Anti-BAG2 antibody and method for treating cancer

Abstract

The present application discloses antibodies or antigen-binding fragments thereof that specifically bind to a BAG2 polypeptide or fragment thereof, and methods of treating cancer.

Description

Technical Field

[0001] Background of the Invention 1. Field of the Invention The present disclosure relates to an antibody or an antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or a fragment thereof. The present application also relates to an anti-cancer therapeutic agent using the composition of the present invention.

Background Art

[0002] 2. General Background and Latest Technology The co-chaperone Bcl-2-associated athanogene (BAG) protein family mediates various physiological processes including intracellular protein folding, stress response, neuronal cell differentiation, apoptosis, and cell proliferation, and functionally binds to various cooperative proteins. BAG2, which is one 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-related ubiquitin ligase. The main role of BAG2 in protein regulation through inhibition of CHIP activity is related to neurodegenerative diseases and autosomal recessive genetic diseases through the stabilization of chaperone-related proteins such as PINK1 and CFTR. It has been reported that BAG2 has apoptosis-promoting activity, such that the expression of BAG2 increases apoptosis induced by proteasome inhibitors, and BAG2 knockdown partially inhibits apoptosis when thyroid cancer cells are exposed to the proteasome inhibitor MG132. BAG proteins, apart from the formation of the BAG2-Hsp70 complex, functionally interact with various binding partners to regulate various cellular processes such as stress signaling, cell division, apoptosis, and cell differentiation. It has also been reported that in tumors induced by various mutant K-Ras, overexpression of BAG2 promotes the stabilization of the STK33 protein, a potent oncogene, thereby promoting tumor development. In addition, it has been suggested that the expression and location of the BAG2 protein may vary according to the histopathological and molecular genetic pathologies of cancer cells during the progression or metastasis of breast cancer. It has been found that the BAG2 protein is secreted from cells through interaction with the protease cathepsin B during tumor formation, and it has been confirmed that loss of the BAG2 protein completely inhibits carcinogenesis and metastasis to the lung in an animal model of breast cancer. As a result, the development of BAG2 protein inhibitors would contribute to the treatment and survival of cancer patients.

Summary of the Invention

Problems 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 tests have not been performed with BAG2 monoclonal antibodies. Therefore, it is necessary to develop an antibody or an antigen-binding fragment thereof that specifically binds to the BAG2 polypeptide or a fragment thereof. **Means for Solving the Problems**

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

[0005] One aspect provides an antibody or an 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 an antigen-binding fragment thereof.

[0007] Another aspect provides a host cell containing this polynucleotide.

[0008] Another aspect provides a method for producing this antibody or an antigen-binding fragment thereof.

[0009] Additional aspects are in part represented in the following description, in part will be apparent from the following description, or may be learned by practice of the embodiments presented in this disclosure.

[0010] One aspect provides an antibody or an 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), a mouse (Mus musculus), a monkey, a cow, or a horse. BAG2 may include the amino acid sequence of SEQ ID NO:69. The amino acid sequence of SEQ ID NO:69 corresponds to the sequence of NCBI reference SEQ ID NO:NM_004282.4. The BAG2 protein includes variants having biological activities equivalent to those of the amino acid sequence of SEQ ID NO:69, although their amino acid sequences may not be identical to the amino acid sequence of SEQ ID NO:69. The BAG2 polypeptide may include an amino acid sequence having at least 60%, such as 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. The 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] An antibody refers to a specific immunoglobulin against an antigenic site. An antibody refers to a polypeptide or a combination of polypeptides that specifically binds to a BAG2 polypeptide or a fragment thereof. The 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. The antibody may include the complete form of an antibody having two full-length light chains and two full-length heavy chains, and may include functional fragments of antibody molecules that retain the antigen-binding function by virtue of including having a specific antigen-binding site, i.e., a binding domain, even though the structure of the intact antibody in the complete form having two light chains and two heavy chains does not exist.

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

[0014] There are five heavy chains γ, δ, α, μ and ε, and the heavy chain may determine the type of antibody. α and γ each contain 450 amino acids, and μ and ε each contain 550 amino acids. The heavy chain has two regions, namely, a variable region and a constant region.

[0015] There are two light chains kappa and lambda, and 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 its antigen-binding fragment comprises a heavy-chain variable region containing complementarity-determining region (VH-CDR) 1 consisting of the amino acid sequence of SEQ ID NO:33, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO:39, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO:45, and a light-chain variable region containing complementarity-determining region (VL-CDR) 1 consisting of the amino acid sequence of SEQ ID NO:51, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO:57, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO:63; a heavy-chain variable region containing VH-CDR1 consisting of the amino acid sequence of SEQ ID NO:34, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO:40, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO:46, and a light-chain variable region containing VL-CDR1 consisting of the amino acid sequence of SEQ ID NO:52, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO:58, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO:64; a heavy-chain variable region containing VH-CDR1 consisting of the amino acid sequence of SEQ ID NO:35, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO:41, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO:47, and a light-chain variable region containing VL-CDR1 consisting of the amino acid sequence of SEQ ID NO:53, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO:59, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO:65; a heavy-chain variable region containing VH-CDR1 consisting of the amino acid sequence of SEQ ID NO:36, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO:42, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO:48, and a light-chain variable region containing VL-CDR1 consisting of the amino acid sequence of SEQ ID NO:54, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO:60, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO:66;A heavy chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO:37, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO:43, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO:49, and a light chain variable region comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO:55, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO:61, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO:67; A heavy chain variable region comprising VH-CDR1 consisting of the amino acid sequence of SEQ ID NO:38, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO:44, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO:50, and a light chain variable region comprising VL-CDR1 consisting of the amino acid sequence of SEQ ID NO:56, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO:62, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO:68; Or a combination thereof may be included.;

[0017] The 6th and 7th Xaa in SEQ ID NO:39 may be Glycine (Gly) or Alanine (Ala). The 2nd Xaa in SEQ ID NO:35 may be Tyrosine (Tyr) or Histidine (His). The 8th Xaa in SEQ ID NO:41 may be Serine (Ser) or Threonine (Thr). The 12th Xaa in SEQ ID NO:47 may be Tyrosine (Tyr) or Histidine (His). The 3rd Xaa in SEQ ID NO:53 may be Methionine (Met) or Isoleucine (Ile). The 2nd Xaa in SEQ ID NO:59 may be Ala or Ser.

[0018] An antibody or an antigen-binding fragment thereof may comprise a heavy chain variable region comprising any one amino acid sequence selected from SEQ ID NO:21-26; a light chain variable region comprising any one amino acid sequence selected from SEQ ID NO:27-32; or a combination of its heavy chain variable region and its light chain variable region.

[0019] The antibody or its antigen-binding fragment 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] The 56th Xaa and 57th Xaa in SEQ ID NO:21 may each be Gly or Ala. In SEQ ID NO:23, the 1st Xaa may be glutamine (G l n) or glutamic acid (Glu), the 7th Xaa may be Ser or pro ン( Pro), the 12th Xaa may be valine (Val) or alanine (Ala), the 27th Xaa may be Tyr or His, the 58th Xaa may be Ser or Thr, the 61st Xaa may be asparagine (Asn) or Ser, the 74th Xaa may be arginine (Arg) or lysine (Lys), the 83rd Xaa may be phenylalanine (Phe) or leucine (Leu), the 92nd Xaa may be Gly or Ala, and the 108th Xaa may be His or Tyr. The 53rd Xaa in SEQ ID NO:27 may be Il e or Phe. In SEQ ID NO:29, the 2 9 nd Xaa may be Met or Il e, 51 th Xaa may be Ala or Ser, 79 th Xaa may be Glu or aspartic acid (Asp), and the 10 6 th Xaa may be Met orIl It may be e.

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

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

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

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

[0025] The antibody or antigen-binding fragment thereof may be produced by hybridoma cells selected from 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 having the ability to form tumors by artificial fusion of two types of cells, and are generally used to continuously produce antibodies by fusing B cells and plasmacytoma cells isolated from an immunized subject. In the present specification, hybridoma cells may also be referred to as hybrid cells or fused cells.

[0027] The inventors identified anti-BAG2 antibodies comprising 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 that produce the same. It was confirmed that the identified anti-BAG2 antibodies exhibit an antigen-antibody reaction in breast cancer cells.

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

[0029] The polynucleotide may be any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 10 encoding a heavy chain variable region and any one nucleotide sequence 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 intracellularly. The cell may be a eukaryotic cell or a 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 the expression vector, the polynucleotide is operably linked to a suitable regulatory region such 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 selectable 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 the antibody, or may include both a polynucleotide encoding the heavy chain variable region and a polynucleotide encoding the light chain variable region.

[0031] The polynucleotide may be conjugated with a detectable label or a label capable of emitting a detectable signal. The label may be a fluorescent dye, a phosphorescent dye, a radioisotope, a chromophore, a quantum dot, a quencher, a magnetic bead nanoparticle, a gold nanoparticle, a nanophosphor, a silicon nanoparticle, semiconductor microparticles having luminescent properties, and the like. For example, the fluorescent dye may be cyanine (cyanine 2), amidomethylcoumarin, fluorescein, indocarbocyanine (cyanine 3), cyanine 3.5, tetramethylrhodamine, rhodamine red, Texas red, indiacarbocyanine (cyanine 5), cyanine 5.5, cyanine 7, Oyster, and the like. For example, the semiconductor microparticles 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 binding the labeling substance to the 3'-end of the polynucleotide, a method of binding the labeling substance to the 5'-end, or a method of including a nucleotide to which the labeling substance is bound in the polynucleotide. The conjugation of the label to the 3'-end or 5'-end may be carried out 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, computed tomography, magnetic resonance imaging, or the like.

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

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

[0034] The host cell may be a bacterial cell, yeast cell, fungal cell, insect cell, animal cell or plant cell, each containing a 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 Drosophila, or Spodoptera Sf9 cells. The animal cell may be Chinese hamster ovary cells (CHO), mouse myeloma (SP2 / 0), human lymphoblastoid, COS, mouse myeloma (NSO), 293T, bovine (bow) melanoma cells, HT-1080, baby hamster kidney cells (BHK), human fetal kidney cells (HEK), or PERC.6 (human retinal cells).

[0035] The polynucleotide may be introduced into the host cell. Introduction refers to a method of delivering a vector containing a polynucleotide encoding an antibody to the host cell. Such introduction may be carried out by various methods known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran mediated transfection, polybrene mediated transfection, electroporation, microinjection, liposome fusion, Lipofectamine and protoplast fusion. In addition, transduction refers to the delivery of the target product into the cell using virus particles by infection. 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 for generating an antibody or an antigen-binding fragment thereof.

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

[0038] The host cell is the same as those described above.

[0039] Cultivation may be carried out according to appropriate 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 cultures.

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

[0041] The carbon source may be, for example, carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, or cellulose; fats such as soybean oil, sunflower oil, castor oil, or coconut oil; fatty acid glycerols such as palmitic acid, stearic acid, and linoleic acid; alcohols such as ethanol; organic acids such as acetic acid; or combinations thereof. The nitrogen source may include, for example, inorganic nitrogen sources such as peptone, yeast extract, meat extract, malt extract, corn steep liquor (CSL), and soybean wheat, urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrogen, or organic nitrogen sources such as combinations 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, appropriate 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, during the period when the host cells are cultured, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid may be added to the bacterial culture by appropriate means to adjust the pH of the culture. In addition, during the culture of the host cells, an antifoaming agent such as a fatty acid polyglycol ester may be used to suppress the formation of foam.

[0043] The cells may be cultured under aerobic, microaerobic, or anaerobic conditions. Microaerobic conditions refer to culture conditions in which a lower level of oxygen than the oxygen level in the atmosphere is dissolved in the culture 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, the microaerobic conditions may include conditions in which the concentration of oxygen dissolved in the culture 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 its antigen-binding fragment reaches the target level.

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

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

[0046] In one aspect, the present invention is directed to a method for treating cancer in an individual, which is either primary or metastatic cancer, the method comprising administering to the individual in need thereof the anti-BAG2 or antigen-binding fragment thereof 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 immunotherapy 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, gastric cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, clear cell sarcoma, melanoma, cerebrospinal tumor, brain cancer, thymus, mesothelioma, esophageal cancer, cholangiocarcinoma, 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] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Patent Office upon request and payment of the necessary fees.

[0048] The present invention will be more fully understood from the detailed description given hereinafter and the accompanying drawings, which are by way of illustration only and thus do not limit the present invention.

Brief Description of the Drawings

[0049]

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Mode for Carrying Out the Invention

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

[0051] As used herein, the terms "about" or "substantially" generally provide a tolerance from strict numerical limitations. For example, when used in the context of the length of a polypeptide sequence, the terms "about" or "substantially" indicate that the polypeptide is not limited to the recited number of amino acids. Two or three amino acids added to or subtracted from the N-terminus or C-terminus may be included as long as functional activity such as binding activity is present.

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

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

[0054] As used herein, the terms "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] As used herein, the term "amino acid sequence variant" generally refers to a molecule having some differences in its amino acid sequence as compared to a reference (e.g., native sequence) polypeptide. The amino acid modifications may be substitutions, insertions, deletions, or any desired combination of such changes in the native amino acid sequence.

[0056] A substitution variant has 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 substitution may be single, in which case only one amino acid in the molecule is substituted, or multiple, in which case two or more amino acids are substituted in the same molecule.

[0057] Substitutions of amino acids within the sequence may be selected from other members of the class to which the amino acid belongs. For example, nonpolar (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, such as by glycosylation, proteolytic cleavage, linkage to an antibody molecule or other cell ligand.

[0058] An insertion variant has one or more amino acids inserted at a position immediately adjacent to an amino acid at a unique location within the native amino acid sequence. Immediately adjacent to an amino acid means being linked to either the α-carboxy or α-amino functional group of the amino acid.

[0059] A deletion mutant is one in which one or more amino acids in the native amino acid sequence have been removed. Usually, a deletion mutant will have one or two amino acids deleted in a specific region of the molecule.

[0060] As used herein, "fragment" or "functional derivative" refers to a covalent modification including the bioactive amino acid sequence or fragment of the polypeptide of the present invention, and derivatives obtained by reaction with an organic derivatizing agent, modification by translational language, derivatives with non-proteinaceous polymers, and immunoadhesins.

[0061] As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient or stabilizer that is non-toxic to the cells or mammals to which it is exposed at the dosages and concentrations used. In many cases, the pharmaceutically acceptable carrier is an aqueous pH buffered solution. Examples of pharmaceutically acceptable carriers include buffers such as phosphates, citrates, 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; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG) and PLURONICS®, but are not limited thereto.

[0062] As used herein, "pharmaceutically acceptable carriers and / or diluents" include any solvent, dispersion medium, coating antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Their use in therapeutic compositions is contemplated, except where any conventional medium or agent is incompatible with the active ingredient. Auxiliary active ingredients may also be incorporated into the compositions.

[0063] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, a dosage unit form refers to physically discrete units suitable as a single dosage for the mammalian subject to be treated; each unit contains a predetermined quantity of the active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are determined by and directly depend on (a) the particular characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such active materials for the treatment of diseases in living subjects having a diseased state where the body's health is impaired.

[0064] The principal active ingredient is compounded with a suitable pharmaceutically acceptable carrier in dosage unit form for convenient and effective administration in an effective amount. The unit dosage form may contain, for example, the principal active compound in an amount ranging from 0.5 μg to about 2000 mg. Expressed as a proportion, the active compound is generally present in a carrier of about 0.5 μg / ml. In examples of compositions containing auxiliary active ingredients, the dosage is determined by reference to the usual dosage and manner of administration of the said ingredients.

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

[0066] As used herein, "host cell" encompasses individual cells or cell cultures that can be or have been recipients of the vectors of the present invention. Host cells include the progeny of a single host cell, which progeny need not be identical (either morphologically or in total complementary DNA) to the original parent cell due to natural, accidental, or deliberate mutations and / or changes.

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

[0068] As used herein, "mammal" for the purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as 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 is a compound that, in a statistical sample, reduces the occurrence of the disorder or disease in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or disease relative to an untreated control sample, or reduces the severity of the disorder or disease.

[0070] The terms "decrease", "reduced", "reduction", or "inhibit" are all used herein to mean a decrease or lowering of a property, level, or other parameter by a statistically significant amount. In some embodiments, "reduce", "reduction" or "decrease" or "inhibit" typically means a reduction of at least 10% compared to a reference level (e.g., the absence of a given treatment), such as 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. "Reduction" or "inhibition" as used herein does not include complete inhibition or reduction compared to the reference level. "Complete inhibition" is 100% inhibition compared to the reference level. The decrease can preferably be a drop to a level that is acceptable within the normal range for an individual without a given disorder.

[0071] The terms "increased", "increasing", "enhanced", or "activated" are all used herein to generally mean an increase in a property, level, or other parameter in a statistically significant amount. To avoid any doubt, the terms "increased", "increasing", "enhanced", or "activated" mean an increase of at least 10% compared to a reference level, such as 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 an increase of 100% or less, or any increase between 10 and 100%, or 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 at least about 1000-fold, or more, compared to the reference level.

[0072] As used herein, "cancer" or "tumor" refers to the uncontrolled growth of cells that interfere with the normal function of body organs and body tissues. A subject having cancer or a tumor is a subject having 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 has metastasized from its original location and seeded in vital organs can potentially lead to the death of the subject through the functional destruction of the ultimately affected organ. Examples of cancer include, but are not limited to, B-cell lymphoma (Hodgkin lymphoma and / or non-Hodgkin lymphoma), brain tumor, breast cancer, colon cancer, lung cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, thyroid cancer, kidney cancer, carcinoma, melanoma, head and neck cancer, brain cancers such as glioblastoma, and prostate cancer, including, but not limited to, androgen-dependent prostate cancer and androgen-independent prostate cancer.

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

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

[0075] As used herein, "subject" means a human or an animal. Usually, the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Examples of primates include chimpanzees, cynomolgus monkeys, rhesus monkeys, and macaques such as the Japanese macaque. Examples of rodents include mice, rats, woodchucks, ferrets, guinea pigs and hamsters. Examples of domestic animals and game animals include cows, horses, pigs, deer, bison, buffalo, feline species such as domestic cats, and canine species such as dogs, foxes, 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, non-human primate, mouse, rat, dog, cat, horse or cow. Additionally, the methods described herein can be used to treat domestic animals and / or pets. In one embodiment, the subject is a human.

[0076] As used herein, "mammal" refers to any member of the class Mammalia, including, without limitation, humans and non-human primates such as chimpanzees and other apes and monkey species; agricultural animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals such as mice, rats and guinea pigs, and the like. This term does not denote a particular age. Thus, adult and neonatal subjects, as well as fetuses, are intended to be included within the scope of this term.

[0077] The subject has a disease that requires treatment (e.g., cancer or autoimmune disease) or one or more complications related to the disease, or has been previously diagnosed or identified as having the disease, and may in some cases have already received treatment for the disease or one or more complications related to the disease. Alternatively, the subject may have never been diagnosed in the past as having the disease or one or more complications related to the disease. For example, the subject may exhibit one or more risks of the disease or one or more complications related to the disease, or may not exhibit risk factors. For example, the subject may exhibit one or more symptoms of the disease or one or more complications related to the disease, or may not exhibit symptoms. A "subject in need of" diagnosis or treatment for a particular disease can be a subject suspected of having the disease, a subject diagnosed as having the disease, a subject who has already been treated or is to be treated for the disease, a subject who is not treated for the disease, or a subject at risk of developing the disease.

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

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

[0080] As used herein, the term "co-administer" refers to the administration of two or more treatments within 24 hours of each other, or two or more therapeutic agents (e.g., an anti-BAG2 antibody and an additional anti-cancer treatment), as part of, for example, a clinical treatment regimen. In other embodiments, "co-administer" 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-administer" refers to simultaneous administration either as part of a single formulation or as multiple formulations administered by the same or different routes. For example, if the anti-BAG2 antibody and the additional anti-cancer treatment are administered in different pharmaceutical compositions or at different times, the route of administration may be the same or different.

[0081] As used herein, "treatment" is an approach for obtaining a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, diminishment of the extent of the disease, stabilization of the disease (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean prolonged survival as compared to expected survival if not receiving treatment. "Treatment" refers to both therapeutic treatment and prophylactic or preventive measures. Those in need of treatment include those already having a disorder as well as those in whom the disorder should be prevented. "Reducing" a disease means that the extent of the disease state and / or the manifestation of the undesirable clinical symptoms are decreased as compared to a situation without treatment, and / or the time course of progression is slowed or extended.

[0082] As used herein, the term "chimeric" antibody refers to an antibody having a variable sequence derived from a non-human immunoglobulin, such as a rat or mouse antibody, and a human immunoglobulin constant region, typically selected from a human immunoglobulin template. Methods for generating chimeric antibodies are known in the art. See, e.g., 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] The "humanized" form of a non-human (e.g., murine) antibody is a chimeric immunoglobulin containing minimal sequences derived from a non-human immunoglobulin. Generally, a humanized antibody contains substantially all, or at least one, and typically two variable domains, where all or substantially all of the CDR regions correspond to those of the non-human immunoglobulin and all or substantially all of the FR regions are of human immunoglobulin sequence. A humanized antibody may also contain at least a portion of the immunoglobulin constant region (Fc), typically of human immunoglobulin consensus sequence. Methods for antibody humanization are known in the art. See, for example, Riechmann et al., 1988, Nature 332:323-7; U.S. Pat. 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 WO91 / 09967; U.S. Pat. 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. Pat. No. 5,565,332.

[0084] "Human antibody" includes antibodies having the amino acid sequences 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 prepared by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Pat. 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 generated using transgenic mice that are unable to express functional endogenous immunoglobulins but can express human immunoglobulin genes. See, for example, PCT Publications WO98 / 24893; WO92 / 01047; WO96 / 34096; WO96 / 33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598. In addition, companies such as LakePharma, Inc. (Belmont, Calif.) or Creative BioLabs (Shirley, N.Y.) may engage in providing human antibodies against selected antigens using techniques similar to those described above. All human antibodies that recognize a selected epitope can be prepared using a technique called "guided selection." In this approach, a selected non-human monoclonal antibody, such as a mouse antibody, is used to guide the selection of fully human antibodies that recognize the same epitope (see Jespers et al., 1988, Biotechnology 12:899-903).

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

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

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

[0088] Removal of hot spots During manufacturing, storage, and in vivo, therapeutic antibodies are at risk of degradation via multiple pathways. In particular, the most frequent degradation reactions in proteins are the chemical degradation of Asn and Asp residues. These reactions can be suppressed by appropriate storage and formulation conditions of the final drug substance and drug product, but degradation during fermentation, downstream processing, and in vivo often cannot be fully controlled. When Asn and Asp residues are involved in antigen recognition, their chemical modification can lead to a severe loss of potency. In multiple examples, these degradation events have been reported to interfere with long-term mAb function. In vivo, proteolytic events are associated with the aging of proteins, cancer by inducing apoptosis, or severe effects on other biological functions, such as a decrease in the stability of human lens betaA3 crystallin, abnormal MAPK signaling, modification of the potential beta-secretase efficacy and specificity in the A beta production process, or an increase in the lysozyme lysis activity against bacterial cells. The identification of drug candidates prone to degradation is ideally carried out early in the drug development process, and the manufacturing and formulation processes are adjusted accordingly, or the problematic candidates are re-engineered to remove such hotspots.

[0089] Asn and Asp residues share a degradation pathway that proceeds through the formation of a cyclic succinimide intermediate. Succinimide results from the deamidation of Asn or the dehydration of Asp by nucleophilic attack of the backbone nitrogen of the consecutive amino acid on the Asn / Asp side-chain γ-carbonyl group. The labile cyclic imide can hydrolyze at either of the two carbonyl groups, depending on the hydrolysis conditions and the steric constraints, to form aspartyl or isoaspartyl linkages in different ratios. In addition, alternative degradation mechanisms for Asn have been proposed, such as nucleophilic attack by the backbone carbonyl oxygen that forms the cyclic isoimide, or direct hydrolysis of Asn to Asp. Multiple analytical methods, most of which are charge-sensitive methods such as ion-exchange chromatography, or isoelectric focusing electrophoresis, have been described for detecting any of the degradation products, namely succinimide, Asp or isoAsp. The analysis by liquid chromatography tandem mass spectrometry (LC-MS / MS) is most suitable for the quantification and localization of the degradation sites in proteins. The 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 as doi: 10.1371 / journal.pone.0100736, is hereby incorporated by reference in its entirety for the disclosure of well-established knowledge regarding the presence of hot spots on the antigen-binding regions of antibodies and the suitability of replacing such hot spot motifs to provide more stability to proteins.

[0090] One such example of hotspot detection and replacement can be observed with antibody 3B5. The VH CDR2 is YIDPYNGGNTYNRKFKG, but the hotspot is detected due to the presence of "NG", and the hotspot is removed and replaced with "NA" such that the sequence with the hotspot removed is YIDPYNAGNTYNRKFKG. The applicant notes that the presence of the Y at the start of the CDR2 must be considered part of the CDR2. It is generally recognized in the art that CDR sequences can maintain binding activity even if some sequences are shortened by only 2 or 3 amino acids at the N-terminus or C-terminus. The length of the CDR for maintaining activity is considered to be well within the scope of reasonable experimentation to determine.

[0091] Humanization of Antibodies The process of humanizing an antibody is well-known, and thus a humanized antibody may be prepared using conventional techniques. One exemplified protocol may be as follows:

[0092] Humanize a murine monoclonal antibody by CDR grafting, identify important residues of the parental murine antibody framework, and introduce them into the humanized sequence. Then, convert the humanized VH / VL to the full-length hIgG1 format. Express the variants by transient transfection and purify them by affinity chromatography. Characterize the purified antibodies by SEC-HPLC, SDS-PAGE, ELISA, and Biacore (affinity characterization). Select the best candidates with a loss of affinity and potency of three-fold or less.

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

[0094] Determine the VH / VL CDR residues and annotate them with the Kabat numbering system. Apply sequence analysis to identify major risk hotspots including unpaired cysteine residues, N - glycosylation sites, and deamination sites within the CDRs. Apply genetic engineering work to remove harmful hotspot motifs if possible. If hotspots exist in the CDRs, check and execute the removal of hotspots.

[0095] Design the removal of hotspots and combine the relative mouse VH and mouse VL into multiple chimeric antibodies. Transiently express each chimeric antibody in 100 mL of 293 cells, capture and purify the supernatant with Mabselect PrismA (GE, 17549801), and record the yield of affinity purification. After testing the purified chimeric antibodies by SDS - PAGE and HPLC - SEC, confirm the affinity by ELISA or Biacore. Perform an affinity ranking of the chimeric antibodies 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; The underlines are the CDR regions after checking for hotspots. QVQLVQSGAEVKKPGASVKVSCKAS GYSFTDYTFY WVRQAPGQRLEWIG YIDPYNAGNTYNRKFKG RVTITVDKSASTAYMELSSLRSEDTAVYYCAR GYYRYGGGGDFDY WGQGTLVTVSS(SEQ ID NO:75)

[0098] 3B5 Subclone 1: VL region; The underlines are the CDR regions after checking for hotspots. DVVMTQSPLSLPVTLGQPASISC RSSQSLVHSNGNTYLH WFQQRPGQSPRLLIH KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SQNTHIPPTFGGGTKVEIK (SEQ ID NO:76)

[0099] 3B5 Subclone 2: VH region; The underlines indicate the CDR regions after checking the hotspots. QVQLVQSGAEVKKPGASVKVSCKAS GYSFTDYTFY WVRQAPGQRLEWIG YIDPYNAGNTYNRKFKG KVTITVDKSASTAYMELNSLRSEDTAVYYCAR GYYRYGGGGDFDY WGQGTLVTVSS (SEQ ID NO:77)

[0100] 3B5 Subclone 2: VL region; The underlines indicate the CDR regions after checking the hotspots. DVVMTQSPLSLPVTLGQPASISC RSSQSLVHSNGNTYLH WFQQRPGQSPRLLIH KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SQNTHIPPT FGGGTKVEIK (SEQ ID NO:78)

[0101] 3B10 Subclone 1: VH region; The underlines indicate the CDR regions after checking the hotspots. QVQLVQSGAEVKKPGASVKVSCKAS GHAFTNYMIE WVRQAPGQGLEWMG VINPGSGGTYNSEKVKG RVTLTADRSISTAYMELSRLRSDDTAVYYCRI YGNYKGYFDH WGQGTLVTVSS (SEQ ID NO:79)

[0102] 3B10 Subclone 1: VL region; The underlines indicate the CDR regions after checking the hotspots. DIQMTQSPSSLSASVGDRVTITC KASQDMNSYLS WFQQKPGKAPKSLIY RSNRLVD GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LQNDEFPFT FGQGTKLEIK (SEQ ID NO:80)

[0103] 3B10 Subclone 2: VH region; The underlines indicate the CDR regions after checking for hotspots. QVQLVQSGSELKKPGASVKVSCKAS GYSFTKYGMN WVKQAPGQGLEWMG WINTNTGEATYGEEVKG RFVFSLDTSVSTAYLQISSLKAEDTAVYYCAR LGLRYLDY WGQGTLVTVSS(SEQ ID NO:81)

[0104] 3B10 Subclone 2: VL region; The underlines indicate the CDR regions after checking for hotspots. DIQMTQSPSSLSASVGDRVTITC RASKSVSTSDYSYMH WYQQKPGKAPKLLIY LASNLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QHNRELPPT FGQGTKLEIK(SEQ ID NO:82)

[0105] 3G8 Subclone 1: VH region; The underlines indicate the CDR regions after checking for hotspots. QVQLVQSGSELKKPGASVKVSCKAS GYSFTKYGMN WVRQAPGQGLEWMG WINTNTGEATYGEEVKG RFVFSLDTSVSTAYLQISSLKAEDTAVYYCAR LGLRYLDY WGQGTLVTVSS(SEQ ID NO:83)

[0106] 3G8 Subclone 1: VL region; The underlines indicate the CDR regions after checking for hotspots. DIQMTQSPSSLSASVGDRVTITC RASKSVSTSDYSYMH WYQQKPGKAPKLLIY LASNLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QHNRELPPT FGQGTKLEIK(SEQ ID NO:84)

[0107] 3G8 Subclone 2: VH region; The underlines indicate the CDR regions after checking for hotspots. QVQLVQSGSELKKPGASVKVSCKAS GYSFTKYGMN WVKQAPGQGLEWMG WINTNTGEATYGEEVKG RFVFSLDTSVSTAYLQISSLKAEDTAVYYCAR LGLRYLDY WGQGTLVTVSS(SEQ ID NO:85)

[0108] 3G8 Subclone 2: VL region; The underlines indicate the CDR regions after checking the hotspots. DIQMTQSPSSLSASVGDRVTITC RASKSVSTSDYSYMH WYQQKPGKAPKLLIY LASNLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QHNRELPPT FGQGTKLEIK(SEQ ID NO:86)

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

[0110] Detection of elevated levels of Bag2 in a patient sample is a diagnosis 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 an indicator that the patient has cancer or is at risk of developing cancer. The levels of BAG2 species can be measured or evaluated by PCR, hybridization schemes, cycling probe technology, FISH, immunocytochemistry, IHC, Western blot, immunoprecipitation, sandwich assay, ELISA assay and the like. The patient sample can be a fluid sample, blood sample, milk, urine, cells, liquid biopsy, biopsy and the like. In patients diagnosed with cancer, elevated levels of BAG2 are an indicator of increased potential for metastasis. Elevated levels of BAG2 are an indicator of prostate cancer. The antibodies of the present invention are used to detect BAG2 and are used as diagnostic tools.

[0111] Since cells and tissues do not normally secrete BAG2, an effective method for diagnosing cancer, or for diagnosing a more aggressive or metastatic form, or for diagnosing a shift to a more aggressive form, is to measure the level of BAG2 in a sample from a patient, from a collection of cells or tissue, or from cultured cells, 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. An increase in the level of BAG2 indicates the presence of cancer, the presence of metastatic cancer, or the onset of metastasis. The sample assayed for the presence of BAG2 can be a collection of cells, a body fluid, a blood sample, a tissue specimen, or a biopsy specimen that can be a cultured cell line or cells from a patient. Therefore, a diagnostic assay for detecting the presence or progression of cancer comprises: 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 the measured BAG2 protein in the test sample to the level in a control patient or control cells; 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 cancer progression if the control to which the test substance was compared was from a donor previously diagnosed with cancer.

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

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

[0114] Immunomodulator Immunomodulators or immunotherapeutic agents can be broadly classified into four categories: checkpoint inhibitors, cytokines, agonists, and adjuvants. Checkpoint inhibitors act by blocking immune checkpoints, i.e., the "brakes" of the immune system, which tumors frequently manipulate to shut down the immune response and protect the tumor itself. As a result, checkpoint inhibitors can elicit a new immune response against cancer and enhance existing responses to promote the elimination of cancer cells. As of 2020, checkpoint inhibitors are perhaps the most well-known and widely successful immunomodulatory substances developed to date.

[0115] For example, the PD-1 / PD-L1 immune checkpoint pathway can shut down cancer-targeted T cells. However, when checkpoint inhibitors block the PD-1 / PD-L1 pathway, they can enable 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 immunotherapy drugs for the treatment of subsets of patients with kidney cancer, leukemia, lymphoma, melanoma, and sarcoma.

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

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

[0119] Examples of such immunomodulatory substances 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 to, or in combination with, other agents or treatments having anti-cancer properties. When used adjunctively, the anti-BAG2 antibody and other agent(s) may be formulated together as a single combined drug formulation, or may be formulated and administered separately in a single coordinated dosing regimen or in different dosing regimens. Agents administered adjunctively to, or in combination with, the anti-BAG2 antibody typically have complementary activity to the anti-BAG2 antibody, such that the antibody or other agent do not adversely affect one another.

[0121] Agents that can be administered adjunctively to an anti-CD40 antibody or together with the antibody include alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotic agents, antiproliferative agents, antiviral agents, aurora kinase inhibitors, activators of the cell death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (bispecific T cell engager) antibodies, antibody-drug conjugates, biological response modifiers, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVD, leukemia virus 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 (IAP), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, inhibitors of mammalian target of rapamycin (mTor), microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, non-steroidal anti-inflammatory drugs (NSAID), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapy 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, retinoids / deltioid plant alkaloids, small inhibitory ribonucleic acids (siRNA), topoisomerase inhibitors, ubiquitin ligase inhibitors and the like, and one or more combinations of these agents, but are not limited thereto.

[0122] Examples of immunotherapeutic agents include, but are not limited to, interferons, immune checkpoint inhibitors, and other immune boosters. Examples of interferons include interferon alpha, interferon alpha-2a, interferon alpha-2b, interferon beta, interferon gamma-1a, interferon gamma-1b, or interferon gamma-n1, combinations thereof, and the like. Examples of immune checkpoint inhibitors include antibodies targeting 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). Examples of immune boosters include anti-OX40 agonist antibodies that activate T cells.

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

[0124] Celanostics Patients diagnosed with an elevated level of secreted BAG2 protein are then treated with a therapeutic agent that specifically binds to BAG2. Therefore, patients diagnosed with an elevated secretion level of BAG2 will benefit from treatment with a therapeutic agent that inhibits BAG2. Thus, assessing the appropriateness of cancer treatment and the administration of an effective amount of a therapeutic agent for the treatment or prevention of cancer consists of: 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, wherein the measured level significantly exceeds that measured in a control sample; 3) determining that the patient has cancer, or has developed a more aggressive or metastatic cancer; and 4) administering to the patient an effective amount of a therapeutic agent that suppresses the expression of BAG2.

[0125] Chemically Modified Peptide Polypeptides or antibody therapeutics may be subject to short circulating half-lives and proteolysis and low solubility. To improve the pharmacokinetic and pharmacodynamic properties of the biopharmaceuticals of the present invention, methods such as manipulation of the amino acid sequence may be carried out to reduce or increase immunogenicity, reduce proteolytic cleavage, and may involve fusion or conjugation of the peptide to serum proteins such as immunoglobulins and albumin; incorporation into drug delivery vehicles for biopharmaceuticals such as the peptides and antibodies of the present invention for protection and sustained release may also be carried out; conjugation to natural or synthetic polymers is also contemplated. Specifically, for synthetic polymer conjugation, pegylation, or acylation, such as N-acylation, S-acylation, etc., is also contemplated.

[0126] Nucleic Acid Construct Also provided is an expression vector comprising a nucleic acid molecule of the present 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 the expression vector of the present invention introduced into a host cell suitable for the 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 present invention also provides a method for producing a polypeptide of the present invention by growing cells of the host-vector system described herein under conditions that allow production of the polypeptide of the present invention and recovering the polypeptide so produced. Polypeptides useful for practicing the present 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, for example, but not limited to, pZErO.

[0129] The polypeptide may be purified by any technique that allows for the formation of a stable bioactive protein. For example, and not by way of limitation, the factor may be recovered from the cells as either a soluble protein or an inclusion body, and they may be extracted quantitatively by 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 of the methods known to those of skill in the art for inserting a DNA fragment into a vector may be used to construct an expression vector encoding the polypeptide of the present invention using appropriate transcription / translation control signals and a protein coding sequence. 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 may be used to control the expression of the polypeptide include 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 within 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. U.S.A. 78:144-1445), the regulatory sequences of the metallothionein gene (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. U.S.A. 75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A.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, ADH (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions that exhibit tissue specificity and have been used in transgenic animals: 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); insulin gene control region active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin gene control region active in lymphocytes (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), testis, mammary gland, lymph, 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 the liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58); alpha1-antitrypsin gene control region active in the liver (Kelsey et al., 1987, Genes and Devel.(Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94); the myelin basic protein gene regulatory region that is active in oligodendrocytes in the brain (Readhead et al., 1987, Cell 48:703-712); the myosin light chain-2 gene regulatory region that is active in skeletal muscle (Shani, 1985, Nature 314:283-286), and the gonadotropin-releasing hormone gene regulatory region that is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378), but are not limited thereto.

[0131] Accordingly, according to the present invention, a host is transfected with an expression vector capable of replicating in a bacterial or eukaryotic host containing a nucleic acid encoding the polypeptide described herein, thereby directing the expression of such nucleic acid to produce a polypeptide, which may then be recovered in a biologically active form. As used herein, a biologically active form includes a form capable of binding to a relevant receptor, 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, but are not limited to, at least three general approaches: (a) DNA-DNA hybridization, (b) the presence or absence of "marker" gene function, and (c) expression of the inserted sequence. In the first approach, the presence of foreign nucleic acid inserted into the 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 specific "marker" gene function (e.g., thymidine kinase activity, antibiotic resistance, transformed phenotype, inclusion body formation in baculovirus, etc.) caused by the insertion of a 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 for foreign nucleic acid products expressed by the recombinant construct. Such assays can be based on the physical or functional properties of the corresponding nucleic acid products, for example, by the binding of a ligand, such as a detectable antibody or a part thereof, to a receptor or a part thereof that can be tagged, or by the binding of an antibody generated against the corresponding protein or a part thereof.

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

[0134] An effective dose useful for treating the diseases or disorders indicated within this application may be determined using methods known to those of ordinary skill in the art (see, e.g., 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 pharmaceutically acceptable liquid, solid, or semi-solid carrier which is linked to a carrier or target molecule (such as an antibody, hormone, growth factor, etc.) and / or incorporated within liposomes, microcapsules, and controlled release preparations prior to in vivo administration. For example, the pharmaceutical composition may comprise the polypeptide in an aqueous solution such as sterile water, physiological saline, phosphate buffer, or dextrose solution. Alternatively, the active agent may be included in a solid (such as wax) or semi-solid (such as gelatinous) formulation that can be implanted into a patient in need of such treatment. Routes of administration may include, without limitation, any mode of administration known in the art, including intravenous, intrathecal, subcutaneous, intrauterine, intra-arterial, intranasal, oral, or via an implant device by injection into the tissue involved.

[0135] Administration can result in the distribution of the active agent of the invention throughout the body or to a local area. For example, in some conditions involving distant regions of the nervous system, intravenous or intrathecal administration of the drug 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, implants based on gelfoam, wax, spray, or microparticles.

[0136] The present invention also provides a pharmaceutical composition comprising the polypeptides described herein in a pharmaceutically acceptable vehicle. The composition 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 a treatment that is carried out by administration of an expressed or expressible nucleic acid to a subject. 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 may be used according to the present invention. Exemplary methods are described below.

[0139] For a general review of the methods of gene therapy, 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 nucleic acids to a patient may be direct, in which case the patient is directly exposed to the nucleic acid or a vector carrying the nucleic acid, or indirect, in which case cells are first transformed with the nucleic acid in vitro and then transplanted into the patient. These two approaches are known as in vivo or ex vivo gene therapy, respectively.

[0141] In a specific embodiment, the nucleic acid sequence is administered directly 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, for example, by constructing the nucleic acid sequence as part of a suitable nucleic acid expression vector and administering the vector such that the nucleic acid sequence enters the cell, for example, 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, or by administering them linked to ligands that have undergone receptor-mediated endocytosis (see, for example, Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (this can be used to target cell types that specifically express the receptor), etc. In another embodiment, a nucleic acid-ligand complex can be formed, in which the ligand contains a fusogenic viral peptide to disrupt the endosome and the nucleic acid is protected from lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell-specific uptake and expression by targeting a specific receptor. Alternatively, the nucleic acid can be introduced into the cell by homologous recombination and integrated into the host cell DNA for expression (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, a viral vector containing a nucleic acid sequence encoding a polypeptide is used. The nucleic acid sequence encoding the polypeptide used in gene therapy is cloned into one or more vectors, which facilitates gene delivery to the patient. Lentiviral vectors such as retroviral vectors, as well as 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 the viral genome and integration into host cell DNA.

[0143] Adenoviruses are particularly attractive vehicles for delivering genes to the respiratory epithelium because they naturally infect the respiratory epithelium and cause mild disease. Other targets for adenovirus-based delivery systems are the 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 introducing genes into cells in tissue culture by methods such as electroporation, lipofection, transfection via calcium phosphate, or viral infection. Typically, the method of introduction involves introducing a selectable marker into the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the introduced gene. Those 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 effected 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, gene transfer via microcells, spheroplast fusion, and the like. Numerous techniques are known in the art for introducing foreign genes into cells and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique must provide stable transfer of the nucleic acid into the cell such that the nucleic acid is expressible by the cell and preferably inheritable and expressible by cell progeny.

[0146] Cells into which the nucleic acid can be introduced for the purposes of gene therapy include any desirable available cell type and include, but are 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; various stem cells or progenitor cells, particularly hematopoietic stem cells or progenitor cells such as those obtained from, for example, bone marrow, umbilical cord blood, peripheral blood, fetal liver, and the like.

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

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

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

[0150] Therapeutic composition Formulations of therapeutic compounds are generally known in the art and can be conveniently referred to in Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., USA. For example, from about 0.05 ng to about 20 mg / kg body weight / day may be administered. The dosing regimen may be adjusted to provide an optimal therapeutic response. For example, multiple divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the urgency of the therapeutic situation. The active compounds may be administered by oral, intravenous (if water-soluble), intramuscular, subcutaneous, intranasal, intraocular, intradermal or suppository routes, or by implantation (e.g., using sustained-release molecules by the intraperitoneal route or by using cells sensitized in vitro and transplanted into the recipient, such as monocytes or dendritic cells). Depending on the route of administration, the peptide may need to be coated with a material to protect it from the action of enzymes, acids and other natural conditions that may inactivate the above components.

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

[0152] The active compounds may also be administered parenterally or intraperitoneally. The dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under normal conditions of storage and use, these preparations contain preservatives that prevent the growth of microorganisms.

[0153] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions (if water-soluble), 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, for example, a solvent or dispersion medium containing water, ethanol, polyols (e.g., 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 maintenance of the required particle size in the case of a dispersion, 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 isotonic agents, for example, sugars or sodium chloride. Sustained absorption of the injectable composition can be brought about by the use of agents that delay absorption in the composition, for example, aluminum monostearate and gelatin.

[0154] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound in an appropriate solvent with various of the other ingredients enumerated hereinabove and then, if necessary, filtering sterilizing. Generally, dispersions are prepared by incorporating various sterilized active ingredients in a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated hereinabove. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient and any additional desired ingredient from previously filtered sterilized solutions thereof.

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

[0156] Tablets, pills, capsules and the like may also contain the following: binders such as tragacanth gum, gum arabic, corn starch or gelatin; excipients such as dicalcium phosphate; disintegrants 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 strawberry flavor may be added. When the dosage unit form is a capsule, it may contain a liquid carrier in addition to the materials of the above type. Various other materials may be present as coatings or in other ways to improve the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Syrups or elixirs may contain the active compound, sucrose as a sweetener, methyl and propyl parabens as preservatives, and dyes and flavorings such as cherry or orange flavor. Of course, any materials used in preparing any dosage unit form must be pharmaceutically 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 A variety of delivery systems are known and can be used to administer the compounds of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, construction of nucleic acids as part of retroviruses or other vectors. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intraocular, epidural, and oral routes. The compound or composition may be administered by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, among others) by any convenient route, such as by infusion or bolus injection, and may be administered together with other bioactive agents. Administration may be systemic or local. In addition, it is desirable to introduce the pharmaceutical compounds or compositions of the present invention into the central nervous system by any suitable route, including intracerebroventricular and intrathecal injection, and intracerebroventricular injection can be facilitated by an intracerebroventricular catheter attached to a reservoir, such as an Ommaya reservoir. Lung administration can also be used, for example, by the use of inhalers or nebulizers, and by formulation of aerosolizing agents.

[0158] In a specific embodiment, it is desirable to locally administer the pharmaceutical compound or composition of the present invention to the area in need of treatment, which can be achieved, for example, but not limited to, by local infusion during surgery, by topical application, for example in combination with a postoperative wound dressing, by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being a porous, non-porous, or gelatinous material containing a membrane or fiber such as a Sialastic membrane. Preferably, when administering a protein containing an antibody or peptide of the present invention, care must be taken to use a material that does not absorb the protein. In another embodiment, the compound or composition can be delivered in vesicles, particularly liposomes. In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, a polymeric material can be used. In yet another embodiment, the controlled release system can be placed near the therapeutic target, thereby requiring only a portion of systemic administration.

[0159] The following examples are provided as an illustration of the present invention and are not limiting.

[0160] Examples Example 1: Selection, Sequencing of Anti-BAG2 Antibody and Antigen-Antibody Reaction 1. Selection of Monoclonal Antibodies Targeting BAG2 and Analysis of Their Amino Acid Sequences The inventors selected antibodies targeting BAG2, analyzed their amino acid sequences, and determined the respective complementarity-determining regions (CDRs) of the antibodies.

[0161] Specifically, 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 recloned into the pCAGGS plasmid to make it linear, and the linearized construct was then inoculated into the muscles of 5 six-week-old female BALB / c mice by applying an electric shock. This construct was inoculated intramuscularly 3 times at 3-week intervals, which consisted of 100 μg of DNA in 100 μl of PBS. At this time, the plasmid of the control group was also subjected to the same method. To generate therapeutic and diagnostic antibodies, an immunization strategy based on DNA vaccines, which is more efficient than antigen injection based on proteins, was implemented. Blood was collected from the posterior vena cava or tail vein of the mice and examined by enzyme immunoassay to show the serum antibody titer, and the spleen was removed 3 days after the last immunization from the mice that showed a sufficient antibody titer. B lymphocytes were isolated from the spleen, and fusion with myeloma cells cultured with the isolated B lymphocytes, i.e., the ATCC's SP2 / 0-Ag14 cell line, continued, thereby obtaining fused cells. The fused cells were cultured in HAT medium containing hypoxanthine, aminopterin, and thymidine, and then approximately 130 clones of hybridoma cells fused only with myeloma and B lymphocytes were obtained by selecting. Through the selection process by immunoblotting, 10 hybridoma cells that produce antibodies specifically binding to the human BAG2 protein were obtained.

[0162] Total RNA of the anti-Bag2 antibody was 5×10 6Hybridoma cells were generated, and 5'-RACE-cDNA was generated from 100 ng of total RNA using the 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, and the amplified genes were inserted into the pGEM-T vector (Promega, USA), cloned, and their nucleotide sequences were analyzed using an automated 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 complementarity-determining regions VH-CDR1, -CDR2, and -CDR3, as well as VL-CDR1, -CDR2, and -CDR3. Determination of the sequences of the complementarity-determining regions was performed using the Kabat database (http: / / www.bioinf.org.uk / abs / ).

[0163] As a result, 10 anti-BAG2 antibodies that specifically bind to BAG2 were obtained from hybridoma cells. The 10 anti-BAG2 antibodies were the 2A11, 4C2, 8C4, 3B5, 9B3, 9B12, 3B10, 10H7, 3GB, and 3F12 antibodies. In addition, the amino acid sequences of the heavy-chain variable region, the 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, the 56th and 57th Xaa of SEQ ID NO:21 are each Gly, and the 53rd Xaa of SEQ ID NO:27 is lie. In the 4C2 antibody, the 56th Xaa and 57th Xaa of SEQ ID NO:21 are Gly and Ala, respectively, and the 53rd Xaa of SEQ ID NO:27 is Phe. In the 8C4 antibody, the 56th Xaa and 57th Xaa of SEQ ID NO:21 are Ala and Gly, respectively, and the 53rd Xaa 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 NO:33, 39, and 45, respectively, and the VL-CDR1, -CDR2, and -CDR3 consist of the amino acid sequences of SEQ ID NO:51, 57, and 63, respectively. In the 2A11 antibody, the 56th and 57th Xaa of SEQ ID NO:21 are each Gly. For the 4C2 antibody, the 56th Xaa of SEQ ID NO:21 is Gly and the 57th Xaa is Ala. For the 8C4 antibody, the 56th Xaa of 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, the 1st Xaa is Glu, the 7th Xaa is Ser, the 12th Xaa is Val, the 27th Xaa is Tyr, the 58th Xaa is Ser, the 61st Xaa is Asn, the 74th Xaa is Lys, the 83rd Xaa is Phe, the 92nd Xaa is Ala, and the 108th Xaa is Tyr. For the 9B3 antibody, in SEQ ID NO:29, the 2 9 nd Xaa is Il e, and 51 the 6th Xaa is Ala, and the 7 9 th Xaa is Glu, and the 10 6 th Xaa is Il e. For the 9B12 antibody, in SEQ ID NO:23, the 1st Xaa is Gin, the 7th Xaa is Ser, the 12th Xaa is Val, the 27th Xaa is Tyr, the 58th Xaa is Ser, the 61st Xaa is Asn, the 74th Xaa is Arg, the 83rd Xaa is Phe, the 92nd Xaa is Gly, and the 108th Xaa is His. For the 9B12 antibody, in SEQ ID NO:29, the 2 9 nd Xaa is Met, and 51 the 6th Xaa is Ala, and the 7 9 th Xaa is Glu, and the 10 6 th Xaa is Met. For the 3B10 antibody, in SEQ ID NO:23, the 1st Xaa is G lwhere n, the 7th Xaa is Pro, the 12th Xaa is Ala, the 27th Xaa is His, the 58th Xaa is Thr, the 61st Xaa is Ser, the 74th Xaa is Arg, the 83rd Xaa is Leu, the 92nd Xaa is Gly, and the 108th Xaa is His. For the 3B10 antibody, at position 2 in SEQ ID NO:29, the Xaa is 9 Met, and 51 at position 4, the Xaa is Ser, at 9 position 7, the Xaa is Asp, at 6 position 10, the Xaa is Il e.

[0167] For the 9B3, 9B12 and 3B10 antibodies, VH - CDR1, - CDR2, and - CDR3 consist of the amino acid sequences of SEQ ID NOS:35, 41, and 47 respectively, and 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 2nd Xaa in SEQ ID NO:35 is Tyr, the 8th Xaa in SEQ ID NO:41 is Ser, the 12th Xaa in SEQ ID NO:47 is Tyr, the 3rd Xaa in SEQ ID NO:53 is Ile, and the 2nd Xaa in SEQ ID NO:59 is Ala. For the 9B12 antibody, the 2nd Xaa in SEQ ID NO:35 is Tyr, the 8th Xaa in SEQ ID NO:41 is Ser, the 12th Xaa in SEQ ID NO:47 is His, the 3rd Xaa in SEQ ID NO:53 is Met, and the 2nd Xaa in SEQ ID NO:59 is Ala. For the 3B10 antibody, the 2nd Xaa in SEQ ID NO:35 is His, the 8th Xaa in SEQ ID NO:41 is Thr, the 12th Xaa in SEQ ID NO:47 is His, the 3rd Xaa in SEQ ID NO:53 is Met, and the 2nd Xaa in SEQ ID NO:59 is Ser.

Table 1

Table 2

Table 3

[0168] 2. Identification of the antigen-antibody response of anti-BAG2 antibody in breast cancer cells Figure 1 shows the results of immunoblotting of anti-BAG2 antibodies generated from 10 mouse hybridoma cells. Specifically, MDA-MB-231 cells, which are human breast cancer 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 / ml aprotinin, and 0.2 mM sodium orthovanadate. After incubation on ice for 15 minutes, the nuclei were removed from the cells by centrifugation, and the supernatant was collected. An appropriate amount of 2X sample buffer consisting of 20% glycerol, 4.6% SOS, 0.125 M Tris pH 6.8, and 0.1% bromophenol blue was added to the supernatant. 10 μg of the protein sample was subjected to SDS-PAGE analysis on a 12% gel under standard conditions using a mini-Protean II system (Bio-Rad, Hercules, CA). For immunoblotting, the protein was transferred onto a Millipore PVDF membrane. A blocking solution consisting of 0.1% Tween 20 and 5% bovine serum albumin (BSA) in TBS was reacted for 1 hour. Next, the primary antibody was a 1 / 2000 dilution of the anti-BAG2 antibody extracted from the hybridoma cell culture, and the goat anti-mouse HRP conjugate (Dako) used as the secondary antibody was diluted to 1 / 5000. Film exposure was performed in the dark using EGL reagent (Amersham Pharmacia Biotech) as the substrate. The exposed bands were compared with a standard molecular marker to identify the band corresponding to the size of BAG2.

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

[0170] Next, for the domain mapping of the BAG2 antigen identified by 10 antibodies in the previous first section, 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 containing a polynucleotide encoding the human BAG2 protein and a polynucleotide encoding a fragment of the BAG2 protein, were transfected into cells. The transfected cells were cultured to express the gene, and then cell lysates were obtained. For the cell lysates, immunoblotting was performed using each of the 10 antibodies. The polynucleotide encoding the human BAG2 protein has the nucleotide sequence of SEQ ID NO:70. The GST-Bag F1, -Bag F2, -Bag F3, and -Bag F4 vectors each consist of the nucleotide sequences of SEQ ID NO:71 to 74.

[0171] Figures 2A and 2B show the results of immunoblotting for the full-length BAG2 polypeptide of the anti-BAG2 antibody or its fragment. In Figure 2, A shows a diagram of the vector and BAG2 protein and its fragments, and B shows the results of immunoblotting. Specifically, immunoblotting was performed as follows: Each of the vectors was introduced into HEK2T cells by the Lipofectamine transfection (Thermo Fisher Scientific, Inc., located in Waltham, Massachusetts, USA) method, and the resulting transformed cells were cultured at a temperature of 37 °C for 30 hours in DMEM (Welgene) medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin, and then 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 reacting with the same blocking solution as described in relation to Figure 1 for 1 hour, each of the 10 purified anti-BAG2 antibodies having a concentration of 2 mg / ml was diluted 1 / 10000 and used as the primary antibody to bind to the cells. The goat anti-mouse HRP conjugate used as the secondary antibody was used at a dilution concentration of 1 / 5000, and film sensitization was performed in the dark using the EGL reagent (Amersham Pharmacia Biotech) as the substrate. Standard molecular markers were 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 fragment. In particular, for each of the 10 anti-BAG2 antibodies, a signal was commonly detected at a position of approximately 50 KDa in the lysate of the GST-Bag Full vector-introduced 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] Figure 3 shows the BAG2 domains that react with each anti-BAG2 antibody. As shown in Figure 3, the 9B3, 9B12, 3B10, and 10H7 antibodies were bound to the N-terminus of the BAG2 protein, the 2A11, 3B5, 4C2, and 8C4 antibodies were bound to the central region of the BAG2 protein, and the 3F12 and 3GB antibodies were bound to the C-terminus of the BAG2 protein. The N-terminus commonly contains a coiled-coil region of amino acids 21-60, and the central region binds to a portion of the BNB region of amino acids 109-189. Therefore, by using a set of antibodies that bind to different sites, the BAG2 protein or its fragments present in a sample can be detected with high sensitivity and specificity.

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

[0175] A polynucleotide encoding an antibody or an antigen-binding fragment thereof according to another embodiment, and a host cell containing the same can be used to produce the antibody or an antigen-binding fragment thereof.

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

[0177] Example 2. Preparation of Drugs and Mouse Models for a Method of Treating Cancer Ten hybridoma cell lines against BAG2 were purified from each culture supernatant 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 (trademark)) was used as an isotype control antibody and obtained from the American Type Culture Collection. Anti-BAG2 mouse antibodies and isotype control antibodies used in in vitro and in vivo tests were generated by Nanotool under endotoxin-free conditions (<0.01 EU / μg).

[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 manufacturer's 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). These mice were housed in a room with a temperature controlled at approximately 22°C, and the mice were freely supplied with feed and water. After one week, 2×10 5 cells of the mouse EMT6 breast cancer cell line (ATCC, CRL-2755 (trademark)), 5×10 5 cells of the mouse Lewis lung cancer (LLC) cell line (ATCC, CRL1642 (trademark)), 5×10 5 cells of the mouse MC38 colon cancer cell line (Kerafast, ENH204-FP), and 3×10 5 cells of the mouse CT26 colon cancer cell line (ATCC, CRL2638 (trademark)) were injected into these 6-week-old mice by subcutaneous injection. 7- to 8-week-old mice were used in the experiment 8 to 12 days after injection of the EMT6, LLC, MC38, and CT26 cell lines. After one week, 2×10 5Cells were injected into these 7-week-old mice by intravenous injection into the tail vein. 9-week-old mice were used in 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) 6 cells were orthotopically transplanted into the pancreatic tail of 7-week-old mice. 10-week-old mice were used in the experiment 16 days after injection of the PANC02-Luc cell line.

[0180] Mechanism of action Tumor-secreted BAG2 protein can bind to lymphocytes, bone marrow, and stromal cells for anti-immunity (adaptive or innate immunity) in the tumor microenvironment. The inventors hypothesized that BAG2 neutralization targets lymph-, marrow-, or stroma-mediated immunosuppression, while immune checkpoint inhibitors restore the function of anergized anti-tumor T cells and the anti-inflammatory properties of bone marrow cells.

[0181] 2.1. Results of breast cancer treatment in mouse models The results are as follows.

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

[0183] Combination therapy with the anti-BAG2 antibody and the anti-PD-L1 antibody has synergistic anti-tumor 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 by tumor size before drug administration. On the 12th day after tumor cell injection, the mice were treated with the anti-BAG2 antibody 3B10. Next, on the 14th day, the mice received a single injection of the anti-PD-L1 antibody.

[0185] As shown in FIGS. 4b and 4c, on day 24, the tumor volumes of the anti-PD-L1 antibody administration group, the anti-BAG2 antibody 3B10 administration group, and the combined administration group of anti-PD-L1 antibody and 3B10 decreased by approximately 20.2%, approximately 39.8%, and approximately 70.1%, respectively, compared to the tumor volume of the isotype control group. Treatment with the anti-PD-1 antibody had a slight 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 single agent.

[0186] As shown in FIG. 4d, the tumor-specific CD3+ / CD8+ T cells (effector memory cells in killing cancer cells) in the anti-PD-L1 antibody administration group, the anti-BAG2 antibody 3B10 administration group, and the combined administration group of anti-PD-L1 antibody and 3B10 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 single agent. The activation can be direct or indirect, i.e., inhibition of suppressor cells.

[0187] 2.2. Results of lung cancer treatment in mouse models The results are as follows.

[0188] Combined therapy with anti-BAG2 antibody and immune checkpoint inhibitors has a synergistic anti-tumor effect in a mouse lung cancer model.

[0189] As shown in FIG. 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 FIGS. 5b and 5c, on day 20, the tumor volumes of the anti-PD-L1 antibody administration group, the anti-BAG2 antibody 3B10 administration group, and the combination administration group of anti-PD-L1 antibody and 3B10 decreased by approximately 8.7%, approximately 17.8%, and approximately 62.2%, respectively, compared to the tumor volume 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 single agent.

[0191] As shown in FIG. 5d, the tumor-specific CD3+ / CD8+ T cells (effector memory cells in killing cancer cells) in the anti-PD-L1 antibody administration group, the anti-BAG2 antibody 3B10 administration group, and the combination administration group of anti-PD-L1 antibody and 3B10 increased by approximately 1.3-fold, 2.8-fold, and 6.6-fold, respectively, compared to the CD3+ / CD8+ T cell levels of 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 single agent. The activation can be direct or indirect, i.e., inhibition of suppressor cells.

[0192] 2.3. Results of melanoma treatment in mouse models The results are as follows.

[0193] The anti-BAG2 antibody has antitumor activity in a mouse melanoma lung metastasis model.

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

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

[0196] As shown in FIGS. 6b and 6c, on day 38, the tumor volumes of the anti-PD-L1 antibody administration group, the anti-BAG2 antibody 3F12 administration group, and the combined administration group of anti-PD-L1 antibody and 3F12 decreased by approximately 19%, approximately 54%, and approximately 92%, respectively, compared to the BLI signal of the isotype control group. Monotherapy with the anti-PD-1 antibody had a slight effect on melanoma tumor growth in the lung, while the anti-BAG2 antibody 3F12 significantly inhibited melanoma tumor growth in the lung. Treatment with the combination of anti-PD-L1 antibody and 3F12 inhibited tumor growth to a greater extent than either single agent.

[0197] 2.4. Treatment of colorectal cancer Background Colorectal cancer (CRC) with high-frequency microsatellite instability (MSI) has defects in mismatch repair (MMR) and increased levels of PD-L1, LAG-3, and IDO, and responds clearly to anti-programmed cell death (PD) therapy. Low-frequency MSI or microsatellite stable (MSS) CRC, which constitutes the majority of tumors in the clinical setting, did not show any benefit from PD-1 inhibition. MSS CRC has a higher proportion of oncogenic KRAS mutations compared to MSI CRC. The combination therapy of anti-BAG2 antibody and anti-PD-1 agent was tested in a synergistic model of C57BL / 6 (MC38; KRASwt, MSI) and BALB / c (CT26; KRASmut, MSS) mice.

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

[0199] The anti-BAG2 antibody alone has anti-tumor activity in a high-frequency MSI type of mouse colorectal cancer model. The combination therapy of anti-BAG2 antibody and anti-PD-1 antibody has a synergistic anti-tumor effect in a mouse CRC model.

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

[0201] As shown in Figs. 7b and 7c, on day 20, the tumor volumes of the anti-PD-1 antibody administration group, the anti-BAG2 antibody 3B10 administration group, and the combined administration group of the anti-PD-1 antibody and 3B10 decreased by approximately 43%, approximately 40.8%, and approximately 86.8%, respectively, compared to the tumor volume of the isotype control group. Treatment with the anti-PD-1 antibody or the anti-BAG2 antibody 3B10 alone significantly inhibited tumor growth. Treatment with the combination of the anti-PD-1 antibody and 3B10 inhibited tumor growth to a greater extent than either single agent.

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

[0203] 2.4.2. Results of treatment of MSS type colorectal cancer in a mouse model The results are as follows.

[0204] The anti-BAG2 antibody alone has antitumor activity in a mouse MSS type colorectal cancer model.

[0205] The combination therapy of the anti-BAG2 antibody and the anti-PD-L1 antibody has a synergistic antitumor effect in a mouse MSS type CRC model.

[0206] As shown in Fig. 8a, CT26 tumor-bearing BALB / c mice were treated on day 11 after tumor cell injection with 250 μg (low dose) or 750 μg (high dose) / mouse of anti-BAG2 antibody 3B10, anti-PD-L1 antibody, a combination of 3B10 and anti-PD-L1 antibody, or a control antibody. The mice were administered by i.p. injection once every two days until the end of the experiment. Tumor growth was monitored and tumor volume was measured at the time points indicated by an electronic caliper.

[0207] As shown in Figs. 8b and 8c, on day 23, the tumor volumes of the low-dose 3B10 administration group, high-dose 3B10 administration group, anti-PD-L1 antibody administration group, combination administration group of anti-PD-L1 antibody and low-dose 3B10, and combination administration group of anti-PD-L1 antibody and high-dose 3B10 decreased by approximately 9.1%, approximately 78.8%, approximately 17.4%, approximately 52.2%, and approximately 95.1%, respectively, compared to the tumor volume of the isotype control group. Treatment with high-dose 3B10 alone strongly inhibited the growth of CT26 tumors. Treatment with the anti-PD-L1 antibody was not very 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 single agent. Moreover, combined treatment with anti-PD-L1 antibody and high-dose 3B10 showed regression of individual tumors to undetectable levels in most of the treated mice.

[0208] As shown in Fig. 8d, the tumor-specific CD3+ / CD8+ T cells (effector memory cells in killing cancer cells) in the low-dose 3B10 administration group, high-dose 3B10 administration group, anti-PD-L1 antibody administration group, combination administration group of anti-PD-L1 antibody and low-dose 3B10, and combination administration group of anti-PD-L1 antibody and high-dose 3B10 increased by approximately 1.4-fold, 3.8-fold, -1.5-fold, 6.6-fold, and 8.7-fold, respectively, compared to the CD3+ / CD8+ T cell levels of 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 single agent. The activation can be direct or indirect, i.e., inhibition of suppressor cells.

[0209] 2.5. Results of Pancreatic Cancer Treatment in Mouse Models The results are as follows.

[0210] Anti-BAG2 antibody alone has an antitumor 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) at 200 μg (low dose) / mouse (days 16 - 32) and 400 μg (high dose) / mouse (three times on days 35 - 39) or a control antibody after tumor cell injection. The mice were administered by i.p. injection three times a week until the end of the study. From days 30 - 39, the mice received an injection of anti-PD-1 antibody or anti-CTLA4 antibody. Tumor growth was monitored by IVIS imaging of bioluminescence (BLI) signals on days 15, 27, and 39 after implantation.

[0213] As shown in Fig. 9b, on day 40, the BLI signals of the 3B10 administration group, 3F12 administration group, 3B5 administration group, and 3G8 administration group, which are the groups administered with anti-BAG2 antibody individually, decreased by approximately 79.3%, approximately 68.4%, approximately 24.8%, and approximately 8.3%, respectively, compared to the isotype control group. The BLI signal intensities of the anti-PD-1 antibody administration group, the combination administration group of anti-PD-1 antibody and 3B10, the combination administration group of anti-PD-1 antibody and 3F12, the combination administration group of anti-PD-1 antibody and 3B5, and the combination administration group of anti-PD-1 antibody and 3G8 decreased by approximately -14.7%, approximately 95.9%, approximately 92.8%, approximately 62.3%, and approximately -10.9%, respectively, compared to the isotype control group. The BLI signal intensities of the administration group of anti-CTLA4 antibody alone, the combination administration group of anti-CTLA4 antibody and 3B10, the combination administration group of anti-CTLA4 antibody and 3F12, the combination administration group of anti-CTLA4 antibody and 3B5, and the combination administration group of anti-CTLA4 antibody and 3G8 decreased 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 Fig. 9c, on the 40th day, the primary tumor weights of the 3B10 administration group, 3F12 administration group, 3B5 administration group, and 3G8 administration group with only anti-BAG2 antibody decreased by approximately 63.8%, approximately 56.7%, approximately 27.8%, and approximately 18.9%, respectively, compared to the isotype control group. The BLI signal intensities of the administration group with only anti-PD-1 antibody, the combined administration group of anti-PD-1 antibody and 3B10, the combined administration group of anti-PD-1 antibody and 3F12, the combined administration group of anti-PD-1 antibody and 3B5, and the combined administration group of anti-PD-1 antibody and 3G8 decreased by approximately 17.8%, approximately 85.7%, approximately 87.4%, approximately 58.1%, and approximately -24.8%, respectively, compared to the isotype control group. The BLI signal intensities of the administration group with only anti-CTLA4 antibody, the combined administration group of anti-CTLA4 antibody and 3B10, the combined administration group of anti-CTLA4 antibody and 3F12, the combined administration group of anti-CTLA4 antibody and 3B5, and the combined administration group of anti-CTLA4 antibody and 3G8 decreased 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 Figs. 9b and 9c, single treatment with 3B10 and 3F12 strongly inhibited the BLI signal intensity and reduced the primary tumor weight in PANC02-Luc tumor-bearing mice, while 3B5 and 3G8 did not significantly inhibit the BLI signal intensity and did not significantly reduce the primary tumor weight in mice. The different phenomena among the four clones may be caused by the affinity for cross-reaction in mice and humans.

[0216] Treatment with anti-PD-1 antibody was not very successful in inhibiting tumor growth as a single agent. Treatment with a combination of anti-BAG2 antibody (3B10, 3F12, or 3B5) and anti-PD-1 antibody or anti-CTLA4 antibody inhibited tumor growth to a greater extent than either single agent. Moreover, combined treatment with anti-BAG2 antibody (3B10, 3F12, or 3B5) and anti-PD-1 antibody or anti-CTLA4 antibody reduced the occurrence of metastasis to the liver, pleura, and diaphragm compared to either single agent (Fig. 9d).

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

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

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

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

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

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

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

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

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

[0226] It should be understood that the embodiments described herein are to be considered in descriptive sense only and not for purposes of limitation. Descriptions of features or aspects in each embodiment should typically be considered as available to other similar features or aspects of 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 detail may be made without departing from the spirit and scope of the disclosure as defined by the following claims.

[0227] It should be understood that the embodiments described in this specification should not be regarded as having only descriptive meaning and should not be regarded for purposes of limitation. The description of features or aspects in each embodiment should typically be regarded as applicable to other similar features or aspects of 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 detail may be made without departing from the spirit and scope of the disclosure as defined by the following claims.

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

[0229] All reference materials cited in this specification are hereby 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 described herein.

Claims

**Claim 1** An antibody or an antigen-binding fragment thereof that specifically binds to a BAG2 polypeptide or a fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region including VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 34, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 40, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 46, and a light chain variable region including VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 52, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 58, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 64; a heavy chain variable region including VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 35 (wherein the second Xaa of SEQ ID NO: 35 is His), VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 41 (wherein the eighth Xaa of SEQ ID NO: 41 is Thr), and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 47 (wherein the twelfth Xaa of SEQ ID NO: 47 is His), and a light chain variable region including VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 53 (wherein the third Xaa of SEQ ID NO: 53 is Met), VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 59 (wherein the second Xaa of SEQ ID NO: 59 is Ser), and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 65; a heavy chain variable region including VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 37, VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 43, and VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 49, and a light chain variable region including VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 55, VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 61, and VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 67; or A heavy chain variable region comprising a VH-CDR1 consisting of the amino acid sequence of SEQ ID NO: 38, a VH-CDR2 consisting of the amino acid sequence of SEQ ID NO: 44, and a VH-CDR3 consisting of the amino acid sequence of SEQ ID NO: 50, and a VL-CDR1 consisting of the amino acid sequence of SEQ ID NO: 56, a VL-CDR2 consisting of the amino acid sequence of SEQ ID NO: 62, and a VL-CDR3 consisting of the amino acid sequence of SEQ ID NO: 68; comprising an antibody or an antigen-binding fragment thereof.

2. A heavy chain variable region of SEQ ID NO: 22 and a light chain variable region of SEQ ID NO: 28; A heavy chain variable region of SEQ ID NO: 23 and a light chain variable region of SEQ ID NO: 29; A heavy chain variable region of SEQ ID NO: 25 and a light chain variable region of SEQ ID NO: 31; or A heavy chain variable region of SEQ ID NO: 26 and a light chain variable region of SEQ ID NO: 32; The antibody or an antigen-binding fragment thereof according to claim 1, comprising

3. The antibody or an antigen-binding fragment thereof according to claim 1, which is a monoclonal antibody.

4. The antibody or an antigen-binding fragment thereof according to claim 1, which is labeled with a detectable label or a label capable of emitting a detectable signal.

5. The antibody or an antigen-binding fragment thereof according to claim 1, wherein the antibody is humanized.

6. The antibody or an antigen-binding fragment thereof according to claim 1, wherein hot spots are removed in the CDR of the antibody.

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

8. The antibody or an antigen-binding fragment thereof according to claim 1, wherein the antibody is bivalent, bispecific, or trispecific.

9. The antibody or an antigen-binding fragment thereof according to claim 1, wherein the antibody is fused to a chemical substance or a protein.

10. The antibody or an antigen-binding fragment thereof according to claim 9, wherein the antibody is fused to a toxin or a cytokine.

11. An antibody or antigen-binding fragment thereof according to claim 1, produced by a hybridoma cell selected from hybridoma cells deposited under accession numbers KCTC 13738BP, KCTC 13739BP, KCTC 13740BP, and KCTC 13741BP.

12. The antibody or antigen-binding fragment thereof according to claim 1, comprising a plurality of antibodies or antigen-binding fragments thereof.

13. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to claim 1.

14. The polynucleotide according to claim 13, which is a vector.

15. The polynucleotide according to 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 according to claim 13.

17. Culturing the host cell according to claim 16; and Separating an antibody or antigen-binding fragment thereof from the resulting culture, A method for producing an antibody or antigen-binding fragment thereof, comprising.

18. The method according to 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 primary cancer or metastatic cancer in an individual, comprising the antibody or antigen-binding fragment thereof according to claim 1.

20. The composition according to claim 19, wherein the method comprises co-administering or sequentially administering an existing therapeutic agent.

21. The composition according to claim 20, wherein the existing therapeutic agent is a cancer immunotherapy agent.

22. The composition according to claim 21, wherein the cancer immunotherapy agent is an inhibitor of an immune checkpoint molecule.

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

24. The composition according to claim 21, wherein the cancer immunotherapy drug 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 according to 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, gastric cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, clear cell sarcoma, melanoma, cerebrospinal tumor, brain cancer, thymus, mesothelioma, esophageal cancer, cholangiocarcinoma, 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 according to claim 25, wherein the cancer is breast cancer, lung cancer, melanoma, colorectal cancer, or pancreatic cancer.

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