagnosed with the disease, a subject that has already been treated or will be treated for the disease, a subject that will not be treated for the disease, or a subject at risk of developing the disease.

[0078] "At risk" shall mean an increased risk compared to a normal subject or compared to a control group, e.g., a patient population. Thus, a subject carrying a unique marker may have an increased risk for a specific disease or disorder and may be identified as needing further testing. "Increased risk" or "elevated risk" refers to any statistically significant increase, e.g., in the probability that a subject has a disorder. The risk is preferably increased by at least 10%, more preferably at least 20%, and even more preferably at least 50% over the control group to which the comparison is made.

[0079] The term "statistically significant" or "significantly" refers to statistical significance, which generally means at least two standard deviations (2SD) away from the reference level. This term refers to statistical evidence that a difference exists. It is defined as the probability of deciding to reject the null hypothesis when the null hypothesis is in fact true.

[0080] The term "co-administered" as used herein refers to administration of two or more therapies or two or more therapeutic agents (e.g., an anti-BAG2 antibody and an additional anti-cancer therapy) within 24 hours of each other, for example, as part of a clinical treatment regimen. In other embodiments, "co-administered" refers to administration within 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes of each other. In other embodiments, "co-administered" refers to administration at the same time, either as part of a single combination or as multiple combinations administered by the same or different routes. For example, when an anti-BAG2 antibody and an additional anti-cancer therapy are administered in different pharmaceutical compositions or at different times, the routes of administration can be the same or different.

[0081] As used herein, "treatment" is an approach to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, attenuation of the extent of disease, a stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, improvement or alleviation of disease state, and remission (whether partial or complete). "Treatment" can also mean prolonged survival compared to expected survival in the absence of treatment. "Treatment" refers to both therapeutic treatment and protective or preventative measures. Those in need of treatment include those already with the disorder as well as those in whom the disorder is to be prevented. "Alleviating" a disease means that the extent of the disease state and / or undesirable clinical manifestations are reduced and / or the time course of progression is slowed or extended compared to the situation without treatment.

[0082] The term "chimeric" antibody as used herein refers to an antibody that has variable sequences derived from non-human immunoglobulins, such as rat or mouse antibodies, and human immunoglobulin constant regions, typically selected from human immunoglobulin templates. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816,397.

[0083] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins that contain minimal sequence derived from non-human immunoglobulin. Generally, a humanized antibody contains substantially all or at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are of a human immunoglobulin sequence. A humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), typically of a human immunoglobulin consensus sequence. Methods for antibody humanization are known in the art. See, e.g., Riechmann et al., 1988, Nature 332:323-7; U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370 to Queen et al.; European Patent No. 239400; PCT Publication No. WO 91 / 09967; U.S. Patent No. 5,225,539; European Patent No. 592106; European Patent No. 519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci. 91:969-973; and U.S. Patent No. 5,565,332.

[0084] "Human antibody" includes antibodies having the amino acid sequence of human immunoglobulins, and includes antibodies isolated from a human immunoglobulin library for one or more human immunoglobulins, or from transgenic animals that do not express endogenous immunoglobulins. Human antibodies can be produced by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT Publications WO98 / 46645; WO98 / 50433; WO98 / 24893; WO98 / 16654; WO96 / 34096; WO96 / 33735; and WO91 / 10741. Human antibodies can also be produced using transgenic mice that cannot express functional endogenous immunoglobulins, but can express human immunoglobulin genes. See, e.g., PCT Publications WO98 / 24893; WO92 / 01047; WO96 / 34096; WO96 / 33735; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598. Additionally, companies such as LakePharma, Inc. (Belmont, Calif.) or Creative BioLabs (Shirley, New York) may be engaged to provide human antibodies against a selected antigen using technology similar to that described above. All-human antibodies that recognize a selected epitope can be generated using a technique called "guided selection," in which a selected non-human monoclonal antibody, e.g., a murine antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope (see Jespers et al., 1988, Biotechnology 12:899-903).

[0085] The term "antibody-like" as used herein refers to molecules that can be engineered to contain portions of antibodies, but are not essentially naturally occurring antibodies. Examples include, but are not limited to, CAR (chimeric antigen receptor) T-cell technology and Ylanthia® technology. CAR technology utilizes antibody epitopes fused to portions of T-cells so that the body's immune system is directed to attack specific target proteins or cells. Ylanthia® technology consists of an "antibody-like" library, a collection of synthetic human fabs that are then screened for binding to peptide epitopes from target proteins. The selected Fab regions can then be engineered into a scaffold or framework so that they resemble antibodies.

[0086] As used herein, an "immunotherapeutic agent" or "immunomodulator" is an agent designed to induce or amplify an immune response, or to reduce or suppress that response.

[0087] As used herein, "high homology" refers to at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% identity in the designed region of overlap between any two polypeptides.

[0088] Remove hot spots During manufacture, storage and in vivo, therapeutic antibodies are at risk of degradation via many pathways. Notably, the most frequent degradation reaction occurring in proteins is the chemical degradation of Asn and Asp residues. Although these reactions can be suppressed by appropriate storage and formulation conditions of the final drug substance and drug product, degradation during fermentation, downstream processing and in vivo often cannot be adequately controlled. When Asn and Asp residues are involved in antigen recognition, their chemical modification can result in severe loss of capacity. In several cases, these degradation events have been reported to interfere with long-term mAb function. In vivo, proteolytic events have been described in association with protein aging and cancer by initiating apoptosis, or with severe effects on other biological functions, such as reduced stability of human lens beta A3 crystallin, abnormal MAPK signaling, potential alteration of beta-secretase efficacy and specificity in the Abeta production process, or increased lysozyme lytic activity against bacterial cells. Identification of drug candidates susceptible to degradation would ideally be performed early in the drug development process in order to adjust manufacturing and formulation processes accordingly or to re-engineer problematic candidates to remove such hot spots.

[0089] Asn and Asp residues share a degradation pathway that proceeds via the formation of a cyclic succinimide intermediate. The succinimide results from deamidation of Asn or dehydration of Asp by nucleophilic attack of the backbone nitrogen of the successive amino acid on the Asn / Asp side chain γ-carbonyl group. The transferable cyclic imide can hydrolyze at either one of the two carbonyl groups to form aspartyl or isoaspartyl bonds in different ratios, depending on the hydrolysis conditions and conformational constraints. In addition, alternative degradation mechanisms of Asn such as nucleophilic attack by the backbone carbonyl oxygen forming a cyclic isoimide or direct hydrolysis of Asn to Asp have been proposed. Multiple analytical methods, mostly charge-sensitive methods such as ion-exchange chromatography or isoelectric focusing, have been described to detect the degradation products, i.e. either succinimide, Asp or isoAsp. The most suitable for quantification and localization of degradation sites in proteins is analysis by liquid chromatography tandem mass spectrometry (LC-MS / MS). Reference Sydow et al., "Structure-Based Prediction of Asparagine and Aspartate Degradation Sites in Antibody Variable Regions", PLoS One. 2014; 9(6):e100736, published online on June 24, 2014 at doi: 10.1371 / journal.pone.0100736, is incorporated herein by reference in its entirety for its disclosure of the well-established knowledge of the presence of hotspots on the antigen-binding regions of antibodies and the desirability of replacing such hotspot motifs to provide more stability to proteins.

[0090] One such example of hotspot detection and replacement can be seen in antibody 3B5. The VH CDR2 is YIDPYNGGNTYNRKFKG, but the hotspot is detected by the presence of "NG", which is removed and replaced with "NA" so that the sequence with the hotspot removed is YIDPYNAGNTYNRKFKG. The applicant notes that the presence of Y at the beginning of CDR2 must be considered as part of the CDR2. It is generally accepted in the art that CDR sequences maintain binding activity even when some sequences are truncated or shortened by a few amino acids at the N-terminus or C-terminus. The length of the CDR to retain activity is believed to be well within reasonable experimentation to determine.

[0091] Antibody Humanization The process of humanizing antibodies is well known and therefore conventional techniques may be used to produce humanized antibodies. One exemplified protocol may be as follows:

[0092] Mouse monoclonal antibodies are humanized by CDR grafting and key residues of the parent mouse antibody framework are identified and introduced into the humanized sequence. The humanized VH / VL are then converted to full-length hIgG1 format. The variants are expressed by transient transfection and purified by affinity chromatography. The purified antibodies are characterized by SEC-HPLC, SDS-PAGE, ELISA, and Biacore (affinity characterization). The best candidates with less than 3-fold loss in affinity and potency are selected.

[0093] Five phases may be included in the antibody humanization process: First, the antibody humanization design (feasibility of hotspot removal and design of antibody humanization) is completed.

[0094] The VH / VL CDR residues are determined and annotated with the Kabat numbering system. Sequence analysis is applied to identify major risk hotspots, including unpaired cysteine ​​residues, N-glycosylation sites, and deamination sites within the CDRs. Genetic engineering procedures may be applied to remove deleterious hotspot motifs. If hotspots exist in the CDRs, the feasibility of hotspot removal is checked and implemented.

[0095] Design hotspot removal and combine the relative mouse VH and mouse VL into multiple chimeric antibodies. Each chimeric antibody is transiently expressed in 100mL 293 cells, the supernatant is captured and purified by Mabselect PrismA (GE, 17549801), and the affinity purification yield is recorded. Purified chimeric antibodies are tested by SDS-PAGE and HPLC-SEC, and then affinity is confirmed by ELISA or Biacore. Affinity ranking of chimeric antibodies is performed to confirm the feasibility of hotspot removal.

[0096] Such humanized sequences may be as follows for the following antibodies:

[0097] 3B5 subclone 1: VH region; underlined are CDR regions after checking for hotspots. QVQLVQSGAEVKKPGASVKVSCKAS GYSFTDYTFY WVRQAPGQRLEWIG YIDPYNAGNTYNRKFKG RVTITVDKSASTAYMELSSLRSEDTAVYYCAR GYYRYGGGGDFDY WGQGTLVTVSS(SEQ ID NO:75)

[0098] 3B5 subclone 1: VL region; underlined are CDR regions after checking for hotspots. DVVMTQSPLSLPVTLGQPASISC RSSQSLVHSNGNTYLH WFQQRPGQSPRLLIH KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC SQNTHIPPTFGGGTKVEIK(SEQ ID NO:76)

[0099] 3B5 subclone 2: VH region; underlined are CDR regions after checking for hotspots. QVQLVQSGAEVKKPGASVKVSCKAS GYSFTDYTFY WVRQAPGQRLEWIG YIDPYNAGNTYNRKFKG KVTITVDKSASTAYMELNSLRSEDTAVYYCAR GYYRYGGGGDFDY WGQGTLVTVSS(SEQ ID NO:77)

[0100] 3B5 subclone 2: VL region; underlined are CDR regions after checking for hotspots. DVVMTQSPLSLPVTLGQPASISC RSSQSLVHSNGNTYLH WFQQRPGQSPRLLIH KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC SQNTHIPPT FGGGTKVEIK(SEQ ID NO:78)

[0101] 3B10 subclone 1: VH region; underlined are CDR regions after checking for hotspots. QVQLVQSGAEVKKPGASVKVSCKAS GHAFTNYMIE WVRQAPGQGLEWMG VINPGSGGTYNSEKVKG RVTLTADRSISTAYMELSRLRSDDTAVYYCRI YGNYKGYFDH WGQGTLVTVSS(SEQ ID NO:79)

[0102] 3B10 subclone 1: VL region; underlined are CDR regions after checking for hotspots. DIQMTQSPSSLSASVGDRVTITC KASQDMNSYLS WFQQKPGKAPKSLIY RSNRLVD GVPSRFSGSGSGTDFLTISSLQPEDFATYYC LQNDEFPFT FGQGTKLEIK(SEQ ID NO:80)

[0103] 3B10 subclone 2: VH region; underlined are CDR regions after checking for hotspots. QVQLVQSGSELKKPGASVKVSCKAS GYSFTKYGMN WVKQAPGQGLEWMG WINTNTGEATYGEEVKG RFVFSLDTSVSTAYLQISSLKAEDTAVYYCAR LGLRYLDY WGQGTLVTVSS(SEQ ID NO:81)

[0104] 3B10 subclone 2: VL region; underlined are CDR regions after checking for hotspots. DIQMTQSPSSLSASVGDRVTITC RASKSVSTSDYSYMH WYQQKPGKAPKLLIY LASNLES GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QHNRELPPT FGQGTKLEIK (SEQ ID NO: 82)

[0105] 3G8 subclone 1: VH region; underlined are CDR regions after checking for hotspots. QVQLVQSGSELKKPGASVKVSCKAS GYSFTKYGMN WVRQAPGQGLEWMG WINTNTGEATYGEEVKG RFVFSLDTSVSTAYLQISSLKAEDTAVYYCAR LGLRYLDY WGQGTLVTVSS(SEQ ID NO:83)

[0106] 3G8 subclone 1: VL region; underlined are CDR regions after checking for hotspots. DIQMTQSPSSLSASVGDRVTITC RASKSVSTSDYSYMH WYQQKPGKAPKLLIY LASNLES GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QHNRELPPT FGQGTKLEIK (SEQ ID NO: 84)

[0107] 3G8 subclone 2: VH region; underlined are CDR regions after checking for hotspots. QVQLVQSGSELKKPGASVKVSCKAS GYSFTKYGMN WVKQAPGQGLEWMG WINTNTGEATYGEEVKG RFVFSLDTSVSTAYLQISSLKAEDTAVYYCAR LGLRYLDY WGQGTLVTVSS(SEQ ID NO:85)

[0108] 3G8 subclone 2: VL region; underlined are CDR regions after checking for hotspots. DIQMTQSPSSLSASVGDRVTITC RASKSVSTSDYSYMH WYQQKPGKAPKLLIY LASNLES GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QHNRELPPT FGQGTKLEIK (SEQ ID NO: 86)

[0109] Use of anti-Bag2 antibodies for the treatment of cancer - Patents.com Those antibodies that inhibit the growth of cancer or its progression to a more metastatic state may be selected for use as anti-cancer therapeutics and administered to patients for the treatment or prevention of cancer. The selected antibodies may be further optimized, for example, by genetic engineering or creating human chimeric or fully human antibodies. To demonstrate the efficacy of this approach,

[0110] Detection of elevated levels of Bag2 in a patient sample is diagnostic of the presence of cancer or progression to a more aggressive or metastatic state. Detection of elevated levels of BAG2 species in a patient sample would be indicative of the patient having cancer or being at risk of developing cancer. The levels of BAG2 species can be measured or assessed by PCR, hybridization schemes, cycling probe technology, FISH, immunocytochemistry, IHC, Western blot, immunoprecipitation, sandwich assays, ELISA assays, and the like. The patient sample can be a fluid sample, blood sample, milk, urine, cell, liquid biopsy, biopsy, and the like. In a patient diagnosed with cancer, elevated levels of BAG2 are indicative of increased metastatic potential. Elevated levels of BAG2 are indicative of prostate cancer. The antibodies of the present invention are used to detect BAG2 and are used as diagnostic tools.

[0111] Because cells and tissues do not normally secrete BAG2, an effective method for diagnosing cancer, or a more aggressive or metastatic form, or a shift to a more aggressive form, is to measure the level of BAG2 in a sample from a patient, from a cell or tissue harvest, or from a cultured cell, compared to the level of BAG2 in a healthy sample, or compared to the level of BAG2 known to be present in healthy adult cells or tissues. Elevated levels of BAG2 indicate the presence of cancer, the presence of metastatic cancer, or the onset of metastasis. The sample assayed for the presence of BAG2 may be a cell harvest, which may be a cultured cell line or cells from a patient, a body fluid, a blood sample, a tissue specimen, or a biopsy specimen. Thus, a diagnostic assay to detect the presence or progression of cancer comprises the steps of: 1) obtaining a sample from a patient having cancer or at risk of developing cancer; 2) subjecting the sample to an assay capable of detecting BAG2 or measuring the level of BAG2; 3) comparing the level of BAG2 protein measured in the test sample to the level in a control patient or control cell; 4) determining that the level of BAG2 is elevated compared to the control; and 5) concluding that the donor of the test sample has cancer or has the progression of cancer if the control to which the test sample is compared is from a donor previously diagnosed with cancer.

[0112] In this assay, the control sample to which the test sample is compared can be a non-cancerous cell, a cultured cell, a sample from a healthy donor, a non-cancerous sample from a donor, or a sample from the donor of the test sample, and the control sample has been collected from the donor at a time in the past.The source of such a sample can be any specimen taken from the patient to be tested for the presence or progression of cancer, including body fluid, cerebrospinal fluid, a bone marrow sample, blood, tissue, cells, biopsy tissue or cells, cultured cells derived from patient cells, and the like.The source of the sample to which the test sample is compared can be body fluid, cerebrospinal fluid, a bone marrow sample, blood, tissue, cells, biopsy tissue or cells, or cultured cells that can be derived from a healthy donor or a test patient, and the sample has been collected at a time in the past.The measurement level to which the test sample is compared can be from pre-recorded data and compiled into a list for comparison to the test sample.

[0113] Combination therapy The first agent is administered in conjunction with another agent. "In conjunction with" refers to administration of one treatment modality in addition to another treatment modality, such as administration of an immunomodulatory agent described herein in addition to administration of an anti-BAG2 antibody or fragment thereof to the same individual. Thus, "in conjunction with" refers to administration of one treatment modality before, during, or after delivery of the other treatment modality to an individual. Such combinations are considered to be part of a single treatment regimen or regimen.

[0114] Immunomodulators Immunomodulators or immunotherapeutic drugs can be broadly divided into four categories: checkpoint inhibitors, cytokines, agonists, and adjuvants. Checkpoint inhibitors work by blocking immune checkpoints, or "brakes" on the immune system, that tumors frequently manipulate to shut down immune responses and protect themselves. As a result, checkpoint inhibitors can elicit new immune responses against cancer as well as enhance existing responses to promote the elimination of cancer cells. As of 2020, checkpoint inhibitors are perhaps the best known and most widely successful immunomodulators ever developed.

[0115] For example, the PD-1 / PD-L1 immune checkpoint pathway can shut down cancer-targeting T cells, but when checkpoint inhibitors block the PD-1 / PD-L1 pathway, they can cause T cells to eliminate cancer cells.

[0116] Cytokines are messenger molecules that regulate the maturation, growth, and responsiveness of immune cells. Currently, there are four FDA-approved cytokine immunotherapeutic drugs for the treatment of a subset of patients with renal cancer, leukemia, lymphoma, melanoma, and sarcoma.

[0117] Agonists activate pathways that promote the adaptive immune response, either by helping activate "killer" T cells that directly attack cancer cells, or by stimulating the activity of innate immune cells, such as dendritic cells, which coordinate the overall immune response against cancer by presenting cancer markers and enhancing T cell activity.

[0118] Adjuvants activate pathways involving the innate immune system that can stimulate a general immune response and ultimately promote an adaptive immune response. One FDA-approved adjuvant immunotherapy is currently available for the treatment of a subset of patients with squamous cell carcinoma, a type of skin cancer.

[0119] Examples of such immunomodulators are granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 12 (IL-12), interleukin 15 (IL-15), B7-1 (CD80), B7-2 (CD86), 4-1BB ligand, GITRL, OX-40L, anti-CD3 antibody, anti-CD27 antibody, anti-CTLA4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-GITR antibody, anti-OX-40 antibody, anti-4-1BB antibody, anti-LAG-3 antibody, and anti-TIM-3 antibody.

[0120] Adjuvant therapy Anti-BAG2 antibodies may be used as an adjunct or in conjunction with other drugs or treatments that have anti-cancer properties. When used adjunctively, anti-BAG2 antibodies and other drug(s) may be combined together in a single combined drug combination, or may be combined and administered separately, in a single coordinated dosing regimen, or in different dosing regimens. Drugs administered adjunctively or together with anti-BAG2 antibodies typically have complementary activities to the anti-BAG2 antibodies, so that the antibodies or other drugs do not adversely affect each other.

[0121] Agents that may be administered adjunctively or in conjunction with anti-CD40 antibodies include alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotics, antiproliferatives, antivirals, Aurora kinase inhibitors, activators of the death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (Bi-specific T cell engager) antibodies, antibody drug conjugates, biological response modifiers, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia viral oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormonal therapy, immunologicals, inhibitors of apoptosis proteins, and the like. These include, but are not limited to, interfering agents (IAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, inhibitors of mammalian target of rapamycin (mTor), microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDs), poly ADP-ribose polymerase (PARP) inhibitors, platinum chemotherapeutic drugs, Bruton's tyrosine kinase (BTK) inhibitors (e.g., ibrutinib, acalabrutinib), polo-like kinase (PlK) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoid / deltoid plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, and the like, as well as combinations of one or more of these agents.

[0122] Examples of immune agents include, but are not limited to, interferons, immune checkpoint inhibitors, and other immune enhancing agents. Interferons include interferon alpha, interferon alpha-2a, interferon alpha-2b, interferon beta, interferon gamma-1a, interferon gamma-1b, or interferon gamma-nl, combinations thereof, and the like. Immune checkpoint inhibitors include antibodies that target PD-1 (e.g., pembrolizumab and nivolumab), PD-L1 (e.g., durvalumab, atezolizumab, avelumab, MEDI4736, MSB0010718C, and MPDL3280A), and CTLA4 (cytotoxic lymphocyte antigen 4; e.g., ipilimumab, tremelimumab). Immune enhancing agents include anti-OX40 agonist antibodies that activate T cells.

[0123] Anti-BAG2 antibodies may also be used to enhance the effectiveness of radiation therapy. Examples of radiation therapy include external beam radiation therapy, internal beam radiation therapy (i.e., brachytherapy), and total body radiation therapy.

[0124] Theranostics Patients diagnosed with elevated levels of secreted BAG2 protein are then treated with a therapeutic agent that specifically binds to BAG2. Thus, patients diagnosed with elevated levels of secreted BAG2 will benefit from treatment with a therapeutic agent that inhibits BAG2. Thus, assessing the suitability of cancer treatment and administration of an effective amount of a therapeutic agent for treating or preventing cancer comprises: 1) obtaining a sample from a patient suspected of having cancer or at risk of developing cancer or at risk of developing metastatic cancer; 2) measuring the amount of secreted BAG2, the measured level being significantly higher than that measured in a control sample; 3) determining that the patient has cancer or develops a more aggressive or metastatic cancer; 4) administering to the patient an effective amount of a therapeutic agent that suppresses the expression of BAG2.

[0125] Chemically Modified Peptides Polypeptide or antibody therapeutics may suffer from short circulating half-life and proteolysis and low solubility.To improve the pharmacokinetic and pharmacodynamic properties of the biopharmaceuticals of the present invention, methods such as amino acid sequence manipulation may be performed to reduce or increase immunogenicity, reduce proteolytic cleavage, and peptide fusion or conjugation to serum proteins such as immunoglobulins and albumins may also be performed; for biopharmaceuticals such as the peptides and antibodies of the present invention, incorporation into drug delivery vehicles for protection and sustained release may also be performed; conjugation to natural or synthetic polymers is also contemplated.In particular, for synthetic polymer conjugation, PEGylation, or acylation, such as N-acylation, S-acylation, etc. are also contemplated.

[0126] nucleic acid construct Also provided is an expression vector comprising a nucleic acid molecule of the invention described herein, wherein the nucleic acid molecule is operably linked to an expression control sequence. Also provided is a host-vector system for the production of a polypeptide comprising an expression vector of the invention introduced into a host cell suitable for expression of the polypeptide. Suitable host cells may be bacterial cells such as E. coli, yeast cells such as Pichia pastoris, insect cells such as Spodoptera frugiperda, or mammalian cells such as COS, HEK, or CHO cells.

[0127] The invention also provides methods for producing a polypeptide of the invention by growing cells of the host-vector system described herein under conditions that allow the production of the polypeptide of the invention, and recovering the polypeptide so produced. Polypeptides useful in practicing the invention may be prepared by expression in prokaryotic or eukaryotic expression systems.

[0128] Any number of methods may be utilized to express the recombinant gene and purify the polypeptide. The gene may be subcloned into a bacterial expression vector, such as, but not limited to, pZErO.

[0129] The polypeptide may be purified by any technique that allows the subsequent formation of a stable biologically active protein.For example, and without limitation, the factor may be recovered from cells as either a soluble protein or inclusion body, which may be quantitatively extracted with 8M guanidine hydrochloride and dialysis.To further purify the factor, any number of purification methods may be used, including, but not limited to, conventional ion exchange chromatography, affinity chromatography, different sugar chromatography, hydrophobic interaction chromatography, reverse phase chromatography or gel filtration.

[0130] Any method known to those skilled in the art for the insertion of DNA fragments into vectors may be used to construct an expression vector encoding the polypeptide of the present invention with appropriate transcriptional / translational control signals and protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques, as well as in vivo recombination (genetic recombination). Expression of the nucleic acid sequence encoding the polypeptide may be regulated by a second nucleic acid sequence, such that the polypeptide of the present invention is expressed in a host transformed with the recombinant DNA molecule. For example, expression of the polypeptides described herein may be controlled by any promoter / enhancer element known in the art. Promoters that can be used to control the expression of a polypeptide include those containing the long terminal repeat described in Squinto et al., (1991, Cell 65:1-20); the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the CMV promoter, the M-MuLV 5' terminal repeat, the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:144-1445), the regulatory sequences of the metallothionein genes (Brinster et al., 1982, Nature 296:39-42); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. USA80:21-25), see also "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; promoter elements from yeast or other fungi, such as the Gal4 promoter, the ADH (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter, the alkaline phosphatase promoter, as well as the following animal transcriptional control regions that exhibit tissue specificity and have been utilized in transgenic animals: the elastase I gene control region, active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulin gene control region, active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin gene control region active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), mouse mammary tumor virus control region active in Sendai virus, lentivirus, albumin gene control region active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58); the alpha 1-antitrypsin gene control region active in the liver (Kelsey et al., 1987, Genes and Devel.1:161-171), the beta-globin gene control region active in bone marrow cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94); the myelin basic protein gene control region active in oligodendrocytes in the brain (Readhead et al., 1987, Cell 48:703-712); the myosin light chain-2 gene control region active in skeletal muscle (Shani, 1985, Nature 314:283-286), and the gonadotropin releasing hormone gene control region active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

[0131] Thus, in accordance with the invention, expression vectors capable of replicating in a bacterial or eukaryotic host containing nucleic acid encoding a polypeptide as described herein are used to transfect the host, thereby directing expression of such nucleic acid to produce a polypeptide which may then be recovered in a biologically active form, which as used herein includes forms capable of binding to an associated receptor and causing a differentiated function and / or affecting the phenotype of a cell expressing the receptor.

[0132] Expression vectors containing nucleic acid inserts can be identified by at least three general approaches, without limitation: (a) DNA-DNA hybridization, (b) the presence or absence of a "marker" gene function, and (c) expression of the inserted sequence. In the first approach, the presence of a foreign nucleic acid inserted into an expression vector can be detected by DNA-DNA hybridization using a probe containing a sequence homologous to the inserted nucleic acid sequence. In the second approach, recombinant vector / host systems can be identified and selected based on the presence or absence of a particular "marker" gene function (e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of the foreign nucleic acid sequence into the vector. For example, if an efl nucleic acid sequence is inserted into the marker gene sequence of the vector, recombinants containing the insert can be identified by the absence of the marker gene function. In the third approach, recombinant expression vectors can be identified by assaying the foreign nucleic acid product expressed by the recombinant construct. Such assays can be based on the physical or functional properties of the nucleic acid product of interest, for example, by binding of a ligand to a receptor or portion thereof, which can be tagged with, for example, a detectable antibody or portion thereof, or by binding to an antibody raised against the protein or portion thereof of interest.

[0133] In particular, modified polypeptides of the invention may be expressed transiently, constitutively or permanently in host cells.

[0134] Effective doses useful for treating the diseases or disorders set forth in this application may be determined using methods known to those skilled in the art (see, for example, Fingl, et al., The Pharmacological Basis of Therapeutics, Goodman and Gilman, eds. Macmillan Publishing Co, New York, pp. 1-46 (1975)). Pharmaceutical compositions for use according to the invention comprise the polypeptides described above in a pharmacologically acceptable liquid, solid or semi-solid carrier, which are linked to carriers or targeting molecules (e.g., antibodies, hormones, growth factors, etc.) prior to in vivo administration, and / or incorporated into liposomes, microcapsules and controlled release preparations. For example, the pharmaceutical composition may comprise the polypeptide in an aqueous solution, such as sterile water, saline, phosphate buffer or dextrose solution. Alternatively, the active agent may be included in a solid (e.g., wax) or semi-solid (e.g., gelatinous) formulation that can be implanted in a patient in need of such treatment. The route of administration may be any mode of administration known in the art, including, but not limited to, intravenous, intrathecal, subcutaneous, intrauterine, by injection into the involved tissue, intraarterial, intranasal, oral, or via an implant device.

[0135] Administration may result in the distribution of the active agent of the present invention throughout the body or in a localized area.For example, in some conditions involving remote regions of the nervous system, intravenous or intrathecal administration of the agent may be desirable.In some situations, an implant containing the active agent may be placed in or near the lesion area.Suitable implants include, but are not limited to, gelfoam, wax, spray, or microparticle-based implants.

[0136] The present invention also provides pharmaceutical compositions comprising the polypeptides described herein in a pharmacologically acceptable vehicle. The compositions may be administered systemically or locally. Any suitable mode of administration known in the art may be used, including but not limited to intravenous, intrathecal, intraarterial, intranasal, oral, subcutaneous, intraperitoneal, or local injection or surgical implant. Sustained release formulations are also provided.

[0137] Gene therapy Gene therapy refers to therapy performed by the administration to a subject of expressed or expressible nucleic acids. In this embodiment of the invention, the nucleic acids produce their encoded proteins that mediate a therapeutic effect.

[0138] Any of the methods for gene therapy available in the art can be used in accordance with the present invention. Exemplary methods are described below.

[0139] For general reviews of gene therapy methods, see Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methods generally known in the art of recombinant DNA technology that may be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

[0140] Delivery of the nucleic acid to the patient can be direct, where the patient is directly exposed to the nucleic acid or a vector carrying the nucleic acid, or indirect, where cells are first transformed with the nucleic acid in vitro and then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.

[0141] In a specific embodiment, the nucleic acid sequence is directly administered in vivo where it is expressed to produce the encoded product. This can be accomplished by any of a number of methods known in the art, such as by constructing the nucleic acid sequence as part of a suitable nucleic acid expression vector and administering the vector so that the nucleic acid sequence is intracellular, such as by infection with a defective or attenuated retrovirus or other viral vector, or by direct injection of naked DNA, or by coating with lipids or cell surface receptors or transfection agents, or by encapsulation in liposomes, microparticles or microcapsules, or by administering them linked to peptides known to enter the nucleus, administering them linked to a ligand that undergoes receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), which can be used to target cell types that specifically express the receptor, and the like. In another embodiment, a nucleic acid-ligand complex can be formed in which the ligand contains a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell-specific uptake and expression by targeting specific receptors. Alternatively, the nucleic acid can be integrated into the host cell DNA for intracellular introduction and expression by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438(1989)).

[0142] In a specific embodiment, viral vectors containing nucleic acid sequences encoding polypeptides are used.The nucleic acid sequences encoding polypeptides used in gene therapy are cloned into one or more vectors, which facilitates gene delivery to patients.Lentiviral vectors, such as retroviral vectors, and other vectors, such as adenoviral vectors and adeno-associated viruses, are examples of viral vectors that can be used.Retroviral vectors contain the components necessary for correct packaging of viral genome and integration into host cell DNA.

[0143] Adenoviruses are particularly attractive vehicles for delivering genes to respiratory epithelia, because they naturally infect respiratory epithelia and cause mild disease.Other targets for adenovirus-based delivery systems are liver, central nervous system, endothelial cells, and muscle.Adenoviruses have the advantage of being able to infect non-dividing cells.In addition, adeno-associated virus (AAV) has also been proposed for use in gene therapy.

[0144] Another approach to gene therapy involves transferring genes into cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer involves the transfer of a selection marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. The cells are then delivered to the patient.

[0145] In this embodiment, the nucleic acid is introduced into the cell prior to in vivo administration of the resulting recombinant cell. Such introduction can be performed by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequence, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, and the like. Numerous techniques are known in the art for the introduction of foreign genes into cells and may be used according to the present invention, provided that the necessary developmental and physiological functions of the recipient cell are not disrupted. The technique must provide for the stable transfer of the nucleic acid into the cell so that the nucleic acid is expressible by the cell, and preferably heritable and expressible by the cell progeny.

[0146] Cells into which nucleic acids can be introduced for purposes of gene therapy include any desired available cell type, including, but not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T-lymphocytes, B-lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes, etc.; various stem or progenitor cells, particularly hematopoietic stem or progenitor cells, such as those obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.

[0147] In a preferred embodiment, the cells used in gene therapy are autologous to the patient.

[0148] In an embodiment in which recombinant cells are used for gene therapy, a nucleic acid sequence encoding a polypeptide is introduced into the cell so that it is expressible by the cell or its progeny, and the recombinant cell is then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and / or progenitor cells that can be isolated and maintained in vitro can potentially be used according to this embodiment of the invention.

[0149] In a specific embodiment, the nucleic acid introduced for purposes of gene therapy contains an inducible promoter operably linked to the coding region, such that expression of the nucleic acid can be controlled by controlling the presence or absence of the appropriate inducer of transcription.

[0150] therapeutic composition Formulations of therapeutic compounds are generally known in the art and can be conveniently found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., USA. For example, about 0.05 ng to about 20 mg per kilogram of body weight per day may be administered. The dosage regimen may be adjusted to provide the optimal therapeutic response. For example, multiple divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The active compound may be administered in a convenient manner, such as by oral, intravenous (if water soluble), intramuscular, subcutaneous, intranasal, intraocular, intradermal or suppository routes, or by implantation (e.g., by intraperitoneal route using sustained release molecules, or by using cells sensitized in vitro and adoptively transferred to the recipient, such as monocytes or dendritic cells). Depending on the route of administration, the peptides may need to be coated in a material to protect them from the action of enzymes, acids and other natural conditions which may inactivate the ingredient.

[0151] For example, the low lipophilicity of peptides will cause them to be destroyed in the digestive tract by enzymes capable of cleaving peptide bonds, and in the stomach by acid hydrolysis. To administer peptides other than parenterally, they will be coated with or administered with materials that prevent inactivation. For example, peptides may be administered in adjuvants, co-administered with enzyme inhibitors, or in liposomes. Adjuvants contemplated herein include resorcinol, non-ionic surfactants such as polyoxyethylene oleyl ether, and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in-water CGF emulsions and conventional liposomes.

[0152] The active compound can also be administered parenterally or intraperitoneally.Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and their mixtures, and in oil.Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

[0153] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, chlorobutanol, phenol, sorbic acid, theomersal, and the like. In many cases, it will be preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the composition of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0154] Sterile injectable solution can be prepared by incorporating the active compound in the required amount in a suitable solvent together with various other ingredients listed above, and then sterilizing by filtration if necessary.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other ingredients required from the ingredients listed above.In the example of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, which produces a powder of active ingredient and any additional desired ingredients from its solution that has been previously sterilized by filtration.

[0155] When the peptide is suitably protected as described above, the active compound may be orally administered, for example, with an inert diluent or an assimilable edible carrier, or it may be enclosed in a hard or soft gelatin capsule, compressed into a tablet, or directly incorporated with dietary food. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 and about 80% by weight. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the invention are prepared so that an oral dosage unit form contains between about 0.1 μg and about 2000 mg of active compound.

[0156] Tablets, pills, capsules and the like may also contain: binders such as gum tragacanth, gum arabic, corn starch or gelatin; excipients such as dicalcium phosphate; disintegrating agents such as corn starch, potato starch, alginic acid and the like; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose or saccharin, or flavoring agents such as peppermint, wintergreen oil or cherry flavor may be added. When the dosage unit form is a capsule, it may contain a liquid carrier in addition to the above types of materials. Various other materials may be present as coatings or to otherwise improve the physical form of the dosage unit. For example, tablets, pills or capsules may be coated with shellac, sugar, or both. A syrup or elixir may contain the active compound, sucrose as a sweetener, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharma- ceutical pure and substantially non-toxic in the amounts used.In addition, the active compounds may be incorporated into sustained-release preparations and formulations.

[0157] Delivery System Various delivery systems, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, construction of the nucleic acid as part of a retrovirus or other vector, are known and can be used to administer the compounds of the present invention. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intraocular, epidural, and oral routes. The compounds or compositions can be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the present invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection, and intraventricular injection can be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, eg, by use of an inhaler or nebulizer, and incorporation of an aerosolizing agent.

[0158] In specific embodiments, it may be desirable to administer the pharmaceutical compounds or compositions of the present invention locally to the area requiring treatment, which may be achieved, for example and without limitation, by local infusion during surgery, by topical application, for example in conjunction with wound dressings after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being a porous, non-porous, or gelatinous material, including membranes such as Sialastic membranes or fibers. Preferably, when administering proteins, including antibodies or peptides of the present invention, care must be taken to use materials to which the proteins do not absorb. In another embodiment, the compounds or compositions may be delivered in vesicles, particularly liposomes. In yet another embodiment, the compounds or compositions may be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, a polymeric material may be used. In yet another embodiment, the controlled release system may be placed in the vicinity of the treatment target, thereby requiring only a fraction of the systemic administration.

[0159] The following examples are offered by way of illustration of the present invention, but not by way of limitation.

[0160] Working Example Example 1: Selection of anti-BAG2 antibodies, sequencing and antigen-antibody reactions 1. Selection of monoclonal antibodies targeting BAG2 and analysis of their amino acid sequences The present inventors selected antibodies targeting BAG2, analyzed their amino acid sequences, and determined the complementarity determining regions (CDRs) of each of the antibodies.

[0161] In detail, the gene consisting of the nucleotide sequence of SEQ ID NO:70 encoding the human BAG2 protein consisting of the amino acid sequence of SEQ ID NO:69 was cloned into pCAGGS plasmid and linearized, and the linearized construct was then inoculated into the muscle of five 6-week-old female BALB / c mice by applying electric shock. The construct was inoculated intramuscularly three times at three-week intervals, consisting of 100 μg of DNA in 100 μl of PBS. At this time, the control plasmid was also subjected to the same procedure. To generate therapeutic and diagnostic antibodies, a DNA vaccine-based immunization strategy was carried out, which is more efficient than protein-based antigen injection. Blood was collected from the vena cava or tail vein of the mice and tested by enzyme immunoassay to show serum antibody titers, and spleens were removed from mice that showed sufficient antibody titers three days after the last immunization. B lymphocytes were isolated from the spleen, followed by fusion of the isolated B lymphocytes, i.e., ATCC's SP2 / 0-Ag14 cell line, with cultured myeloma cells, thereby obtaining fused cells. After culturing the fused cells in HAT medium containing hypoxanthine, aminopterin, and thymidine, hybridoma cells fused only with myeloma and B lymphocytes were obtained by selecting approximately 130 clones. From the hybridoma cells obtained through the selection process by immunoblotting, 10 hybridoma cells were obtained that produce antibodies that specifically bind to human BAG2 protein.

[0162] Anti-Bag2 antibody total RNA was added at 5 × 10 65'-RACE-cDNA was generated from 100ng of total RNA by using SMART RACE cDNA Amplification kit (Clontech) according to the manufacturer's instructions. The regions encoding the heavy chain variable region (VH) and the light chain variable region (VL) were amplified by PCR, the amplified genes were inserted and cloned into pGEM-T vector (Promega, USA), and their nucleotide sequences were analyzed by using an automatic gene analyzer (ABI Prism 310, Applied Biosystem Co.). The nucleotide sequences of the analyzed genes were identified by comparison with previously reported nucleotide sequences, and the identified nucleotide sequences were artificially translated for use in determining the sequences of the complementarity determining regions VH-CDR1, -CDR2, and -CDR3, and VL-CDR1, -CDR2, and -CDR3. Complementarity determining region sequences were determined by using the Kabat database (http: / / www.bioinf.org.uk / abs / ).

[0163] As a result, ten anti-BAG2 antibodies that specifically bind to BAG2 were obtained from the hybridoma cells. The ten anti-BAG2 antibodies were 2A11, 4C2, 8C4, 3B5, 9B3, 9B12, 3B10, 10H7, 3GB, and 3F12 antibodies. In addition, the amino acid sequences of the heavy chain variable region, light chain variable region, and their complementarity determining regions shown in Tables 1 to 3, as well as the nucleotide sequences of the genes encoding the antibodies, were determined.

[0164] The 2A11, 4C2, and 8C4 antibodies comprise a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:21 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO:27. In the 2A11 antibody, Xaa at positions 56 and 57 of SEQ ID NO:21 are Gly, respectively, and Xaa at position 53 of SEQ ID NO:27 is lie. In the 4C2 antibody, Xaa at positions 56 and 57 of SEQ ID NO:21 are Gly and Ala, respectively, and Xaa at position 53 of SEQ ID NO:27 is Phe. In the 8C4 antibody, Xaa at positions 56 and 57 of SEQ ID NO:21 are Ala and Gly, respectively, and Xaa at position 53 of SEQ ID NO:27 is Phe.

[0165] The VH-CDR1, -CDR2 and -CDR3 of the 2A11, 4C2 and 8C4 antibodies consist of the amino acid sequences of SEQ ID NOs:33, 39 and 45, respectively, and the VL-CDR1, -CDR2 and -CDR3 consist of the amino acid sequences of SEQ ID NOs:51, 57 and 63, respectively. For the 2A11 antibody, the 56th and 57th Xaa in SEQ ID NO:21 are Gly, respectively. For the 4C2 antibody, the 56th Xaa in SEQ ID NO:21 is Gly and the 57th Xaa is Ala. For the 8C4 antibody, the 56th Xaa in SEQ ID NO:21 is Ala and the 57th Xaa is Gly.

[0166] The 9B3, 9B12 and 3B10 antibodies comprise a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:23 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO:29. For the 9B3 antibody, in SEQ ID NO:23, Xaa at position 1 is Glu, Xaa at position 7 is Ser, Xaa at position 12 is Val, Xaa at position 27 is Tyr, Xaa at position 58 is Ser, Xaa at position 61 is Asn, Xaa at position 74 is Lys, Xaa at position 83 is Phe, Xaa at position 92 is Ala and Xaa at position 108 is Tyr. For the 9B3 antibody, in SEQ ID NO:29, Xaa at position 29 is Ile, Xaa at position 51 is Ala, Xaa at position 79 is Glu, and Xaa at position 106 is Ile. For the 9B12 antibody, in SEQ ID NO:23, Xaa at position 1 is Gin, Xaa at position 7 is Ser, Xaa at position 12 is Val, Xaa at position 27 is Tyr, Xaa at position 58 is Ser, Xaa at position 61 is Asn, Xaa at position 74 is Arg, Xaa at position 83 is Phe, Xaa at position 92 is Gly, and Xaa at position 108 is His. For the 9B12 antibody, in SEQ ID NO:29, Xaa at position 29 is Met, Xaa at position 51 is Ala, Xaa at position 79 is Glu, and Xaa at position 106 is Met. For the 3B10 antibody, in SEQ ID NO:23, Xaa at position 1 is Gln, Xaa at position 7 is Pro, Xaa at position 12 is Ala, Xaa at position 27 is His, Xaa at position 58 is Thr, Xaa at position 61 is Ser, Xaa at position 74 is Arg, Xaa at position 83 is Leu, Xaa at position 92 is Gly, and Xaa at position 108 is His. For the 3B10 antibody, in SEQ ID NO:29, Xaa at position 29 is Met, Xaa at position 51 is Ser, Xaa at position 79 is Asp, and Xaa at position 106 is Ile.

[0167] For the 9B3, 9B12 and 3B10 antibodies, the VH-CDR1, -CDR2 and -CDR3 consist of the amino acid sequences of SEQ ID NOS:35, 41 and 47, respectively, and the VL-CDR1, -CDR2 and -CDR3 consist of the amino acid sequences of SEQ ID NOS:53, 59 and 65, respectively. For the 9B3 antibody, the second Xaa of SEQ ID NO:35 is Tyr, the eighth Xaa of SEQ ID NO:41 is Ser, the twelfth Xaa of SEQ ID NO:47 is Tyr, the third Xaa of SEQ ID NO:53 is lie and the second Xaa of SEQ ID NO:59 is Ala. For the 9B12 antibody, the second Xaa of SEQ ID NO:35 is Tyr, the eighth Xaa of SEQ ID NO:41 is Ser, the twelfth Xaa of SEQ ID NO:47 is His, the third Xaa of SEQ ID NO:53 is Met, and the second Xaa of SEQ ID NO:59 is Ala. For the 3B10 antibody, the second Xaa of SEQ ID NO:35 is His, the eighth Xaa of SEQ ID NO:41 is Thr, the twelfth Xaa of SEQ ID NO:47 is His, the third Xaa of SEQ ID NO:53 is Met, and the second Xaa of SEQ ID NO:59 is Ser. [Table 1] [Table 2] [Table 3]

[0168] 2. Identification of antigen-antibody response of anti-BAG2 antibody in breast cancer cells FIG. 1 shows the results of immunoblotting of anti-BAG2 antibodies generated from 10 kinds of mouse hybridoma cells. Specifically, human breast cancer cells, MDA-MB-231 cells, were cultured at a temperature of 37° C. in DMEM (Welgene) medium containing 10% FBS, 100 U / ml penicillin and 100 μg / ml streptomycin. The cells were detached from the wells, washed with PBS, and lysed in a lysis buffer solution containing 1% Brij97, 5 mM EDTA, 0.02 M HEPES pH 7.3, 0.15 M NaCl, 1 mM PMSF, 0.5 mM NaF, 10 μg / rni aprotinin, and 0.2 mM sodium orthovanadate. After 15 minutes of incubation on ice, the nuclei were removed from the cells by centrifugation, and the supernatant was collected. 2X sample buffer consisting of 20% glycerol, 4.6% SOS, 0.125M Tris pH 6.8, 0.1% bromophenol blue was added to the appropriate amount of supernatant. 10 μg protein samples were subjected to SOS-PAGE analysis on 12% gel under standard conditions by using mini-Protean II system (Bio-Rad, Hercules, CA). For immunoblotting, proteins were transferred onto Millipore PVDF membrane. Blocking solution consisting of 0.1% Tween 20 and 5% bovine serum albumin (BSA) in TBS was reacted for 1 hour. The primary antibody was then 1 / 2000 dilution of anti-BAG2 antibody extracted from hybridoma cell culture, and goat anti-mouse HRP conjugate (Dako) used as secondary antibody was diluted 1 / 5000. Film exposure was performed in the dark using EGL reagent (Amersham Pharmacia Biotech) as a substrate. The exposed bands were compared with standard molecular markers to identify the band corresponding to the size of BAG2.

[0169] As a result, as shown in Figure 1, antibodies 2A11, 3B5, 3B10, 3F12, 3GB, 4C2, 8C4, 9B3, 9B12 and 10H7 showed antigen-antibody reactions targeting BAG2, compared with ab58682 (Abeam), a commercially available polyclonal anti-BAG2 antibody used as a positive control.

[0170] Then, for domain mapping of the BAG2 antigen against which the ten antibodies were identified in the previous section 1, cells transfected with a GST-empty vector (pcDNA3.1+ / GST vector, NovoPro Bioscience Inc., China) having a molecular weight of about 26 kDa were used as a negative control. GST-Bag Full vector, GST-Bag F1 vector, GST-Bag F2 vector, GST-Bag F3 vector, and GST-Bag F4 vector, each of which contains a polynucleotide encoding human BAG2 protein and a polynucleotide encoding a fragment of BAG2 protein, were transfected into cells, and the cells transfected with the vectors were cultured to express the genes, and then cell lysates were obtained. For the cell lysates, immunoblotting was performed with each of the ten antibodies. The polynucleotide encoding human BAG2 protein has the nucleotide sequence of SEQ ID NO:70. The GST-Bag F1, -Bag F2, -Bag F3, and -Bag F4 vectors consist of the nucleotide sequences of SEQ ID NOs:71 to 74, respectively.

[0171] Figures 2A and 2B show the results of immunoblotting of the full-length BAG2 polypeptide with anti-BAG2 antibodies or fragments thereof. In Figure 2, A shows the diagram of the vector and BAG2 protein and its fragments, and B shows the results of immunoblotting. In detail, immunoblotting was performed as follows: each of the vectors was introduced into HEK2T cells by Lipofectamine transfection (Thermo Fisher Scientific, Inc., Waltham, Massachusetts, USA) method, and the resulting transformed cells were cultured in DMEM (Welgene) medium containing 10% FBS, 100 U / ml penicillin and 100 μg / ml streptomycin at a temperature of 37°C for 30 hours, after which the cells were isolated. The isolated cells were disrupted using the same method as described in relation to Figure 1 and subjected to SOS-PAGE analysis on a 12% gel. For immunoblotting, after 1 hour of reaction in the same blocking solution as described in relation to Figure 1, ten purified anti-BAG2 antibodies with a concentration of 2 mg / ml were used as primary antibodies at a dilution of 1 / 10000 to bind to the cells. Goat anti-mouse HRP conjugate was used as secondary antibody at a dilution of 1 / 5000, and film exposure was performed in the dark using EGL reagent (Amersham Pharmacia Biotech) as substrate. Standard molecular marker size was expressed to confirm the size of BAG2.

[0172] As a result, as shown in Figure 2, each anti-BAG2 antibody differentially bound to the full-length BAG2 polypeptide or its fragments. In particular, for each of the 10 anti-BAG2 antibodies, a signal was commonly detected at a position of about 50 KDa in the lysate of GST-Bag Full vector-transfected cells. This result indicates that all of these antibodies can bind to the full-length BAG2 antibody. Finally, the domain region of BAG2 to which each BAG2 antibody reacts was identified.

[0173] FIG. 3 shows the BAG2 domains that react with each anti-BAG2 antibody. As shown in FIG. 3, 9B3, 9B12, 3B10, and 10H7 antibodies were bound to the N-terminus of BAG2 protein, 2A11, 3B5, 4C2, and 8C4 antibodies were bound to the central region of BAG2 protein, and 3F12 and 3GB antibodies were bound to the C-terminus of BAG2 protein. The N-terminus commonly contains a coiled-coil region of 21 to 60 amino acids, and the central region is bound to a part of the BNB region of 109 to 189 amino acids. Therefore, by using a set of antibodies that bind to different sites, the BAG2 protein or its fragments present in a sample may be detected with high sensitivity and specificity.

[0174] An antibody or antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or fragment thereof according to one embodiment may elicit an antigen-antibody reaction with BAG2 polypeptides or fragments thereof of various lengths.

[0175] Polynucleotides encoding antibodies or antigen-binding fragments thereof according to another embodiment, and host cells containing same, can be used to produce antibodies or antigen-binding fragments thereof.

[0176] According to the method for producing an antibody or antigen-binding fragment thereof according to another embodiment, the antibody or antigen-binding fragment thereof can be efficiently produced.

[0177] Example 2. Preparation of drugs and mouse models for cancer treatment methods Ten hybridoma cell lines of anti-BAG2 were purified from their respective culture supernatants by affinity chromatography on a Sepharose protein A / G column according to the manufacturer's instructions (GE Healthcare Biosciences AB). Control Mu-IgG2a (ATCC, CCL-167™) was used as an isotype control antibody and was obtained from the American Type Culture Collection. Anti-BAG2 mouse antibodies and isotype control antibodies used in in vitro and in vivo studies were produced by Nanotool under endotoxin-free conditions (<0.01EU / ug).

[0178] Anti-PD-L1 monoclonal antibody (BioXCell, cat. no. BE0101), anti-PD-1 monoclonal antibody (BioXCell, cat. no. BE0033-2), and anti-CTLA4 monoclonal antibody (BioXCell, cat. no. BE0131) were prepared according to the manufacturers' instructions.

[0179] To verify the in vivo antitumor effect, male 5-week-old SPF C57BL / 6 and BALB / c mice were purchased from Koatech Co. (Korea). The mice were kept in a temperature-controlled room at approximately 22°C, and the mice were provided with food and water ad libitum. After one week, 2 × 10 mouse EMT6 breast cancer cell line (ATCC, CRL-2755™) was transfected into the mice. 5 Cells, mouse Lewis Lung Carcinoma (LLC) cell line (ATCC, CRL1642™) 5×10 5 Cells, mouse MC38 colon cancer cell line (Kerafast, ENH204-FP) 5 × 10 5 cells, and mouse CT26 colon cancer cell line (ATCC, CRL2638™) 3×10 5 Cells were injected into these 6-week-old mice by subcutaneous injection. 7-8 week-old mice were used for the experiments 8-12 days after injection of EMT6, LLC, MC38, and CT26 cell lines. One week later, 2×10 5The cells were injected into these 7-week-old mice via tail vein injection. Nine-week-old mice were used for the experiment 15 days after injection of B16-F10-Luc2. One week later, 2 × 10 mouse PANC02-Luc pancreatic adenocarcinoma cell line (Professor Kyu Lim, Chungnam National University, Republic of Korea) were injected into the mice. 6 The cells were orthotopically transplanted into the pancreatic tail of 7-week-old mice. 10-week-old mice were used for the experiment 16 days after injection of the PANC02-Luc cell line.

[0180] Mechanism of action Tumor secreted BAG2 protein can bind to lymphatic, myeloid and stromal cells for anti-immunity (adaptive or natural immunity) in tumor microenvironment. We hypothesize that BAG2 neutralization targets lymphatic, myeloid or stromal mediated immunosuppression, while immune checkpoint inhibitors restore the function of anergized anti-tumor T cells and the anti-inflammatory properties of myeloid cells.

[0181] 2.1. Results of breast cancer treatment in mouse models The results show the following:

[0182] Anti-BAG2 antibody alone has anti-tumor activity in a mouse breast cancer model.

[0183] Combination therapy of anti-BAG2 antibody and anti-PD-L1 antibody has synergistic antitumor activity in a mouse breast cancer model.

[0184] As shown in Figure 4a, 12 days after tumor inoculation, EMT6 tumor-bearing BALB / c mice were randomized according to tumor size before drug administration was performed. On day 12 after tumor cell injection, the mice were treated with anti-BAG2 antibody 3B10. Then, on day 14, the mice received a single injection of anti-PD-L1 antibody.

[0185] As shown in Figures 4b and 4c, on day 24, the tumor volumes of the anti-PD-L1 antibody, anti-BAG2 antibody 3B10, and anti-PD-L1 antibody plus 3B10 groups were reduced by approximately 20.2%, approximately 39.8%, and approximately 70.1%, respectively, compared to the tumor volumes of the isotype control group. Treatment with anti-PD-1 antibody had a marginal effect on tumor growth, but treatment with 3B10 significantly inhibited tumor growth. Treatment with the combination of anti-PD-L1 antibody and 3B10 inhibited tumor growth to a greater extent than either agent alone.

[0186] As shown in Figure 4d, the tumor-specific CD3+ / CD8+ T cells (effector memory cells in killing cancer cells) in the anti-PD-L1 antibody group, the anti-BAG2 antibody 3B10 group, and the combination of anti-PD-L1 antibody and 3B10 group were increased by approximately 2.4-fold, 2.8-fold, and 8.2-fold, respectively, compared to the amount of CD3+ / CD8+ T cells in the isotype control group. Treatment with the combination of anti-PD-L1 antibody and 3B10 activated tumor-specific CD3+ / CD8+ T cells to a greater extent than either agent alone. Activation could be direct or indirect, i.e., inhibition of suppressor cells.

[0187] 2.2. Results of lung cancer treatment in mouse models The results show the following:

[0188] Combination therapy of anti-BAG2 antibody and immune checkpoint inhibitor has synergistic antitumor effects in a mouse lung cancer model.

[0189] As shown in Figure 5a, LLC tumor-bearing C57BL / 6 mice were first treated with anti-BAG2 antibody 3B10 on day 8 after tumor cell injection. Then, on day 14, the mice received a single injection of anti-PD-L1 antibody.

[0190] As shown in Figures 5b and 5c, on day 20, the tumor volumes of the anti-PD-L1 antibody, anti-BAG2 antibody 3B10, and anti-PD-L1 antibody in combination with 3B10 groups were reduced by approximately 8.7%, approximately 17.8%, and approximately 62.2%, respectively, compared to the tumor volumes of the isotype control group. Treatment with the combination of anti-PD-L1 antibody and 3B10 inhibited tumor growth to a greater extent than either agent alone.

[0191] As shown in Figure 5d, tumor-specific CD3+ / CD8+ T cells (effector memory cells in killing cancer cells) in the anti-PD-L1 antibody group, the anti-BAG2 antibody 3B10 group, and the combination of anti-PD-L1 antibody and 3B10 group were increased by approximately 1.3-fold, 2.8-fold, and 6.6-fold, respectively, compared to the CD3+ / CD8+ T cell levels in the isotype control group. Treatment with the combination of anti-PD-L1 antibody and 3B10 activated tumor-specific CD3+ / CD8+ T cells to a greater extent than either agent alone. Activation could be direct or indirect, i.e., inhibition of suppressor cells.

[0192] 2.3. Results of melanoma treatment in mouse models The results show the following:

[0193] Anti-BAG2 antibodies have anti-tumor activity in a mouse melanoma lung metastasis model.

[0194] Combination therapy of anti-BAG2 and anti-PD-L1 antibodies has synergistic antitumor activity in a mouse melanoma lung metastasis model.

[0195] As shown in Figure 6a, B16-F10-Luc2 tumor-bearing C57BL / 6 mice were first treated with anti-BAG2 antibody 3F12 on day 15 after intravenous tumor cell injection, and then on day 23, the mice received a single injection of anti-PD-L1 antibody.

[0196] As shown in Figures 6b and 6c, at day 38, the tumor volumes of the anti-PD-L1 antibody, anti-BAG2 antibody 3F12, and anti-PD-L1 antibody plus 3F12 groups were reduced by about 19%, about 54%, and about 92%, respectively, compared to the BLI signal of the isotype control group. Treatment with anti-PD-1 antibody alone had little effect on melanoma tumor growth in the lungs, while anti-BAG2 antibody 3F12 significantly inhibited melanoma tumor growth in the lungs. Treatment with the combination of anti-PD-L1 antibody and 3F12 inhibited tumor growth to a greater extent than either agent alone.

[0197] 2.4. Treatment of colon cancer background Microsatellite instability-high (MSI) colorectal cancer (CRC) has defective mismatch repair (MMR) and increased levels of PD-L1, LAG-3, and IDO, and responds distinctly to anti-programmed cell death (PD) therapy. MSI-low or microsatellite stable (MSS) CRC, which constitute the majority of tumors in the clinical setting, did not see any benefit from PD-1 blockade. MSS CRC harbor a higher proportion of KRAS oncogenic mutations compared to MSI CRC. Combination therapy of anti-BAG2 antibody and anti-PD-1 agents was tested in a synergistic model in C57BL / 6 (MC38; KRASwt, MSI) and BALB / c (CT26; KRASmut, MSS) mice.

[0198] 2.4.1. Results of treatment of MSI-high frequency colon cancer in mouse models The results are shown below.

[0199] Anti-BAG2 antibody alone has antitumor activity in a murine MSI-high colorectal cancer model. Combination therapy with anti-BAG2 antibody and anti-PD-1 antibody has synergistic antitumor effects in a murine CRC model.

[0200] As shown in Figure 7a, MC38 tumor-bearing C57BL / 6 mice were first treated with anti-BAG2 antibody 3B10 on day 8 after tumor cell injection. Then, on day 10, the mice received a single injection of anti-PD-1 antibody.

[0201] As shown in Figures 7b and 7c, on day 20, the tumor volumes of the anti-PD-1 antibody, anti-BAG2 antibody 3B10, and anti-PD-1 antibody plus 3B10 combination treatment groups were reduced by about 43%, about 40.8%, and about 86.8%, respectively, compared to the tumor volumes of the isotype control group. Treatment with anti-PD-1 antibody or anti-BAG2 antibody 3B10 alone significantly inhibited tumor growth. Treatment with the combination of anti-PD-1 antibody and 3B10 inhibited tumor growth to a greater extent than either agent alone.

[0202] As shown in Figure 7d, tumor-specific CD3+ / CD8+ T cells (effector memory cells in killing cancer cells) in the anti-PD-1 antibody group, the anti-BAG2 antibody 3B10 group, and the combination of anti-PD-1 antibody and 3B10 group were increased by about 4.4-fold, 3.9-fold, and 36.2-fold, respectively, compared with CD3+ / CD8+ T cells in the isotype control group. Treatment with the combination of anti-PD-1 antibody and 3B10 activated tumor-specific CD3+ / CD8+ T cells to a greater extent than either agent alone. Activation could be direct or indirect, i.e., inhibition of suppressor cells.

[0203] 2.4.2. Results of treatment of MSS type of colon cancer in mouse models The results show the following:

[0204] Anti-BAG2 antibody alone has anti-tumor activity in a mouse MSS-type colon cancer model.

[0205] Combination therapy of anti-BAG2 antibody and anti-PD-L1 antibody has synergistic antitumor effects in a mouse MSS CRC model.

[0206] As shown in Figure 8a, CT26 tumor-bearing BALB / c mice were treated with anti-BAG2 antibody 3B10, anti-PD-L1 antibody, combination of 3B10 and anti-PD-L1 antibody, or control antibody at 250μg (low dose) or 750μg (high dose) / mouse on day 11 after tumor cell injection. Mice were dosed by ip injection every 2 days until the end of the study. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points.

[0207] As shown in Figures 8b and 8c, on day 23, the tumor volumes of the low-dose 3B10 group, the high-dose 3B10 group, the anti-PD-L1 antibody group, the combination of anti-PD-L1 antibody and low-dose 3B10 group, and the combination of anti-PD-L1 antibody and high-dose 3B10 group were reduced by about 9.1%, about 78.8%, about 17.4%, about 52.2%, and about 95.1%, respectively, compared to the tumor volumes of the isotype control group. Treatment with high-dose 3B10 alone potently inhibited the growth of CT26 tumors. Treatment with anti-PD-L1 antibody was less successful in inhibiting tumor growth as a single agent. Treatment with the combination of anti-PD-L1 antibody and low-dose 3B10 inhibited tumor growth to a greater extent than either agent alone. Moreover, combined treatment with anti-PD-L1 antibodies and high-dose 3B10 showed regression of individual tumors to undetectable levels in the majority of treated mice.

[0208] As shown in Figure 8d, tumor-specific CD3+ / CD8+ T cells (effector memory cells in killing cancer cells) were increased by approximately 1.4-fold, 3.8-fold, -1.5-fold, 6.6-fold, and 8.7-fold in the low-dose 3B10 group, the high-dose 3B10 group, the anti-PD-L1 antibody group, the combination of anti-PD-L1 antibody and low-dose 3B10 group, and the combination of anti-PD-L1 antibody and high-dose 3B10 group, respectively, compared to the CD3+ / CD8+ T cell levels in the isotype control group. Treatment with the combination of anti-PD-L1 antibody and 3B10 activated tumor-specific CD3+ / CD8+ T cells to a greater extent than either agent alone. Activation could be direct or indirect, i.e., inhibition of suppressor cells.

[0209] 2.5. Results of pancreatic cancer treatment in mouse models The results show the following:

[0210] Anti-BAG2 antibody alone has an anti-tumor effect in a mouse pancreatic cancer model.

[0211] Combination therapy of anti-BAG2 antibody and immune checkpoint inhibitors (anti-PD-1 antibody and anti-CTLA4 antibody) has synergistic antitumor activity in a mouse pancreatic cancer model.

[0212] As shown in Figure 9a, PANC02-Luc tumor-bearing C57BL / 6 mice were treated with four anti-BAG2 antibodies (3B10, 3F12, 3B5, and 3G8) or control antibodies at 200 μg (low dose) / mouse (days 16-32) and 400 μg (high dose) / mouse (three times on days 35-39) after tumor cell injection. Mice were dosed by ip injection three times a week until the end of the study. On days 30-39, mice received injections of anti-PD-1 or anti-CTLA4 antibodies. Tumor growth was monitored by IVIS imaging of bioluminescence (BLI) signals on days 15, 27, and 39 after implantation.

[0213] As shown in Figure 9b, on day 40, the BLI signals of the anti-BAG2 antibody-treated groups (3B10, 3F12, 3B5, and 3G8) were decreased by about 79.3%, about 68.4%, about 24.8%, and about 8.3%, respectively, compared to the isotype control group. The BLI signal intensities of the anti-PD-1 antibody-treated group, the anti-PD-1 antibody and 3B10 combination group, the anti-PD-1 antibody and 3F12 combination group, the anti-PD-1 antibody and 3B5 combination group, and the anti-PD-1 antibody and 3G8 combination group were decreased by about -14.7%, about 95.9%, about 92.8%, about 62.3%, and about -10.9%, respectively, compared to the isotype control group. The BLI signal intensities in the groups administered anti-CTLA4 antibody alone, in the combination of anti-CTLA4 antibody and 3B10, in the combination of anti-CTLA4 antibody and 3F12, in the combination of anti-CTLA4 antibody and 3B5, and in the combination of anti-CTLA4 antibody and 3G8 were reduced by approximately -14.5%, approximately 91.1%, approximately 85.3%, approximately 66.3%, and approximately 10.9%, respectively, compared to the isotype control group.

[0214] As shown in Figure 9c, on day 40, the primary tumor weights of the anti-BAG2 antibody alone 3B10, 3F12, 3B5, and 3G8 treatment groups were reduced by about 63.8%, about 56.7%, about 27.8%, and about 18.9%, respectively, compared to the isotype control group. The BLI signal intensities of the anti-PD-1 antibody alone treatment group, the anti-PD-1 antibody and 3B10 combination treatment group, the anti-PD-1 antibody and 3F12 combination treatment group, the anti-PD-1 antibody and 3B5 combination treatment group, and the anti-PD-1 antibody and 3G8 combination treatment group were reduced by about 17.8%, about 85.7%, about 87.4%, about 58.1%, and about -24.8%, respectively, compared to the isotype control group. The BLI signal intensities in the groups administered anti-CTLA4 antibody alone, the group administered in combination with anti-CTLA4 antibody and 3B10, the group administered in combination with anti-CTLA4 antibody and 3F12, the group administered in combination with anti-CTLA4 antibody and 3B5, and the group administered in combination with anti-CTLA4 antibody and 3G8 were reduced by approximately 3.4%, approximately 76.1%, approximately 74.2%, approximately 54.8%, and approximately 29.7%, respectively, compared to the isotype control group.

[0215] As shown in Figures 9b and 9c, single treatment with 3B10 and 3F12 strongly inhibited the BLI signal intensity and reduced the primary tumor weight of PANC02-Luc tumor-bearing mice, while 3B5 and 3G8 did not significantly inhibit the BLI signal intensity or significantly reduce the primary tumor weight of mice. The different phenomena of the four clones may be caused by the affinity for cross-reaction in mice and humans.

[0216] Treatment with anti-PD-1 antibodies was less successful in inhibiting tumor growth as a single agent. Treatment with a combination of anti-BAG2 antibodies (3B10, 3F12, or 3B5) and anti-PD-1 or anti-CTLA4 antibodies inhibited tumor growth to a greater extent than either agent alone. Moreover, combined treatment with anti-BAG2 antibodies (3B10, 3F12, or 3B5) and anti-PD-1 or anti-CTLA4 antibodies reduced the incidence of metastases to the liver, pleura, and diaphragm compared to either agent alone (Figure 9d).

[0217] As shown in Figure 9e, tumor-specific CD3+ / CD8+ T cells (effector memory cells for killing cancer cells) were increased by approximately 1.8-, 3.5-, and 14.2-fold, respectively, in the anti-PD-1 antibody-, 3F12-, and anti-PD-1 antibody and 3F12 combination-treated groups compared to the isotype control group.

[0218] As shown in Figure 9e, tumor-specific CD45+ / CD3- / CD19+ B cells in the anti-PD-1 antibody-treated group, the 3F12-treated group, and the combination of anti-PD-1 antibody and 3F12-treated group were increased by approximately -1.1-fold, -1.05-fold, and 1.04-fold, respectively, compared to the isotype control group.

[0219] As shown in Figure 9e, tumor-specific CD45+ / CD3- / CD49b+ NK (NK cell) cells in the anti-PD-1 antibody-treated group, the 3F12-treated group, and the combination of anti-PD-1 antibody and 3F12-treated group were increased by approximately 1.3-, 2.1-, and 3.6-fold, respectively, compared to the isotype control group.

[0220] As shown in Figure 9e, tumor-specific CD45+ / CD11b+ / Gr1- / F4 / 80+ macrophages in the anti-PD-1 antibody-treated group, the 3F12-treated group, and the combination of anti-PD-1 antibody and 3F12-treated group were reduced by approximately 1.1-, 1.7-, and 2.7-fold, respectively, compared to the isotype control group.

[0221] As shown in Figure 9e, tumor-specific CD45+ / CD11b+ / Gr1+ MDSCs (myeloid-derived suppressor cells) in the anti-PD-1 antibody-treated group, the 3F12-treated group, and the combination of anti-PD-1 antibody and 3F12-treated group were reduced by approximately 1.07-, 1.15-, and 1.9-fold, respectively, compared to the isotype control group.

[0222] As shown in Figure 9e, tumor-specific CD45- / CD90.2+ stromal cells in the anti-PD-1 antibody-treated group, the 3F12-treated group, and the combination of anti-PD-1 antibody and 3F12-treated group were reduced by approximately -1.06-, 1.35-, and 1.86-fold, respectively, compared to the isotype control group.

[0223] Treatment with the combination of anti-PD-L1 antibody and 3F12 activated tumor-specific CD3+ / CD8+ T cells and CD45+ / CD3- / CD49b+ NK cells to a greater extent than either agent alone, but did not increase the CD45+ / CD3- / CD19+ B cell population. Treatment with the combination of anti-PD-L1 antibody and 3F12 reduced CD45+ / CD11b+ / Gr1- / F4 / 80+ macrophages, CD45+ / CD11b+ / Gr1+ MDSC, and CD45- / CD90.2+ stromal cells to a greater extent than either agent alone.

[0224] Activation can be direct or indirect, ie, inhibition of suppressor cells.

[0225] Therefore, 1) anti-BAG2 antibody was found to have a significant tumor growth and metastasis inhibitory effect compared to the isotype control group. 2) The combination of anti-PD-L1 antibody, anti-PD-1 antibody, and anti-CTLA4 antibody as immune checkpoint inhibitors with anti-BAG2 antibody was found to have a significant tumor inhibitory effect compared to anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA4 antibody, and anti-BAG2 antibody alone in mouse breast cancer, lung cancer, melanoma, colon cancer, and pancreatic cancer models.

[0226] It should be understood that the embodiments described herein should be regarded as merely illustrative and not restrictive. The description of a feature or aspect in each embodiment should typically be regarded as applicable to other similar features or aspects in other embodiments. Although one or more embodiments have been described with reference to the drawings, it will be understood by those skilled in the art that various changes in form and details may be made without departing from the spirit and scope of the present disclosure as defined by the following claims.

[0227] It should be understood that the embodiments described herein should be regarded as merely illustrative and not restrictive. The description of a feature or aspect in each embodiment should typically be regarded as applicable to other similar features or aspects in other embodiments. Although one or more embodiments have been described with reference to the drawings, it will be understood by those skilled in the art that various changes in form and details may be made without departing from the spirit and scope of the present disclosure as defined by the following claims.

[0228] The following deposits are international deposits under the Budapest Treaty: [Accession number] Deposited at: Korea Research Institute of Bioscience and Biotechnology Accession number: KCTC13737BP Deposit date: November 28, 2018 Deposit: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13738BP Deposit date: November 28, 2018 Deposit: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13739BP Deposit date: November 28, 2018 Deposit: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13740BP Deposit date: November 28, 2018 Deposit: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13741BP Deposit date: November 28, 2018 Deposit: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13742BP Deposit date: November 28, 2018 Deposit: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13743BP Deposit date: November 28, 2018 Deposit: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13744BP Deposit date: November 28, 2018 Deposit: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13745BP Deposit date: November 28, 2018 Deposit: Korea Institute of Bioscience and Biotechnology Accession number: KCTC13746BP Deposit date: November 28, 2018

[0229] All references cited herein are incorporated by reference in their entirety.

[0230] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or a fragment thereof, The antibody or antigen-binding fragment thereof a heavy chain variable region comprising a complementarity determining region (VH-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 33, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 (wherein the 6th and 7th Xaas of SEQ ID NO: 39 are Gly), and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 45; and a light chain variable region comprising a complementarity determining region (VL-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 51, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 57, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 63; a heavy chain variable region comprising a complementarity determining region (VH-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 33, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 (wherein the 6th and 7th Xaas of SEQ ID NO: 39 are Gly and Ala, respectively), and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 45, and a light chain variable region comprising a complementarity determining region (VL-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 51, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 57, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 63; or a heavy chain variable region comprising a complementarity determining region (VH-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 33, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 39 (wherein the 6th and 7th Xaas of SEQ ID NO: 39 are Ala and Gly, respectively), and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 45; and a light chain variable region comprising a complementarity determining region (VL-CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 51, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 57, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 63; An antibody or antigen-binding fragment thereof.

2. A heavy chain variable region of SEQ ID NO: 21 and a light chain variable region of SEQ ID NO: 27; The antibody or antigen-binding fragment thereof of claim 1, comprising:

3. An antibody or antigen-binding fragment thereof described in claim 1, which is a monoclonal antibody.

4. An antibody or antigen-binding fragment thereof described in claim 1, which is attached with a detectable label or a label capable of emitting a detectable signal.

5. The antibody or antigen-binding fragment thereof described in claim 1, wherein the antibody is humanized.

6. An antibody or antigen-binding fragment thereof described in claim 1, wherein hot spots have been removed in the CDRs of the antibody.

7. The antibody or antigen-binding fragment thereof described in claim 1, wherein the antibody is a monovalent, Fab, or single-chain variable region fragment antibody (scFv).

8. The antibody or antigen-binding fragment thereof described in claim 1, wherein the antibody is bivalent, bispecific, or trispecific.

9. An antibody or antigen-binding fragment thereof described in claim 1, wherein the antibody is fused to a chemical or protein.

10. An antibody or antigen-binding fragment thereof described in claim 9, wherein the antibody is fused to a toxin or cytokine.

11. The antibody or antigen-binding fragment thereof described in claim 1, produced by a hybridoma cell selected from the hybridoma cells deposited under accession numbers KCTC 13737BP, KCTC 13742BP, and KCTC 13743BP.

12. An antibody or antigen-binding fragment thereof described in claim 1, comprising multiple antibodies or antigen-binding fragments thereof.

13. A polynucleotide encoding the antibody or antigen-binding fragment thereof described in claim 1.

14. The polynucleotide described in claim 13, which is a vector.

15. The polynucleotide of claim 14, wherein a detectable label or a label capable of emitting a detectable signal is conjugated to the polynucleotide.

16. A host cell comprising the polynucleotide described in claim 13.

17. Culturing the host cell of claim 16; and isolating the antibody or antigen-binding fragment thereof from the resulting culture; 1. A method for producing an antibody or antigen-binding fragment thereof, comprising:

18. The method of claim 17, further comprising labeling the antibody or antigen-binding fragment thereof.

19. A composition for use in a method for treating cancer, which is a primary or metastatic cancer, in an individual, comprising an antibody or antigen-binding fragment thereof described in claim 1.

20. The composition described in claim 19, wherein the method includes co-administering or sequentially administering an existing therapeutic agent.

21. The composition described in claim 20, wherein the existing therapeutic agent is a cancer immunotherapy drug.

22. The composition described in claim 21, wherein the cancer immunotherapy drug is an inhibitor of an immune checkpoint molecule.

23. The composition described in claim 22, wherein the immune checkpoint molecule is PD-1, PD-L1, or CTLA-4.

24. The composition of claim 21, wherein the cancer immunotherapy agent is selected from the group consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 12 (IL-12), interleukin 15 (IL-15), B7-1 (CD80), B7-2 (CD86), 4-1BB ligand, GITRL, OX-40L, anti-CD3 antibody, anti-CD27 antibody, anti-CTLA4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-GITR antibody, anti-OX-40 antibody, anti-4-1BB antibody, anti-LAG-3 antibody, and anti-TIM-3 antibody.

25. The composition of claim 19, wherein the cancer is selected from the group consisting of breast cancer, colorectal cancer, head and neck cancer, colon cancer, skin cancer, pancreatic cancer, lung cancer, stomach cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, clear cell sarcoma, melanoma, cerebrospinal tumor, brain cancer, thymus, mesothelioma, esophageal cancer, bile duct cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndrome (MOS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, Hodgkin's disease, endocrine cancer, and sarcoma.

26. The composition of claim 25, wherein the cancer is breast cancer, lung cancer, melanoma, colon cancer, or pancreatic cancer.

Images