Antibodies against PSMA and uses thereof

Abstract

The present invention relates to: anti-PSMA antibodies or antigen-binding fragments thereof; nucleic acids encoding same; recombinant expression vectors comprising the nucleic acids; host cells transfected with the recombinant expression vectors; methods for preparing the antibodies or antigen-binding fragments thereof; chimeric receptors (CARs) comprising the antibodies or antigen-binding fragments thereof; immune cell therapeutic agents comprising the chimeric receptors; antibody-drug conjugates (ADC) having the antibody or antigen-binding fragment thereof conjugated to a drug; compositions comprising the antibodies or antigen-binding fragments thereof, for diagnosing, preventing, or treating cancer; methods for diagnosing, preventing, or treating cancer; and uses thereof. The anti-PSMA antibodies or antigen-binding fragments thereof, according to the present invention, exhibit superior binding affinity compared to conventional anti-PSMA antibodies, and can be usefully employed in diagnosing, preventing, or treating target tumors or cancers.

Description

Antibodies to PSMA and their uses

[0001] The present invention relates to an anti-PSMA antibody or an antigen-binding fragment thereof, a nucleic acid encoding the same, a recombinant expression vector comprising the nucleic acid, a host cell transfected with the recombinant expression vector, a method for producing the antibody or an antigen-binding fragment thereof, a chimeric receptor (CAR) comprising the antibody or an antigen-binding fragment thereof, an immune cell therapeutic agent comprising the chimeric receptor, an antibody-drug conjugate (ADC) in which the antibody or an antigen-binding fragment thereof is linked to a drug, a composition for diagnosing, preventing, or treating cancer comprising the antibody or an antigen-binding fragment thereof, and a method and use for diagnosing, preventing, or treating cancer.

[0002]

[0003] Prostate cancer is the most common cancer in men, excluding skin cancer, and is the leading cause of cancer-related death in men (World Cancer Report, World Health Organization, 2014). It most commonly occurs in men over the age of 50 and is accompanied by symptoms such as pain, difficulty urinating, and blood in the urine. Worldwide, more than one million new cases are diagnosed annually, and hundreds of thousands die from prostate cancer (CA: A Cancer Journal for Clinicians, 68 (6): 394-424). Approximately 89% of prostate cancers are diagnosed in the local or regional stage, in the prostate or nearby organs, and in these cases, the five-year survival rate is nearly 100%, at 99%. However, some prostate cancer patients are diagnosed after the cancer has metastasized to other organs, at which point the five-year survival rate drops sharply to approximately 30% (Cancer Facts & Figures 2021).

[0004] Glutamate carboxypeptidase II (GCPII), also known as prostate-specific membrane antigen (PSMA), is a membrane glycoprotein encoded by the human FOLH1 (folate hydrolase 1) gene. The function of PSMA is still being studied, and it has recently been reported that it has an internalization signaling system that can internalize cell surface proteins into endosomes (Mol Biol Cell. 2003;14:4835-4845.). PSMA is mainly expressed in the prostate, and its expression in prostate cancer is reported to be 8-12 times higher than that in non-cancerous cells (The Prostate. 58 (2): 200-10.). PSMA overexpression in prostate cancer has been reported as a key biomarker for the diagnosis, prognosis, and prediction of recurrence potential, as well as for targeted therapy of prostate cancer (Journal of Cellular Biochemistry. 102 (3): 571-9). Recently, PSMA has been expressed not only in prostate cancer but also in other malignant tumors, especially in cancer-related neovascularization, and has been reported to be useful for the diagnosis and targeted therapy of various cancers (Clin Cancer Res. 1997;3:81-85; Cancer Res. 1997;57:3629-3634; Clin Cancer Res. 1999;5:2674-2681).

[0005] In the clinical setting of prostate cancer, efforts are ongoing to develop antibodies that specifically bind to PSMA due to its importance. 7E11 is the first anti-PSMA antibody that specifically binds to an intracellular or cytoplasmic epitope of PSMA, developed from the LNCaP prostate cancer cell line (Anticancer Res. 1987;7:927-936). ProstaScint (Cytogen Corporation, Princeton, NJ), a radioimmunoconjugate of the anti-PSMA antibody 7E11, has been approved by the FDA for use as a diagnostic tool in prostate cancer.

[0006] Furthermore, PSMA is also very useful as a therapeutic antigen target for prostate cancer, and recently, it has been reported that compounds combining anti-PSMA antibodies and radioisotopes can treat various cancers such as metastatic prostate cancer and renal cell carcinoma (J Nucl Med. 2003; 44: 610-617; J Urol. 2003; 170(6 part 2): S84-S88. discussion S88-S89). In addition, it has been reported that CAR-T cells containing PSMA-specific single-chain antibodies can successfully lyse PSMA-positive prostate cancer cells (Prostate. 1998; 35: 144-151), and it has been reported that recombinant anti-PSMA antibodies (Abgenix, Fremont, CA) can directly participate in tumor death by inducing antibody-dependent cytotoxicity of prostate tumor cells (J Clin Oncol. 2004; 22(14S): 2546).

[0007] Although various treatments for solid tumors, including immunotherapy targeting PSMA for prostate cancer, are being developed, there are several limitations in the treatment of solid tumors. First, the tumor microenvironment (TME) is formed around tumor cells, consisting of various cells other than immune cells and stromal cells, blood vessels, and extracellular factors (cytokines, chemokines, growth factors, etc.). The tumor microenvironment is mainly characterized by oxidative stress, nutrient depletion, low pH, and the presence of immunosuppressive cells such as tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs), as well as an extracellular matrix that blocks immune cell infiltration. As a result, various anticancer agents, including CAR-T cell therapy, do not work properly (Front. Immunol. 2018;9:1104).

[0008] Furthermore, unlike hematological malignancies, it is difficult to identify specific tumor-specific antigens that are highly and uniformly expressed in solid tumors. For example, PSMA is expressed not only in cancer tissues but also in normal tissues such as the prostate and kidney, which can lead to adverse effects of immunotherapy, destroying normal tissues (Stem Cell Res Ther. 2021;12:81).

[0009] The PSMA-specific antibodies reported to date are not completely specific to prostate cancer and can also bind to PSMA expressed in normal prostate tissues (Clin Cancer Res. 1999;5:4034-4040). In particular, in order to eliminate the side effects of antibody therapeutics on normal tissues expressing PSMA for the treatment of prostate cancer, it is essential to develop antibodies that maintain binding affinity to PSMA protein but are active in the tumor microenvironment of prostate cancer tissues and various antibodies that specifically recognize and bind to various epitopes of PSMA in order to exhibit a potent and precisely targeted effect (Urology. 1998;51:657-662).

[0010] Most normal human tissues, including the prostate, maintain a pH of 7.4. However, in the microenvironment surrounding tumors, various factors (such as abnormal glycolysis, high lactate production, and proton accumulation) lead to a relatively low pH (6.5–6.8). This interferes with the proper functioning of various immune / cellular therapies (Cancer Res. 2012;72(16):3938-3947).

[0011] In the present invention, the STACT (Solid Tumor Adapted CAR-T therapy) technology is intended to develop an antibody and immune cell therapeutic agent that can exhibit activity in an acidified tumor microenvironment while eliminating side effects that occur when binding to PSMA in normal tissues. In this regard, an antibody that binds to PSMA in the low pH environment of the tumor microenvironment while not binding to the same PSMA protein in a normal pH environment was developed.

[0012] Under these backgrounds, the present inventors have made efforts to develop a novel anti-PSMA antibody with improved binding affinity for PSMA and specificity for prostate cancer, and as a result, have identified a novel anti-PSMA antibody exhibiting high PSMA affinity, and in particular, have selected an antibody with differentiated characteristics that have a more sensitive reactivity to prostate cancer cells than known anti-PSMA antibodies, and have confirmed that the selected antibody can be used for the treatment of the desired cancer, thereby completing the present invention.

[0013]

[0014] Summary of the invention

[0015] An object of the present invention is to provide a novel PSMA antibody or antigen-binding fragment thereof that specifically binds to prostate-specific membrane antigen (PSMA).

[0016] Another object of the present invention is to provide a nucleic acid encoding the antibody or an antigen-binding fragment thereof.

[0017] Another object of the present invention is to provide a recombinant expression vector comprising the nucleic acid.

[0018] Another object of the present invention is to provide a recombinant host cell into which the nucleic acid or the recombinant expression vector has been introduced.

[0019] Another object of the present invention is to provide a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to the PSMA.

[0020] Another object of the present invention is to provide a chimeric antigen receptor (CAR) comprising an antibody or an antigen-binding fragment thereof that specifically binds to the PSMA.

[0021] Another object of the present invention is to develop a pH-dependent PSMA binding antibody and provide an antibody that specifically binds to PSMA of tumor cells.

[0022] Another object of the present invention is to provide an immune cell therapeutic agent comprising the chimeric antigen receptor.

[0023] Another object of the present invention is to provide an antibody-drug conjugate (ADC) in which the antibody or an antigen-binding fragment thereof is bound to a drug.

[0024] Another object of the present invention is to provide a composition for diagnosing, preventing or treating cancer, or a method and use for diagnosing, preventing or treating cancer, comprising an antibody or an antigen-binding fragment thereof, or an antibody-drug conjugate comprising the antibody or an antigen-binding fragment thereof.

[0025] To achieve the above object, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to prostate-specific membrane antigen (PSMA), comprising:

[0026] A light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 26;

[0027] A light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 to 30;

[0028] A light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31 to 34 and 45;

[0029] A heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 37, and 46;

[0030] A heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 38 to 41, 47, and 48; and

[0031] A heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 42 to 44.

[0032] The present invention provides a nucleic acid encoding the antibody or an antigen-binding fragment thereof.

[0033] The present invention also provides a recombinant expression vector comprising the nucleic acid.

[0034] The present invention also provides a recombinant host cell into which the nucleic acid or the recombinant expression vector has been introduced.

[0035] The present invention also provides a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to PSMA, comprising the steps of culturing the recombinant host cell to produce an antibody; and isolating and purifying the produced antibody.

[0036] The present invention also provides a chimeric antigen receptor (CAR) comprising an antibody or an antigen-binding fragment thereof that specifically binds to the PSMA.

[0037] The present invention also provides an immune cell expressing the chimeric antigen receptor on its surface.

[0038] The present invention also provides a composition for preventing or treating cancer, including the chimeric antigen receptor or an immune cell expressing the same on its surface, and a use therefor.

[0039] The present invention also provides an antibody-drug conjugate (ADC) in which the antibody or an antigen-binding fragment thereof is bound to a drug.

[0040] The present invention also provides a composition for diagnosing, preventing or treating cancer, comprising the antibody or an antigen-binding fragment thereof, or an antibody-drug conjugate comprising the antibody or an antigen-binding fragment thereof.

[0041] The present invention also provides a method for treating cancer, comprising administering to a subject the antibody or an antigen-binding fragment thereof, or an antibody-drug conjugate comprising the antibody or an antigen-binding fragment thereof.

[0042] The present invention also provides the use of the antibody or an antigen-binding fragment thereof, or an antibody-drug conjugate comprising the antibody or an antigen-binding fragment thereof, for the manufacture of a composition for diagnosing, preventing or treating cancer.

[0043] The present invention also provides a use of the antibody or an antigen-binding fragment thereof, or an antibody-drug conjugate comprising the antibody or an antigen-binding fragment thereof, for the diagnosis, prevention, or treatment of cancer.

[0044] The present invention also provides a bispecific or multispecific antibody comprising the antibody or an antigen-binding fragment thereof.

[0045]

[0046] Figure 1 shows the results of an enzyme-linked immunosorbent assay (ELISA) to select hybridoma clones that show reactivity to PSMA.

[0047] Figure 2 shows the results of flow cytometry analysis to confirm the binding ability to PSMA using a cancer cell line expressing PSMA.

[0048] Figure 3 shows the results of purifying the clones identified through Figures 1 and 2.

[0049] Figure 4 shows the results of confirming the binding ability of the purified antibody to PSMA through flow cytometry using a cancer cell line expressing PSMA.

[0050] Figure 5 shows the results of flow cytometry analysis to confirm the antibody binding ability of human antibodies 41B11, 42E4, 45C3, and 46D3 to prostate cancer cell lines.

[0051] Figure 6 shows the results of confirming the antibody binding ability of human antibody 44H8 to prostate cancer cell lines through flow cytometry.

[0052] Figure 7 shows the results of flow cytometry analysis to confirm the introduction efficiency of scFv-CAR after production of five types of PSMA CAR-T cells.

[0053] Figure 8 shows the results of confirming the anticancer effect of CAR-T cells by co-culturing five types of PSMA CAR-T cells with prostate cancer cell lines LNCaP cells and PC3 cells.

[0054] Figure 9 shows the results of confirming the secretion of interferon gamma (IFN-γ) and granzyme B in CAR-T cells when PSMA CAR-T cells PB41B11, PB42E4, and PB46D3 cells were co-cultured with prostate cancer cells, respectively.

[0055] Figure 10 shows the results of confirming the anticancer effect of PSMA CAR-T cells in vivo by subcutaneously injecting prostate cancer cells (LNCaP) into mice and intravenously injecting PSMA CAR-T cells, PB-CTA-42E4, on the 13th day after cancer cell administration.

[0056] Figure 11 shows the results of ELISA to confirm the binding affinity of the parent antibody, 42E4 antibody, to the PSMA antigen at pH 7.4 and pH 6.0.

[0057] Figure 12 shows the results of ELISA to confirm the binding affinity of PSMA36, a STACT antibody, to the PSMA antigen at pH 7.4 and pH 6.0.

[0058] Figure 13 shows the results of ELISA to confirm the binding ability of PSMA37, a STACT antibody, to the PSMA antigen at pH 7.4 and pH 6.0.

[0059] Figure 14 shows the results of ELISA to confirm the binding ability of PSMA38, a STACT antibody, to the PSMA antigen at pH 7.4 and pH 6.0.

[0060] Figure 15 shows the results of flow cytometry analysis of the PSMA antigen binding capacity of STACT CAR-T cells at pH 6.0 and pH 7.4.

[0061] Figure 16 shows the results of confirming the anticancer effect of STACT CAR-T cells in vivo by subcutaneously injecting prostate cancer cells (LNCaP) into mice and intravenously injecting STACT CAR-T cells, PB-CTA-PSMA36, on the 13th day after cancer cell administration.

[0062]

[0063] Detailed description of the invention and preferred embodiments

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, unless otherwise defined herein. Generally, the nomenclature used herein is well known and commonly used in the art.

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0066] Antibodies or antigen-binding fragments thereof

[0067] In one aspect, the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to prostate-specific membrane antigen (PSMA), comprising:

[0068] A light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 26;

[0069] A light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 to 30;

[0070] A light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31 to 34 and 45;

[0071] A heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 37, and 46;

[0072] A heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 38 to 41, 47, and 48; and

[0073] A heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 42 to 44.

[0074] One aspect of the present invention is an antibody or antigen-binding fragment thereof that specifically binds to prostate-specific membrane antigen (PSMA), comprising:

[0075] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23;

[0076] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27;

[0077] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 31;

[0078] A heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 35;

[0079] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 38; and

[0080] A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 42.

[0081] Another aspect of the present invention is an antibody or antigen-binding fragment thereof that specifically binds to prostate-specific membrane antigen (PSMA), comprising:

[0082] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23;

[0083] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27;

[0084] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 31;

[0085] A heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 35;

[0086] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 38; and

[0087] A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 42.

[0088] Another aspect of the present invention is an antibody or antigen-binding fragment thereof that specifically binds to prostate-specific membrane antigen (PSMA), comprising:

[0089] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23;

[0090] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27;

[0091] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 31;

[0092] A heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 35;

[0093] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 39; and

[0094] A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 42.

[0095] Another aspect of the present invention is an antibody or antigen-binding fragment thereof that specifically binds to prostate-specific membrane antigen (PSMA), comprising:

[0096] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 24;

[0097] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 28;

[0098] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 32;

[0099] A heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 36;

[0100] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 40; and

[0101] A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 43.

[0102] Another aspect of the present invention is an antibody or antigen-binding fragment thereof that specifically binds to prostate-specific membrane antigen (PSMA), comprising:

[0103] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 25;

[0104] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 29;

[0105] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 33;

[0106] A heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 36;

[0107] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 40; and

[0108] A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 43.

[0109] Another aspect of the present invention is an antibody or antigen-binding fragment thereof that specifically binds to prostate-specific membrane antigen (PSMA), comprising:

[0110] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26;

[0111] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 30;

[0112] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 34;

[0113] A heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 37;

[0114] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 41; and

[0115] A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 44.

[0116] Another aspect of the present invention is an antibody or antigen-binding fragment thereof that specifically binds to prostate-specific membrane antigen (PSMA), comprising:

[0117] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26;

[0118] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 30;

[0119] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 45;

[0120] A heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 46;

[0121] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 41; and

[0122]

[0123] A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 44.

[0124] Another aspect of the present invention is an antibody or antigen-binding fragment thereof that specifically binds to prostate-specific membrane antigen (PSMA), comprising:

[0125] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26;

[0126] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 30;

[0127] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 45;

[0128] A heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 37;

[0129] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 47; and

[0130] A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 44.

[0131] Another aspect of the present invention is an antibody or antigen-binding fragment thereof that specifically binds to prostate-specific membrane antigen (PSMA), comprising:

[0132] A light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26;

[0133] A light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 30;

[0134] A light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 45;

[0135] A heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 37;

[0136] A heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 48; and

[0137] A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 44.

[0138] As used herein, the term "antibody" refers to a collection of antibody protein molecules comprising one or more complementary determination regions (CDRs), a complete antibody, or a derivative thereof. In the present invention, an antibody that specifically binds to the prostate-specific membrane antigen (PSMA) may be used interchangeably with the terms "anti-PSMA antibody" or "PSMA antibody." The scope of the present invention includes not only a complete antibody form that specifically binds to PSMA, but also an antigen-binding fragment of the antibody molecule.

[0139] A complete antibody typically has two full-length light chains and two full-length heavy chains, with each light chain linked to a heavy chain by a disulfide bond. The heavy chain constant regions are of the gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, with subclasses of gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), and alpha2 (α2). The light chain constant regions are of the kappa (κ) and lambda (λ) types.

[0140] As used herein, the term "heavy chain" refers to both a full-length heavy chain and fragments thereof, which comprises a variable domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and three constant domains CH1, CH2, and CH3. In addition, the term "light chain" as used herein refers to both a full-length light chain and fragments thereof, which comprises a variable domain VL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and a constant domain CL.

[0141] An antigen-binding fragment of an antibody or an antibody fragment refers to a fragment that possesses an antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv, etc. In the present invention, the antigen-binding fragment of an antibody refers to a fragment that possesses the function of recognizing and specifically binding to a prostate-specific membrane antigen.

[0142] Among antibody fragments, Fab has a structure that has a single antigen-binding site with the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (CH1) of the heavy chain. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the CH1 domain of the heavy chain. F(ab')2 is generated when the cysteine ​​residues in the hinge region of Fab' form a disulfide bond.

[0143] Fv is the smallest antibody fragment that contains only the heavy chain variable region and the light chain variable region. A two-chain Fv has a heavy chain variable region and a light chain variable region non-covalently linked, while a single-chain Fv (single-chain Fv, scFv) has a heavy chain variable region and a light chain variable region covalently linked, usually via a peptide linker, or directly linked at the C-terminus, so that it can form a dimer-like structure like a two-chain Fv. These antibody fragments can be produced using proteolytic enzymes (for example, restriction digestion of a complete antibody with papain yields Fab, and digestion with pepsin yields F(ab')2) or using genetic recombination technology.

[0144] An "Fv" fragment is an antibody fragment containing the complete antibody recognition and binding site. This region is a dimer composed of one heavy chain variable domain and one light chain variable domain.

[0145] The "Fab" fragment comprises the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. An F(ab')2 antibody fragment typically comprises a pair of Fab' fragments covalently linked by cysteines in the hinge region at the C-terminus of the Fab' fragment.

[0146] A "single-chain Fv (scFv)" antibody fragment is a construct composed of a single polypeptide chain comprising the VH and VL domains of an antibody. The scFv may additionally comprise a polypeptide linker between the VH and VL domains that enables it to form the desired structure for antigen binding.

[0147] In one embodiment, the antibodies of the present invention include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, scFvs, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs) and anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of the foregoing antibodies.

[0148] The heavy chain constant region can be selected from any one of the gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) isotypes. For example, the constant region is gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), or gamma 4 (IgG4). The light chain constant region can be kappa or lambda type.

[0149] The term "monoclonal antibody" refers to antibodies obtained from a substantially homogeneous population of antibodies, i.e., antibodies that are identical except for possible naturally occurring mutations that may be present in trace amounts in individual antibodies within the population. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to conventional (polyclonal) antibodies, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0150] A “polyclonal antibody” refers to a composition of different antibody molecules that bind to or react with one or more immunogenic determinants or epitopes of the same or different antigens. Each individual antibody in the composition may be characterized differently and each can bind to or react with a specific epitope. For example, the diversity of a polyclonal antibody may generally be determined by the variable region of the antibody, such as the complementarity determining regions (CDR1, CDR2, and CDR3) of the light or heavy chain. The polyclonal antibody may also be due to differences between individual antibody molecules in the constant region. For example, a polyclonal antibody may be a mixture of antibodies comprising two or more different antibody isotypes, such as human isotypes IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE, or murine isotypes IgG1, IgG2a, IgG2b, IgG3, and IgA. In the present invention, the polyclonal antibody may be isolated or identified and purified from mammalian blood, secretions, other body fluids, germ cells, etc., may include a mixture of different monoclonal antibodies, and may be produced as a recombinant polyclonal antibody.

[0151] The above “recombinant polyclonal antibody” refers to a polyclonal antibody produced by recombinant technology, wherein each antibody molecule in the polyclonal antibody exhibits the desired binding activity to a target antigen composed of one or more epitopes.

[0152] An "epitope" is a protein determinant to which an antibody can specifically bind. Epitopes typically consist of chemically active surface molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural features as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished by the fact that binding to the former is lost in the presence of denaturing solvents, while binding to the latter is not.

[0153] The above "humanized" forms of non-human (e.g., mouse) antibodies are chimeric antibodies that contain minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region of the recipient antibody have been replaced with residues from the hypervariable region of a non-human species (donor antibody) that retains the desired specificity, affinity, and ability, such as mouse, rat, rabbit, or non-human primate.

[0154] The above "human antibody" means a molecule derived from human immunoglobulin, and the entire amino acid sequence constituting the antibody, including the complementarity determining region and structural region, is composed of human immunoglobulin.

[0155] "Chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies that exhibit the desired biological activity.

[0156] As used herein, “antibody variable region” or “antibody variable domain” refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and the framework regions (FR). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

[0157] "Complementarity determining regions" (CDRs; i.e., CDR1, CDR2, and CDR3) refer to the amino acid residues of an antibody variable domain that are necessary for antigen binding. Each variable domain typically has three CDR regions, identified as CDR1, CDR2, and CDR3.

[0158] "Framework regions (FR)" are variable domain residues other than CDR residues. Each variable domain typically has four FRs: FR1, FR2, FR3, and FR4.

[0159] In another aspect, the present invention relates to a bispecific or multispecific antibody comprising the antibody or an antigen-binding fragment thereof.

[0160] In the present invention, the antibody or antigen-binding fragment thereof may comprise at least two, preferably two, heavy chains and light chains each comprising CDR1 to CDR3. At this time, the complementarity determining regions included in each heavy chain and light chain may be independent and different. Preferably, the antibody or antigen-binding fragment thereof may be in the form of a homodimer comprising two identical heavy chains and two light chains, but may be in the form of a heterodimer comprising two or more different heavy chains and light chains.

[0161] The term "multispecific antibody" above refers to an antibody capable of recognizing and specifically binding to two or more antigens. In some cases, the multispecific antibody may comprise two or more heavy and light chains, each of which includes complementarity-determining regions capable of specifically binding to different antigens.

[0162] Multispecific antibodies include tetraspecific, trispecific, or bispecific antibodies. For example, a bispecific antibody is an antibody that can bind to two different antigens (target proteins) and is produced through genetic engineering or any other method.

[0163] Multispecific antibodies are antibodies that have binding specificities for at least two different antigens. Antibodies belonging to multispecific antibodies can be classified into scFv-based antibodies, Fab-based antibodies, and IgG-based antibodies. In the case of bispecific antibodies, because they can simultaneously suppress or amplify two signals, they can be more effective than cases where one signal is suppressed / amplified. Compared to cases where each signal is treated with a separate signal inhibitor, lower dosages are possible, and two signals can be suppressed / amplified in the same time and space.

[0164] Methods for producing bispecific antibodies are widely known. Traditionally, recombinant production of bispecific antibodies relies on the co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy chains possess different specificities.

[0165] In the case of bispecific antibodies based on scFv, the VL and VH of different scFvs can be combined with each other to produce a hybrid scFv in a heterodimeric form to produce a diabody, different scFvs can be linked to each other to produce a tendem ScFv, CH1 and CL of Fab can be expressed at the ends of each scFv to produce a heterodimeric miniantibody, and some amino acids of the CH3 domain, which is a homodimeric domain of Fc, can be substituted to change it into a heterodimeric structure in the form of a 'knob into hole', and these changed CH3 domains can be expressed at the ends of different scFvs to produce a minibody in the form of a heterodimeric scFv.

[0166] In the case of a bispecific antibody based on Fab, individual Fabs for specific antigens can be combined with each other using a disulfide bond or a mediator to produce a heterodimeric Fab form, and by expressing scFvs for different antigens at the terminals of the heavy or light chains of a specific Fab, it can be produced to have two antigen-binding valencies, or by providing a hinge region between the Fab and scFv, it can be produced to have four antigen-binding valencies in a homodimeric form. In addition, a dual-target bibody in which the antigen-binding valencies are made three by fusing scFvs for different antigens to the light and heavy chain terminals of the Fab, and a triple-target bibody in which the antigen-binding valencies are made three by fusing different scFvs to the light and heavy chain terminals of the Fab, respectively, can be obtained by chemically conjugating three different Fabs.

[0167] For IgG-based bispecific antibodies, a method has been known for producing bispecific antibodies by crossbreeding mouse and rat hybridomas to produce hybrid hybridomas, also known as quadromas, by Trion Pharma. In addition, bispecific antibodies can be produced in the so-called 'Holes and Knob' form, which is produced by modifying some amino acids in the CH3 homodimeric domain of the Fc for different heavy chains while sharing the light chain portion, thereby producing a heterodimeric form. In addition to heterodimeric bispecific antibodies, two different scFvs can be fused and expressed in the constant domains of the light and heavy chains of IgG instead of the variable domains, thereby producing (scFv)4-IgG in a homodimeric form.

[0168] A wide variety of recombinant antibody formats have been developed, including bivalent, trivalent, or tetravalent bispecific or multispecific antibodies. Examples include the bivalent, trivalent, or tetravalent antibodies described in International Patent Application Publications WO2001 / 077342, WO2009 / 080251, WO2009 / 080252, WO2009 / 080253, WO2009 / 080254, WO2010 / 112193, WO2010 / 115589, WO2010 / 136172, WO2010 / 145792, WO2010 / 145793, and WO2011 / 117330. An antibody being bivalent, trivalent, or tetravalent indicates that two or more binding domains, three or more binding domains, or four or more binding domains are present in the antibody molecule, respectively.

[0169] The anti-PSMA antibody or antigen-binding fragment thereof according to the present invention is an antibody selected and identified having high binding affinity to a prostate-specific membrane antigen, and can be characterized by having high specificity for prostate cancer cells.

[0170] In the present invention, the antibody or antigen-binding fragment thereof may be characterized by including at least one of the following light chain variable regions including light chain CDR1 to light chain CDR3:

[0171] A light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 26;

[0172] A light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 to 30;

[0173] A light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31 to 34 and 45.

[0174] In the present invention, preferably, the antibody or antigen-binding fragment thereof may be characterized by including at least one of the following heavy chain variable regions including heavy chain CDR1 to heavy chain CDR3:

[0175] A heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 37, and 46;

[0176] A heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 38 to 41, 47, and 48; and

[0177] A heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 42 to 44.

[0178] An antibody or antigen-binding fragment thereof according to the present invention may be characterized by comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 14, 16, 18, 20, 21 and 53.

[0179] In the present invention, the antibody or antigen-binding fragment thereof may be characterized by including a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, 17, 19, 22, and 54 to 56.

[0180] In the present invention, the antibody may be characterized by comprising at least one combination of a light chain variable region and a heavy chain variable region comprising:

[0181] i) a light chain variable region represented by SEQ ID NO: 12 and a heavy chain variable region represented by SEQ ID NO: 13;

[0182] ii) a light chain variable region represented by SEQ ID NO: 14 and a heavy chain variable region represented by SEQ ID NO: 15;

[0183] iii) a light chain variable region represented by SEQ ID NO: 16 and a heavy chain variable region represented by SEQ ID NO: 17;

[0184] iv) a light chain variable region represented by SEQ ID NO: 18 and a heavy chain variable region represented by SEQ ID NO: 19;

[0185] v) a light chain variable region represented by SEQ ID NO: 20 and a heavy chain variable region represented by SEQ ID NO: 22;

[0186] vi) a light chain variable region represented by SEQ ID NO: 21 and a heavy chain variable region represented by SEQ ID NO: 22;

[0187] vii) a light chain variable region represented by SEQ ID NO: 53 and a heavy chain variable region represented by SEQ ID NO: 54;

[0188] viii) a light chain variable region represented by SEQ ID NO: 53 and a heavy chain variable region represented by SEQ ID NO: 55; and

[0189] ix) A light chain variable region represented by SEQ ID NO: 53 and a heavy chain variable region represented by SEQ ID NO: 56.

[0190] An scFv is an antibody fragment, consisting of a single polypeptide chain containing the VH and VL domains of an antibody. The scFv may additionally comprise a polypeptide linker between the VH and VL domains, enabling it to form the desired structure for antigen binding.

[0191] In one embodiment, in a single-chain Fv (scFv) comprising the VH and VL domains of an antibody, the VH and VL domains may be linked via a linker. A light chain variable region comprising the light chain CDRs of SEQ ID NOs: 23 to 34 may be linked to a heavy chain variable region comprising the heavy chain CDRs of SEQ ID NOs: 35 to 44.

[0192] In the present invention, the antibody or antigen-binding fragment thereof may be characterized by including the light chain and heavy chain variable regions of each monoclonal antibody disclosed in Table 2 of the examples.

[0193] The linker may be a peptide linker and may have a length of about 10-25 aa. For example, it may include, but is not limited to, hydrophilic amino acids such as glycine and / or serine.

[0194] Specifically, the linker may be, for example, (GS) n , (GGS) n , (GSGGS) n or (G n S) m (n, m are 1 to 10 respectively) may be included, but the linker may be, for example, (G n S) m (n and m can be 1 to 10, respectively).

[0195] The antibodies of the present invention or antigen-binding fragments thereof may include not only the sequence of the anti-PSMA antibodies of the present invention described herein, but also biological equivalents thereof, as long as they can specifically recognize PSMA. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody. Such amino acid mutations are constructed based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; Alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

[0196] Considering the mutations having the above-described biological equivalent activity, the sequences represented by each sequence number described in the antibody or antigen-binding fragment thereof of the present invention are interpreted to also include sequences showing substantial identity therewith. The substantial identity means a sequence showing at least 80% homology, preferably at least 90% homology, most preferably at least 95% homology, 96% or more, 97% or more, 98% or more, or 99% or more homology when the sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequences are analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NCBI, etc., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet. BLAST is available at www.ncbi.nlm.nih.gov / BLAST / . Instructions for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov / BLAST / blast_ help.html.

[0197] Based on this, the antibody or antigen-binding fragment thereof of the present invention may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology with all or part of the sequences described in the specification. Such homology can be determined by sequence comparison and / or alignment using methods known in the art. For example, the percent sequence homology of the nucleic acid or protein of the present invention can be determined using a sequence comparison algorithm (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection.

[0198] From another perspective, the present invention relates to a nucleic acid encoding the antibody or antigen-binding fragment thereof. The nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention can be isolated to recombinantly produce the antibody or antigen-binding fragment thereof.

[0199] "Nucleic acid" is a comprehensive term encompassing DNA (gDNA and cDNA) and RNA molecules. Nucleotides, the basic structural units of nucleic acids, include not only natural nucleotides but also analogues with modified sugar or base moieties. The sequence of the nucleic acid encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include additions, deletions, non-conservative substitutions, or conservative substitutions of nucleotides.

[0200] Nucleic acids encoding the above antibodies or antigen-binding fragments thereof can be readily isolated or synthesized using conventional molecular biological techniques (e.g., by using oligonucleotide probes capable of specifically binding to nucleic acids (e.g., DNA) encoding complete antibodies, heavy chains and / or light chains), and the nucleic acids can be isolated and further cloned (amplified) or further expressed by inserting them into replicable vectors.

[0201] In the present invention, the nucleic acid may be characterized by including at least one sequence selected from the group consisting of SEQ ID NOs: 1 to 11 and SEQ ID NOs: 49 to 52.

[0202] Based on this, the present invention relates to a recombinant expression vector comprising the nucleic acid from another perspective.

[0203] The term "vector" as used herein refers to a means for expressing a target gene in a host cell, and includes viral vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors.

[0204] Components of a vector typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more antibiotic resistance marker genes, an enhancer element, a promoter, and a transcription termination sequence. A nucleic acid encoding an antibody may be operably linked to the promoter and transcription termination sequence, among other elements.

[0205] "Operably linked" means a functional association between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, whereby the regulatory sequence regulates transcription and / or translation of the other nucleic acid sequence.

[0206] In the case of a prokaryotic cell as a host, it is common to include a strong promoter that can drive transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter), a ribosome binding site for translation initiation, and a transcription / translation termination sequence. In addition, for example, when a eukaryotic cell is used as a host, promoters derived from the genome of mammalian cells (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, promoter of Epstein-Barr virus (EBV), and promoter of Rous sarcoma virus (RSV)) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

[0207] In some cases, the vector may be fused with other sequences to facilitate purification of the antibody expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA).

[0208] The above vector contains antibiotic resistance genes commonly used in the art as selectable markers, for example, resistance genes for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

[0209] In another aspect, the present invention relates to a host cell into which the nucleic acid or the recombinant expression vector has been introduced. The cell used to produce the antibody of the present invention may be a prokaryotic, yeast, or higher eukaryotic cell, but is not limited thereto.

[0210] The above vector can be introduced into a host cell by a method such as transformation or transfection. The term "transformation" as used herein refers to the introduction of DNA into a host cell so that the DNA becomes replicable as an extrachromosomal element or by chromosomal integration. The term "transfection" as used herein refers to the acceptance of an expression vector by a host cell, regardless of whether any coding sequence is actually expressed. To introduce the above vector, various techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells can be used, such as, but not limited to, electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection.

[0211] It should be understood that not all vectors and expression control sequences are equally effective in expressing the DNA sequences of the present invention. Similarly, not all hosts function equally well in the same expression system. However, those skilled in the art can appropriately select from among various vectors, expression control sequences, and hosts without undue experimental burden and without departing from the scope of the present invention. For example, when selecting a vector, consideration should be given to the host, as the vector must replicate within it. The vector's copy number, its ability to control copy number, and the expression of other proteins encoded by the vector, such as antibiotic markers, should also be considered. When selecting an expression control sequence, various factors should be considered, such as the relative strength of the sequence, its modulation potential, and its compatibility with the DNA sequences of the present invention, particularly with respect to potential secondary structures. The unicellular host should be selected by considering factors such as the selected vector, the toxicity of the product encoded by the DNA sequence of the present invention, secretion characteristics, the ability to accurately fold the protein, culture and fermentation requirements, and the ease of purifying the product encoded by the DNA sequence of the present invention from the host. Within the scope of these variables, those skilled in the art can select various vector / expression control sequence / host combinations that can express the DNA sequence of the present invention in fermentation or large-scale animal culture. When attempting to clone cDNA by expression cloning, screening methods such as the binding method, the panning method, and the film emulsion method can be applied.

[0212] Prokaryotic host cells can be used, such as strains of the genus Bacillus, such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, and Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis and Staphylococcus (e.g., Staphylococcus carnosus).

[0213] However, animal cells are of greatest interest, and examples of useful host cell lines include, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0214] In the present invention, the nucleic acid encoding the anti-PSMA antibody or antigen-binding fragment thereof can be directly introduced into the genome of a host cell and exist as a chromosomal element. It will be apparent to those skilled in the art that inserting the gene into the host cell's genomic chromosome will have the same effect as introducing a recombinant vector into the host cell.

[0215] In another aspect, the present invention relates to a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to a prostate-specific membrane antigen, comprising the steps of (a) culturing the host cell; and (b) recovering the antibody or an antigen-binding fragment thereof from the cultured host cell.

[0216] The above cells can be cultured in various media. Any commercially available medium can be used as a culture medium. Any other essential supplements known to those skilled in the art may be included at appropriate concentrations. Culture conditions, such as temperature and pH, are already used with the host cells selected for expression and will be readily apparent to those skilled in the art.

[0217] The antibody or antigen-binding fragment thereof can be recovered by removing impurities, for example, by centrifugation or ultrafiltration, and purifying the resultant product, for example, by using affinity chromatography. Additional purification techniques, for example, anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, etc., can be used.

[0218]

[0219] Chimeric Antigen Receptor (CAR)

[0220] In another aspect, the present invention relates to a chimeric antigen receptor comprising the antibody or an antigen-binding fragment thereof.

[0221] In the present invention, it is preferable that the chimeric antigen receptor comprises a single chain variable fragment (scFv) composed of the antibody or an antigen-binding fragment thereof.

[0222] The term "chimeric antigen receptor (CAR)" of the present invention refers to a genetically engineered receptor protein capable of inducing a specific immune response against a target antigen and cells expressing the antigen. CAR comprises an extracellular domain, a transmembrane domain, and a signaling domain. By artificially introducing a CAR gene containing a receptor that specifically recognizes a cancer cell surface antigen expressed on the surface of cancer cells into an immune cell, cancer cells can be killed. Immune cells expressing CARs can be produced through genetic methods. These immune cells may include, but are not limited to, T cells, natural killer (NK) cells, and macrophages. Immune cells introduced with CARs can target cancer cells expressing antigens that can bind to the antigen-binding domain of the CAR and induce an immune response.

[0223] First-generation CARs comprised an extracellular domain containing an antigen recognition site specifically expressed in cancer cells, a transmembrane domain, and a signaling domain, and utilized only CD3ζ as the signaling domain. However, they had minimal therapeutic effects against cancer and had a short duration of action. This first-generation CAR is specifically described in U.S. Patent No. 6,319,494, which is incorporated herein by reference.

[0224] To enhance responsiveness to immune cells, second-generation CARs were manufactured by combining costimulatory domains (CD28 or CD137 / 4-1BB) and CD3ζ, and the number of immune cells expressing the CAR gene remaining in the body was significantly increased compared to the first-generation CAR. While the second-generation CAR used one costimulatory domain, the third-generation CAR used two or more costimulatory domains. 4-1BB, CD28, or OX40 can be used as costimulatory domains to achieve expansion and persistence of immune cells containing the CAR in vivo. Second-generation CARs are specifically described in U.S. Patent Nos. 7,741,465, 7,446,190, or 9,212,229, and third-generation CARs are specifically described in U.S. Patent No. 8,822,647, which are incorporated herein by reference.

[0225] The fourth-generation CAR includes additional genes encoding cytokines such as IL-12 or IL-15, allowing these cytokines to be additionally expressed in immune cells, thereby increasing the activity of T cells and simultaneously attracting and activating innate immune cells.

[0226] Fifth-generation CARs additionally include an interleukin receptor chain, such as IL-2Rβ, to enhance immune cell function. The fourth-generation CAR is specifically described in U.S. Patent No. 10,316,102, and the fifth-generation CAR is specifically described in U.S. Patent No. 10,336,810, both of which are incorporated herein by reference.

[0227] In the present invention, the chimeric antigen receptor may be characterized by including a transmembrane domain (TM).

[0228] Any transmembrane domain that can be included in the chimeric antigen receptor according to the present invention can be used as long as it can connect the extracellular domain and the signal transduction domain across the cell membrane. Preferably, the transmembrane domain is composed of a CD28-derived transmembrane domain and / or a CD8-derived transmembrane domain, and may include all or part of the CD28-derived transmembrane domain and / or the CD8-derived transmembrane domain.

[0229] In the present invention, the chimeric antigen receptor may be characterized by including a signaling domain.

[0230] In the present invention, the signal transduction domain is a portion located inside the cell membrane of an immune cell, i.e., in the cytoplasm, and refers to a portion that transmits a signal within the cell when the antigen binding domain included in the extracellular domain binds to a target antigen, thereby activating the immune response of the immune cell.

[0231] In the present invention, the signal transduction domain may be characterized by including a signal transduction domain and / or a co-stimulatory signal transduction region.

[0232] The signal transduction domain can induce activation of the function of the immune cell in which the CAR is located. For example, it can induce cytolytic T cell activation or helper T cell activation through the secretion of cytokines. The signal transduction domain can include a truncated fragment of the signal transduction domain sufficient to transduce an effector function signal.

[0233] The signal transduction domain may include cytoplasmic T cell receptors (TCRs) and co-receptors that act cooperatively to initiate signal transduction after antigen receptor engagement.

[0234] It is also known that signals generated through the TCR alone are insufficient for full T cell activation and that co-stimulatory signals are required. Therefore, T cell activation may involve both initiating antigen-dependent primary activation through the TCR and acting in an antigen-dependent manner to provide secondary or co-stimulatory signals. Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex in a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAMs containing primary cytoplasmic signaling sequences may include CD3 zeta (ζ), TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0235] The costimulatory signaling domain refers to a portion of a CAR that contains the signaling domain of a costimulatory molecule. For example, it may include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0236] The chimeric antigen receptor according to the present invention may be characterized by comprising two or more signal transduction domains. When comprising two or more signal transduction domains, the signal transduction domains may be connected to each other in series. Alternatively, they may be connected via an oligopeptide linker or polypeptide linker consisting of 2 to 10 amino acids. An example of such a linker sequence is a continuous sequence of glycine-serine.

[0237] In the present invention, the signal transduction domain may be characterized by including at least one selected from the group consisting of a costimulatory signal transduction region of CD28 or 4-1BB and a signal transduction domain of CD3 zeta, but is not limited thereto.

[0238] In the present invention, the chimeric antigen receptor may additionally include an immune function promoting factor of an immune cell, and the immune function promoting factor of the immune cell may be characterized as being an interleukin signal sequence. The interleukin signal sequence may be characterized as inducing the expression of IL (interleukin)-12, IL-8, or IL-2, but is not limited thereto. In addition, examples of the immune function promoting factor of T cells among the immune cells include IL-7 or CCL19, and with regard to the immune function promoting factor of T cells, reference may be made to WO 2016 / 056228 A.

[0239] In the present invention, the chimeric antigen receptor may additionally include an interleukin receptor chain comprising a JAK binding motif and a STAT 3 / 5 association motif, such as, but not limited to, IL-2Rβ. In this regard, reference may be made to WO 2016 / 127257 A.

[0240] In another aspect, the present invention relates to a nucleic acid encoding the chimeric antigen receptor.

[0241] In another aspect, the present invention relates to an expression vector comprising the chimeric antigen receptor encoding nucleic acid and a virus comprising the expression vector.

[0242] In the present invention, "virus" refers to a virus genetically modified to express the chimeric antigen receptor of the present invention, for use in the treatment of cancer, etc. Genetically modified means adding foreign genetic material in the form of DNA or RNA to the entire genetic material in a cell.

[0243] From another perspective, the present invention relates to an immune cell expressing the chimeric antigen receptor on its surface.

[0244] In the present invention, the immune cell can induce immunity to induce a desired cancer treatment effect, and may be selected from the group consisting of, for example, T cells, NK cells, NKT cells, cytokine-induced killer cells (CIK), macrophages, and dendritic cells, but is not limited thereto. Preferably, it may be characterized as being a T cell.

[0245] Accordingly, the immune cell expressing the chimeric antigen receptor according to the present invention may be a CAR-T cell (Chimeric Antigen Receptor T Cell), a CAR-NK cell (Chimeric Antigen Receptor Natural Killer Cell), or a CAR-NKT cell (Chimeric Antigen Receptor Natural killer T Cell).

[0246] In the present invention, the T cell may be characterized by being selected from the group consisting of cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes (TILs), and T cells isolated from peripheral blood mononuclear cells (PBMCs).

[0247] In the present invention, “cancer” and “tumor” are used interchangeably and refer to or mean a physiological condition of a mammal that is typically characterized by unregulated cell growth / proliferation.

[0248] The cancer or carcinoma that can be treated with the composition of the present invention is not particularly limited and includes both solid cancer and blood cancer. Preferably, it may be a solid cancer, and examples of such cancer may be selected from the group consisting of prostate cancer, skin cancer such as melanoma, liver cancer, hepatocellular carcinoma, hepatocellular cancer, gastric cancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, colon cancer, pancreatic cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, biliary tract cancer, testicular cancer, rectal cancer, head and neck cancer, cervical spine cancer, ureteral cancer, osteosarcoma, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma and glioma, but are not limited thereto.

[0249] More preferably, the cancer is characterized by expression or overexpression of PSMA protein, and may be, but is not limited to, prostate cancer, breast cancer, pancreatic cancer, lung cancer, thyroid cancer, stomach cancer, ovarian cancer, colon cancer, liver cancer, gallbladder cancer, kidney cancer, cervical cancer, bladder cancer, acute myeloid leukemia, or multiple myeloma. The cancer may be primary cancer or metastatic cancer. In another aspect, the present invention relates to a method for treating cancer, comprising a step of administering the chimeric antigen receptor or an immune cell expressing the same to a subject.

[0250] In another aspect, the present invention relates to the use of the above immune cells for cancer treatment.

[0251] In another aspect, the present invention relates to the use of the above immune cells for the manufacture of a drug for treating cancer.

[0252] The subject may be a mammal having a tumor, specifically, but not limited to, a human.

[0253] The immune cells expressing the chimeric antigen receptor according to the present invention or the composition containing the same may be administered by oral administration, infusion, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injectoon, intrarectal administration, topical administration, intranasal injection, etc., but are not limited thereto.

[0254] The dosage of the active ingredient can be appropriately selected depending on various factors such as the route of administration, the patient's age, sex, weight, and severity of the condition, and the therapeutic composition according to the present invention can be administered in combination with a known compound having an effect of preventing, improving, or treating cancer symptoms.

[0255]

[0256] Antibodies for STACT

[0257] In one aspect of the present invention, for the STACT (solid tumor adapted CAR-T therapy) form, an antibody was developed that has low binding to PSMA protein at normal pH and can maintain specific binding to PSMA at the low pH of the tumor microenvironment (TME).

[0258] To develop antibodies that bind in an acidic environment (low pH), specific amino acids in the heavy and light chain CDR sequences of the PSMA CAR-T cell clone PB-CTA-42E4 were substituted with histidine to produce STACT antibodies (PSMA36, PSMA37, and PSMA38). The binding affinity to the PSMA antigen was confirmed at pH 7.4 and pH 6.0. As a result, it was confirmed that the STACT antibodies PSMA36, PSMA37, and PSMA38 showed higher binding affinity to the PSMA antigen at pH 6.0 (Figs. 12-14). In addition, STACT CAR-T cells were produced for the above antibody, and the PSMA antigen binding ability of the STACT CAR-T cells was confirmed by flow cytometry at pH 6.0 and pH 7.4. As a result, it was confirmed that the STACT CAR-T cells did not specifically bind to the PSMA antigen at pH 7.4, but specifically bound to the PSMA antigen only at pH 6.0 (Fig. 15).

[0259] Therefore, the STACT antibody according to the present invention exhibits excellent binding affinity to PSMA in a tumor microenvironment, and thus, excellent cancer treatment effects can be expected.

[0260] Therefore, the STACT antibodies (PSMA36, PSMA37 and PSMA38) of the present invention are characterized by having binding specificity to the PSMA antigen at pH 4 to 7, preferably by having high antigen binding specificity at pH 5.5 to 6.8, and more preferably by having binding specificity at pH 5.8 to 6.5.

[0261]

[0262] Antibody-drug conjugate (ADC)

[0263] In another aspect, the present invention relates to an antibody-drug conjugate (ADC) in which the antibody or an antigen-binding fragment thereof is bound to a drug.

[0264] PSMA, to which the antibody or antigen-binding fragment thereof of the present invention specifically recognizes and binds, is reported to be highly upregulated in prostate cancer cells and some cancer cells, and therefore, the antibody-drug conjugate of the present invention can target cells expressing PSMA, particularly cancer cells in which the expression of PSMA is upregulated (overexpressed).

[0265] Antibody-drug conjugates require that the anticancer drug be stably bound to the antibody before it can be delivered to target cancer cells. Once delivered to the target, the drug can be released from the antibody and induce target cell death. To achieve this, the drug must be stably bound to the antibody and possess sufficient cytotoxicity to induce target cell death upon release.

[0266] In one embodiment, the antibody may be linked to a drug via a linker. The linker serves as a linking moiety between the anti-PSMA antibody and the drug. The linker must be cleavable under intracellular conditions, i.e., allow the drug to be released from the antibody in the intracellular environment. Given the antibody's long half-life, the antibody must be stable in systemic circulation, and the linker-drug binding must not affect the stability or pharmacokinetics of the antibody.

[0267] The linker may include, for example, a cleavable linker or a non-cleavable linker. In the case of a cleavable linker, such as a peptide linker, the linker may be cleaved by an intracellular peptidase or protease enzyme, such as a lysosomal or endosomal protease, and in the case of a non-cleavable linker, such as a thioether linker, the drug may be released after the antibody is non-selectively degraded by intracellular hydrolysis.

[0268] In one embodiment, the cleavable linker may comprise a peptide linker. The peptide linker has a length of at least two amino acids. For example, the peptide linker may comprise a Val-Cit, Val-Ala, or Val-Cit dipeptide, or Phe-Leu or Gly-Phe-Leu-Gly. Examples of linkers are specifically described in International Patent Application Publication No. WO2004 / 010957, which is incorporated herein by reference.

[0269] The above antibody-drug conjugate can be internalized into cancer cells via the endosome-lysosome pathway by binding the antibody region of the ADC to an antigen on the target cancer cell, forming an ADC-antigen complex. In this case, the intracellular release of the cytotoxic drug is controlled by the internal environment of the endosome / lysosome.

[0270] In one embodiment, the cleavable linker may be pH-sensitive, meaning that it is susceptible to hydrolysis at a specific pH value. Generally, a pH-sensitive linker indicates that it can be hydrolyzed under acidic conditions. For example, an acid-labile linker capable of being hydrolyzed in lysosomes may be a hydrazone, a semicarbazone, a thiosemicarbazone, a cis-aconitic amide, an orthoester, an acetal, a ketal, etc.

[0271] In another embodiment, the linker may be cleaved under reducing conditions, for example, a disulfide linker. Various disulfide bonds may be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene). These disulfide linkers can be cleaved by disulfide exchange with the thiol of intracellular glutathione.

[0272] The drug and / or drug-linker may be randomly conjugated via lysine of the antibody, or via cysteines exposed upon reduction of the disulfide bond chain. In some cases, the linker-drug may be conjugated via cysteine ​​present in a genetically engineered tag, for example, a peptide or protein. The genetically engineered tag, for example, a peptide or protein, may comprise an amino acid motif that can be recognized by an isoprenoid transferase. The peptide or protein may have a deletion at the carboxyl terminus, or may have an addition via covalent bonding of a spacer unit to the carboxyl (C) terminus of the peptide or protein.

[0273] The above peptide or protein may be directly covalently linked to the amino acid motif or may be covalently linked to a spacer unit to be linked to the amino acid motif. The amino acid spacer unit is composed of 1 to 20 amino acids, and among them, a glycine unit is preferred.

[0274] The above isoprenoid transferase may be, for example, farnesyl transferase (FTase, farnesyl protein transferase) or geranylgeranyl transferase (GGTase, geranylgeranyl transferase), and FTase and GGTase I can recognize the CAAX motif in the aforementioned chemical formula 1, and GGTase II can recognize the XXCC, XCXC or CXX motif (wherein C is cysteine, A is an aliphatic amino acid, and X is an amino acid that determines the substrate specificity of the isoprenoid transferase).

[0275] In another embodiment, the linker may comprise a beta-glucuronide linker that is recognized and hydrolyzed by beta-glucuronidase, which is abundant in lysosomes or overexpressed in some tumor cells. Unlike peptide linkers, this linker has a high hydrophilicity, which has the advantage of increasing the solubility of the antibody-drug complex when combined with a highly hydrophobic drug.

[0276] In this regard, a beta-glucuronide linker disclosed in International Patent Application Publication No. WO2015 / 182984, for example a beta-glucuronide linker including a self-immolative group, may be used, the disclosure of which is incorporated herein by reference.

[0277] In some cases, the linker may be, for example, a non-cleavable linker, allowing the drug to be released through a single step of antibody hydrolysis within the cell, producing, for example, an amino acid-linker-drug conjugate. This type of linker may be a thioether group or a maleimidocaproyl group, and may maintain stability in the bloodstream.

[0278] According to one embodiment of the present invention, the linker-drug can be linked by introducing a random linker-drug or an antibody-terminal binding peptide having the sequence GGGGGGGCVIM through a cysteine ​​exposed when the disulfide bond chain of the antibody is reduced.

[0279] The drug (including D of Formula (1)) may be conjugated to an antibody as a pharmacologically effective agent, and may be, in particular, a chemotherapeutic agent, a toxin, microRNA (miRNA), siRNA, shRNA, or a radioactive isotope. The chemotherapeutic agent may be, for example, a cytotoxic agent or an immunosuppressant. Specifically, it may include a microtubulin inhibitor, a mitotic inhibitor, a topoisomerase inhibitor, or a chemotherapeutic agent that can function as a DNA intercalator. It may also include an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an anthelmintic agent, or a combination thereof.

[0280] These drugs include, for example, maytansinoids, auristatins (including MMAE and MMAF), aminopterins, actinomycins, bleomycins, talisomycins, camptothecins, N8-acetyl spermidine, 1-(2 chloroethyl)-1,2-dimethyl sulfonyl hydrazide, esperamycins, etoposide, 6-mercaptopurines, dolastatins, trichothecenes, calicheamicins, taxol, taxanes, paclitaxel, docetaxel, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, duocarmycin, L-asparaginase, mercaptopurine, Thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustard, cytoxan, etoposide, 5-fluorouracil, bischloroethylnitrosourea, irinotecan, camptothecin, bleomycin, idarubicin, daunorubicin, Dactinomycin, plicamycin, mitoxantrone, asparaginase, vinorelbine, chlorambucil, melphalan, carmustine, lomustine,Busulfan, treosulfan, decarbazine, etoposide, teniposide, topotecan, 9-aminocamptothecin, crisnatol, mitomycin C, trimetrexate, mycophenolic acid, tiazofurin, ribavirin, EICAR (5-ethynyl-1-beta-Dribofuranosylimidazole-4-carboxamide), hydroxyurea, deferoxamine, fluxuridine, doxifluridine, raltitrexed, Cytarabine (ara C), cytosine arabinoside, fludarabine, tamoxifen, raloxifene, megestrol, goserelin, leuprolide acetate, flutamide, bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4, demethoxy-hypocrellin A, interferon-α, interferon-γ, tumor necrosis factor, Gemcitabine, Velcade, Revamid, Thalamide, Lovastatin,It may be at least one selected from the group consisting of 1-methyl-4-phenylpyridinium ion, staurosporine, actinomycin D, dactinomycin, bleomycin A2, bleomycin B2, peplomycin, epirubicin, pirarubicin, zorubicin, mitoxantrone, verapamil, and thapsigargin, nucleases, and toxins of bacterial, animal, or plant origin, but is not limited thereto.

[0281] In some cases, the drug may comprise one or more nucleophilic groups selected from the group consisting of amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups that can react to form a covalent bond with electrophilic groups on the linker and linker reagent.

[0282]

[0283] Composition for diagnosing, preventing and / or treating cancer

[0284] In another aspect, the present invention relates to a composition for diagnosing cancer comprising the antibody or an antigen-binding fragment thereof.

[0285] Prostate-specific membrane antigen (PSMA) has been reported in numerous studies as a biomarker for the diagnosis of various cancers, including prostate cancer (Journal of Cellular Biochemistry. 102 (3): 571-9.; Clin Cancer Res. 1997;3:81-85; Cancer Res. 1997;57:3629-3634; Clin Cancer Res. 1999;5:2674-2681). In addition, it has been reported that PSMA can accurately predict the likelihood of prostate cancer recurrence.

[0286] In the present invention, the term "diagnosis" refers to accurately determining the condition of a subject with respect to a specific disease or condition. For example, the condition of a subject with respect to a specific disease or condition is used in a broad sense, including not only susceptibility to a specific disease or condition, determination of the disease the subject is currently suffering from, but also confirmation of the characteristics of the disease, such as prognosis, identification of cancer status, determination of cancer stage, or prediction of cancer sensitivity and responsiveness to treatment, obtaining a basis for appropriate treatment according to the patient's disease and condition, such as confirming the condition of the subject to confirm the therapeutic efficacy of a specific drug, and further predicting and confirming the presence or absence of recurrence in a subject cured from a specific disease or condition. In the present invention, the diagnosis preferably refers to confirming the presence or possibility of developing a disease. In the present invention, the disease refers to cancer, including both solid cancer and hematological cancer. Preferably, it may be a solid cancer, and examples of such cancer may be selected from the group consisting of, but are not limited to, prostate cancer, skin cancer such as melanoma, liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, gastric cancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, colon cancer, pancreatic cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, biliary tract cancer, testicular cancer, rectal cancer, head and neck cancer, cervical spine cancer, ureteral cancer, osteosarcoma, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma.

[0287] More preferably, the cancer is characterized by expression or overexpression of PSMA protein, and may be, but is not limited to, prostate cancer, breast cancer, pancreatic cancer, lung cancer, thyroid cancer, stomach cancer, ovarian cancer, colon cancer, liver cancer, gallbladder cancer, kidney cancer, cervical cancer, bladder cancer, acute myeloid leukemia, or multiple myeloma. The cancer may be primary cancer or metastatic cancer.

[0288] In the present invention, the term "prognosis" means an expectation of medical outcome (e.g., long-term survival possibility, disease-free survival rate, etc.), and includes a positive prognosis (positive prognosis) or a negative prognosis (negative prognosis), wherein the negative prognosis includes disease progression or mortality such as recurrence, tumor growth, metastasis, drug resistance, etc., and the positive prognosis includes disease remission such as disease-free state, improvement or stabilization such as tumor regression, etc.

[0289] In the present invention, the term "prediction" means to guess in advance about the medical outcome, and for the purpose of the present invention, it means to guess in advance the course of the disease (progression, improvement, recurrence of gastric cancer, tumor growth, drug resistance) of a patient diagnosed with ovarian cancer.

[0290] In the present invention, the antibody or antigen-binding fragment thereof may be characterized in that it is linked to a signal molecule such as a radioactive isotope or fluorescent dye for detection.

[0291] In another aspect, the present invention relates to a composition for preventing or treating cancer, comprising an antibody or an antigen-binding fragment thereof that specifically binds to the prostate-specific membrane antigen, a bispecific or multispecific antibody comprising the antibody or an antigen-binding fragment thereof, an antibody-drug conjugate comprising the antibody or an antigen-binding fragment thereof, a chimeric antigen receptor comprising the antibody or an antigen-binding fragment thereof, or an immune cell comprising the chimeric antigen receptor.

[0292] In the present invention, the composition for preventing or treating cancer may be characterized as being a pharmaceutical composition.

[0293] The present invention may be a pharmaceutical composition for preventing or treating cancer, comprising, for example, (a) a pharmaceutically effective amount of an antibody or an antigen-binding fragment thereof that specifically binds to a prostate-specific membrane antigen according to the present invention, a bispecific or multispecific antibody comprising the antibody or an antigen-binding fragment thereof, an antibody-drug conjugate comprising the antibody or an antigen-binding fragment thereof, a chimeric antigen receptor comprising the antibody or an antigen-binding fragment thereof, or an immune cell comprising the chimeric antigen receptor; and (b) a pharmaceutically acceptable carrier.

[0294] In another aspect, the present invention relates to a method for preventing or treating cancer, comprising administering to a cancer patient an antibody or an antigen-binding fragment thereof, a bispecific or multispecific antibody comprising the antibody or an antigen-binding fragment thereof, an antibody-drug conjugate comprising the antibody or an antigen-binding fragment thereof, a chimeric antigen receptor comprising the antibody or an antigen-binding fragment thereof, or an immune cell comprising the chimeric antigen receptor.

[0295] “Prevention” means any action that inhibits the growth or delays the progression of cancer by administering a composition according to the present invention, and “treatment” means inhibition of cancer development, reduction of tumor, or elimination of cancer.

[0296] In the present invention, the term "cancer" is used with the same meaning as "tumor," and the preventive or therapeutic composition according to the present invention can be applied to all types of cancer, including solid cancer and blood cancer. Unlike blood cancer, solid cancer refers to cancer that forms in a mass in an organ, and most cancers that occur in organs fall under this category.

[0297] The cancer or carcinoma that can be treated with the composition of the present invention is not particularly limited and includes both solid cancer and blood cancer. Preferably, it may be a solid cancer, and examples of such cancer may be selected from the group consisting of prostate cancer, skin cancer such as melanoma, liver cancer, hepatocellular carcinoma, hepatocellular cancer, gastric cancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, colon cancer, pancreatic cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, biliary tract cancer, testicular cancer, rectal cancer, head and neck cancer, cervical spine cancer, ureteral cancer, osteosarcoma, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma and glioma, but are not limited thereto.

[0298] More preferably, the cancer is characterized by expression or overexpression of PSMA protein, and may be, but is not limited to, prostate cancer, breast cancer, pancreatic cancer, lung cancer, thyroid cancer, stomach cancer, ovarian cancer, colon cancer, liver cancer, gallbladder cancer, kidney cancer, cervical cancer, bladder cancer, acute myeloid leukemia, or multiple myeloma. The cancer may be primary cancer or metastatic cancer.

[0299] Pharmaceutically acceptable carriers included in the composition of the present invention are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0300] The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration.

[0301] Since proteins or peptides are digested upon oral administration, oral compositions must be formulated to coat the active agent or protect it from degradation in the stomach. Furthermore, the pharmaceutical composition can be administered by any device capable of transporting the active agent to the target cell.

[0302] The appropriate dosage of the composition according to the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. A skilled physician can easily determine and prescribe a dosage effective for the desired treatment or prevention. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to prevent or treat cancer or an autoimmune disease.

[0303] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0304] The preventive or therapeutic composition according to the present invention may be administered in combination with a known compound having an effect of preventing, improving, or treating symptoms of cancer, immune disease, or infectious disease, and may be administered in combination with, for example, one or more other immunotherapeutic agents, chemotherapeutic agents, antibody therapeutic agents, etc.

[0305] The pharmaceutical composition of the present invention may be characterized in that it is administered in combination with various treatment therapies, such as one or more anticancer therapies, for example, radiation therapy, direct surgical therapy, and dietary therapy.

[0306] In the present invention, the pharmaceutical composition may be characterized in that it is used in combination with one or more anticancer agents.

[0307]

[0308] [Example]

[0309] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0310]

[0311] Example 1: Development of antibodies against human PSMA protein

[0312] PSMA protein (Acrobiosystems, Catalog #PSA-H52H3, Human PSMA / FOLH1 Protein, His Tag) was used as an antigen for antibody production against human PSMA. 100 μg of the antigen was mixed with Freund's Adjuvant (Sigma-Aldrich, Catalog #F5506, Freund's Adjuvant, Incomplete) and injected intraperitoneally into H2L2 mice (Harbour BioMed). At 2-week intervals, 100 μg of the antigen was diluted in phosphate-buffered saline (PBS) and injected intraperitoneally three times. Three days after the third injection, the spleens were removed, and mouse B cells were isolated. The isolated lymphocytes were mixed with the mouse myeloma cell line SP2 / 0-Ag14 (ATCC, Catalog #CRL-1581) at a 5:1 ratio and fused with the SP2 / 0-Ag14 cells and mouse B cells using PEG-1500 (Roche, Catalog #10783641001, Polyethylene Glycol 1500). The fused cells were cultured in a medium containing HAT supplement (Sigma-Aldrich, Catalog #H0262, HAT Media Supplement (50Х) Hybri-Max) to selectively select and culture the fused cells (hybridomas). The obtained hybridomas were confirmed to be cells that produce antibodies that bind to the antigen through enzyme-linked immunosorbent assay (ELISA). The experimental method is as follows.Human PSMA protein (Acrobiosystems, Catalog #PSA-H52H3, Human PSMA / FOLH1 Protein, His Tag) or purified His tag protein was immobilized at a concentration of 1 μg / ml in a Costar 96-well plate (Corning, Catalog #3590, 96-well Clear Polystyrene Microplates) at room temperature for 2 hours. The His tag protein was used to select antibodies that bind only to PSMA but not to the His tag protein, since the antigen used for antibody production is His tag-conjugated. After immobilization, the plate was washed three times with 300 μl of PBS-T (PBS containing 0.05% Tween 20) buffer, and then blocked with 300 μl of 1% BSA / PBS buffer for 1 hour at room temperature. The blocked plate was washed three times, hybridoma culture medium was added, and the plate was reacted with the antigen for 2 hours at room temperature. After washing three times, goat anti-Rat IgG-HRP antibody (Invitrogen, Catalog #31470, Goat anti-Rat IgG (H+L) Secondary Antibody, HRP) was added as a secondary antibody at a 1:5,000 dilution in 1% BSA / PBS solution and reacted at room temperature for 1 hour. After washing three times, TMB (SurModics, Catalog #TMBC-1000-01, TMB Conductivity One Component HRP Microwell Substrate) was added and color development was performed at room temperature for 5 minutes, and 1 N sulfuric acid (Duksan, Catalog #255) was added to stop color development. The absorbance was measured at 450 nm using a microplate reader (TECAN, Catalog #Infinite F50), and 14 clones with an OD450 value of 0.7 or higher were selected (Fig. 1).

[0313]

[0314] Example 2: Evaluation of cell binding capacity of developed antibodies: Flow cytometry

[0315] Since human PSMA is a protein expressed on the cell surface, whether the developed antibodies bind to LNCaP (Lymph Node Carcinoma of the Prostate) cells derived from human prostate adenocarcinoma cells with lymph node metastasis expressing PSMA was confirmed through flow cytometry using hybridoma cultures of 14 clones selected in Figure 1. The experimental method was to react 5x105 cells with 1 mL of hybridoma culture at 4°C for 1 hour, and then wash twice with buffer (BD, Catalog #554656, Stain Buffer (FBS)).

[0316] The samples were reacted with a fluorescence-conjugated secondary antibody (Invitrogen, catalog #A-11006, Goat anti Rat IgG(H+L) Cross-Absorbed secondary antibody, Alexa Fluor 488) for 1 hour. After washing twice with a buffer (BD Biosciences, Catalog #554656, Stain Buffer(FBS)), the samples were analyzed using a flow cytometer (BD FACSAriaII). Among the 14 clones, 8 clones that strongly bind to PSMA were selected, and the selection criteria were a median fluorescence value of 5000 or higher. In the experiment, samples in which only cells were analyzed and cells in which only the secondary antibody was reacted were used as negative controls, and a commercially available antibody (LSBio, Calog # LS-C150527, Monoclonal Mouse antiHuman FOLH1 / PSMA Antibody (clone 107.1A4)) was used as a positive control (Fig. 2).

[0317] Eight clones were selected by single-cell cloning through cell serial dilution. Six clones that specifically bind to human PSMA protein were selected from a single cell through five cell serial dilutions. The six selected monoclonal hybridoma cultures were purified using Protein G Agarose beads (Amicogen, Catalog #2010100, rProtein G Agarose Resin) and confirmed by SDS-PAGE (Fig. 3). The binding of the six purified antibodies to PSMA was confirmed by flow cytometry using a PSMA-expressing cell line (LNCaP). 5 x 105 LNCaP cells and 2 μg of purified antibodies were reacted at 4 °C for 1 hour and then washed twice with buffer (BD Biosciences, Catalog #554656, Stain Buffer (FBS)). The cells were reacted with a fluorescence-conjugated secondary antibody (Invitrogen, catalog #A-11006, Goat anti Rat IgG(H+L) Cross-Absorbed secondary antibody, Alexa Fluor 488) for 1 hour. After washing twice with buffer (BD Biosciences, Catalog #554656, Stain Buffer (FBS)), the samples were analyzed using a flow cytometer (BD FACSAriaII). As a result, it was confirmed that all five clones bound to LNCaP cells expressing PSMA, and among them, 46H11 was confirmed to have lower affinity for PSMA expressed in LNCaP cells compared to the other clones. The criterion for affinity was selected based on the median fluorescence value of 5000.In this experiment, samples in which only cells were analyzed and cells reacted only with the secondary antibody were used as negative controls, and commercially available antibodies (LSBio, Catalog # LS-C150527, Monoclonal Mouse antiHuman FOLH1 / PSMA Antibody (clone 107.1A4)) were used as positive controls. Based on these results, the sequences of five clones excluding 46H11 were analyzed (Fig. 4).

[0318]

[0319] Example 3: Sequence analysis of each clone antibody

[0320] The sequences of the five antibody clones selected in Example 2 were analyzed.

[0321] Sequence analysis was performed by extracting RNA from each monoclonal hybridoma, synthesizing cDNA, and then following the protocol provided by the mouse supplier, Harbour Biomed. The VH and VL regions were amplified by PCR, followed by gel extraction and TA cloning. TA cloning was performed in the pJet1.2 cloning vector (Thermo, Catalog #K1232, CloneJET PCR Cloning Kit), and the sequence was confirmed through Cosmogenetec. The confirmed sequence was analyzed and compared with the CDR region on the IMGT site (imgt.org), resulting in the results shown in Table 1. The specific amino acid sequences and CDR sequences are as follows in Tables 2 to 4.

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328] Example 4: Confirmation of antibody binding affinity of five anti-PSMA antibodies to prostate cancer cell lines.

[0329] The binding affinity of five antibodies to pancreatic cancer cell lines was confirmed.

[0330] Human antibodies were produced and purified using HEK293 cell line (ATCC CRL-1573).

[0331] The VL region of five human antibodies (41B11, 44H8, 45C3, 46D3, and 42E4) whose sequences were confirmed were inserted into pFUSE2ss-CLIg-hK (InvivoGen, Catalog #pfuse2ss-hclk) to produce five DNA plasmid-VL, and the VH region was inserted into pFusess-CHIg-hG1 (InvivoGen, #pfusess-hchg1) to produce DNA plasmid-VH, which were used for antibody production. Antibodies were produced using the five light chain and heavy chain DNA plasmids using the ExpiCHO Expression system kit (Gibco, Catalog #A29133), and the produced antibodies were purified using Protein G Agarose beads (Amicogen, Catalog #2010100, rProtein G Agarose Resin).

[0332] The purified human antibodies 41B11, 44H8, 45C3, 46D3, and 42E4 (2 μg each) were reacted with PSMA-positive LNCaP cells (ATCC, CRL-1740-LUC2) and PSMA-negative PC3 cells (ATCC, CRL-1435-LUC2) at 4°C for 1 hour, respectively. After washing twice with buffer (BD Biosciences, Catalog#554656, Stain Buffer (FBS)), each cell was reacted with a fluorescence-conjugated secondary antibody (Biolegend, catalog #366903, PE anti-human IgG Fc Recombinant Antibody) for 1 hour. After washing twice with buffer (BD Biosciences, Catalog#554656, Stain Buffer (FBS)), the sample was analyzed using a flow cytometer (BD FACSAriaII).

[0333] As a result, as shown in Figures 5 and 6, all five clones strongly bound to LNCaP cells (PSMA positive) and did not bind to PC3 cells.

[0334]

[0335] Example 5: Production of PSMA CAR-T cells

[0336] A lentiviral vector was constructed that expresses an antibody (scFv) that binds to PSMA on the cell surface and has a signaling domain inside the cell. Human peripheral blood mononuclear cells (PBMC, Lonza) were suspended in TexMACS medium (Miltenyi Biotec.) containing IL7 and IL15, and TransAct (Miltenyi Biotec, polymeric nanomatrix conjugated to CD3 and CD28 agonists) was added. The cells were seeded in G-REX 6-well plates (WILSONWOLF) to activate them, and the lentiviral vector was treated after 24 hours. After 48 hours, TexMACS medium containing IL7 and IL15 was additionally added, and the cells were cultured for an additional 10 days, with fresh medium being replaced every 3–4 days. Afterwards, 500 ng of FITC-PSMA protein (Acro Biosystems, Catalog#. PSA-HF244, FITC-Labeled Human PSMA / FOLH1 Protein, His Tag) was treated to 5x105 CAR-T cells and incubated at 4°C for 1 hour. Afterwards, the cells were washed twice with buffer (BD Biosciences, Catalog#554656, Stain Buffer (FBS)) and analyzed using a flow cytometer (BD FACSAriaII). As a result, the ratio of lentivirus-derived scFv-CAR genes introduced into CAR-T cells produced for five types of antibodies was confirmed as shown in Fig. 7.

[0337]

[0338] Example 6: Confirmation of the cell killing effect of PSMA CAR-T cells against prostate cancer cell lines.

[0339] The anticancer effect of the CAR-T cells was confirmed by co-culturing the five types of PSMA CAR-T cells (PB-CTA-41B11, PB-CTA-44H8, PB-CTA-45C3, PB-CTA-42E4, and PB-CTA-46D3) produced in Example 5 with the prostate cancer cell lines LNCaP cells and PC3 cells.

[0340] As a result, as shown in Fig. 8, all five types of PSMA CAR-T cells showed a killing effect against the prostate cancer cell line LNCaP that expresses PSMA on its surface, and did not show an anticancer effect against PC3 cells, which are prostate cancer cells that do not express PSMA on their surface.

[0341]

[0342] Example 7: Confirmation of cytokine secretion by PSMA CAR-T cells

[0343] When PSMA CAR-T cells PB-CTA-41B11, PB-CTA-42E4, and PB-CTA-46D3 were co-cultured with prostate cancer cells, the secretion of interferon gamma (IFN-γ) and granzyme B was confirmed in the CAR-T cells. As a positive control, PSMA-PC, a CAR-T cell containing anti-PSMA scFv (produced directly by citing SEQ. NO. 1 and 2 of patent WO2019 / 191728A1; the remaining sequences are identical to those of the company's CAR-T cells), was used.

[0344] As a result, as shown in Figure 9, it was confirmed that the three types of CAR-T cells had superior IFN-γ and granzyme B secretion capabilities than the positive control group, PSMA-PC.

[0345]

[0346] Example 8: Anticancer effect of PSMA CAR-T cells in vivo

[0347] Mice (NOD.Cg-Prkdc scid Il2rg tm1sug / JicKoat, male, 5 weeks old, Coretec) were incubated with 5x 10 prostate cancer cells (LNCaP). 6 Dogs were injected subcutaneously, and on day 13 after cancer cell administration, 5x 10 PSMA CAR-T cells, PB-CTA-42E4 6 The dogs were administered intravenous injection and observed for 28 days.

[0348] As a result, as shown in Fig. 10, it was confirmed that the tumor completely disappeared in the PB-CTA-42E4 administration group (individuals 2 and 3).

[0349] In the negative control group, subject 1, the tumor persisted, and in the negative control group, subject 4, the initial cancer engraftment was not proper, so the ivis image signal decreased over time.

[0350]

[0351] Example 9: Improvement of PSMA antibodies through additional mutations

[0352] For STACT (solid tumor adapted CAR-T therapy), we developed an antibody that maintains high binding affinity to the target antigen in the low pH of the tumor microenvironment (TME) while having low binding affinity to the target antigen in a normal pH environment.

[0353] To develop an antibody with high binding affinity to PSMA specifically at low pH, a STACT antibody was produced by substituting specific amino acids in the heavy and light chain CDR sequences of the PSMA CAR-T cell clone PB-CTA-42E4 with histidine.

[0354] Three types of STACT antibodies (PSMA36, PSMA37, and PSMA38) were produced by substituting the R residue at position 3 of CDR3 of the DNA plasmid-VL of the 42E4 clone with a H residue, and the DNA plasmid-VH of PSMA36 was produced by substituting the S residue at position 6 of CDR1 of the DNA plasmid-VH of 42E4 with a H residue, the DNA plasmid-VH of PSMA37 was produced by substituting the Y residue at position 4 of CDR2 of the DNA plasmid-VH of 42E4 with a H residue, and the DNA plasmid-VH of PSMA38 was produced by substituting the S residue at position 5 of CDR2 of the DNA plasmid-VH of 42E4 with a H residue, and used for antibody production. Residue substitutions in the sequence were performed using the Phusion Site-Directed Mutagenesis Kit (Thermo, Catalog #F541).

[0355] The antibody sequences mutated from PB-CTA-42E4 are shown in Table 5.

[0356]

[0357] Tables 6 and 7 show the CDR sequences of STACT antibodies PSMA36, PSMA37, and PSMA38.

[0358]

[0359]

[0360]

[0361]

[0362]

[0363] Example 10: Confirmation of binding affinity to PSMA antigen using STACT antibody at low pH

[0364] The binding affinity of the STACT antibodies (PSMA36, PSMA37, and PSMA38) produced in Example 9 to the PSMA antigen was confirmed at pH 7.4 and pH 6.0. The parent antibody, 42E4 antibody, was used as a positive control.

[0365] The binding affinity of the STACT antibodies (PSMA36, PSMA37, and PSMA38) produced to the PSMA antigen was confirmed through an enzyme-linked immunosorbent assay (ELISA). The experimental method is as follows. First, HPR was conjugated to the produced STACT antibodies (PSMA36, PSMA37, PSMA39) and the parent antibody 42E4 using the EZ-LINK plus activated peroxidase kit (Thermo, Catalog #31489, EZ-LINK plus activated peroxidase kit). Human PSMA protein (Acrobiosystems, Catalog #PSA-H52H3, Human PSMA) as an antigen was fixed at 25 ng / well, 5 ng / well, and 1 ng / well in Costar 96-well half plates (Corning, Catalog #3690, 96-well Half Area Clear Polystyrene Microplates) at 4°C for 16 h. After fixation, the plates were washed three times with 180 μl of PBS-T (PBS containing 0.05% Tween 20) buffer and blocked with 100 μl of 1% BSA / PBS buffer for 1 h at room temperature. After washing the blocked plate three times, 50 μl of the HRP-conjugated STACT antibodies (PSMA36, PSMA37, PSMA39) and the parent antibody 42E4 were diluted in 1% BSA / PBS buffer titrated to pH 6.0 and pH 7.4, respectively, at a concentration gradient of 1 / 10 from 10,000 ng / ml to 1 ng / ml, and reacted with the antigen at room temperature for 2 hours. The plates after the reaction were incubated in PBS-T (0.After washing three times with 180 µl of PBS buffer containing 0.5% Tween 20, TMB (SurModics, Catalog #TMBC-1000-01, TMB Conductivity One Component HRP Microwell Substrate) was added and color development was performed at room temperature for 5 minutes. 1N sulfuric acid (Duksan, Catalog #255) was added to the plate after the color reaction was completed to stop the color development. The absorbance was measured at 450 nm using a microplate reader (TECAN, Catalog #Infinite F50), and the binding affinity of the STACT antibody to the PSMA antigen at pH 7.4 and pH 6.0 was confirmed by comparing each OD450 value.

[0366] ELISA experimental conditions:

[0367] -Ag coating: 25ng / well, 5ng / well, 1ng / well

[0368] -Ab Con.(ng / ml): 1 / 3 Serial dilution (20,000ng / ml~0.339ng / ml)

[0369] As a result, as shown in Fig. 11, the parent antibody 42E4 showed no difference in binding affinity to PSMA according to pH change, and as shown in Figs. 12 to 14, the STACT antibodies PSMA36, PSMA37, and PSMA38 showed higher binding affinity to the PSMA antigen at pH 6.0.

[0370]

[0371] Example 11: Confirmation of antigen binding capacity of STACT cells

[0372] STACT cells PB-CTA-PSMA36, PB-CTA-PSMA37, and PB-CTA-PSMA38 were produced using the scFv sequences of PSMA36, PSMA37, and PSMA38 antibodies. The same process as that used to produce PSMA CAR-T cells in Example 5 was followed.

[0373] Lentiviral vectors containing scFv sequences of PSMA36, PSMA37, and PSMA38 antibodies and CARs were constructed, and the same process for PSMA CAR-T cell production and flow cytometry analysis for measuring lentivirus transduction efficiency as in Example 5 was followed. However, to confirm the pH-dependent binding affinity of STACT cells, PSMA antigen binding was confirmed at pH 6.0 and pH 7.4, respectively, during flow cytometry. Parental PB-CTA-42E4 cells were used as a positive control.

[0374] As a result, as shown in Fig. 15, it was confirmed that STACT cells specifically bind to the PSMA antigen only at pH 6.0.

[0375]

[0376] Example 12: Anticancer effect of STACT cells in vivo

[0377] Mice (NOD.Cg-Prkdc scid Il2rg tm1sug / JicKoat, male, 5 weeks old, Coretec) were injected with 5x10 prostate cancer cells (LNCaP). 7 The dogs were injected subcutaneously, and on the 13th day after cancer cell administration, 5x10 STACT CAR-T cells, PB-CTA-PSMA36 6 After intravenous injection, the dogs were observed for 32 days.

[0378] As a result, as shown in Fig. 16, complete remission was confirmed in which the tumor completely disappeared on the 28th day in subject No. 1, which was in the PB-CTA-PSMA36 administration group, and complete remission was confirmed on the 14th day in subject No. 2, which was in the PB-CTA-PSMA36 administration group.

[0379] In the negative control groups, subjects 3 and 4, it was confirmed that the tumor continued to grow.

[0380]

[0381] The anti-PSMA antibody or antigen-binding fragment thereof according to the present invention exhibits not only excellent binding affinity for the PSMA antigen but also tumor tissue-specific binding, and has been confirmed to have an excellent anticancer effect in animal experiments, and thus can be usefully used for the diagnosis, prevention, or treatment of a target tumor or cancer.

[0382]

[0383] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0384]

[0385] Electronic file attached.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to prostate-specific membrane antigen (PSMA) comprising: A light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 26; A light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 to 30; A light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31 to 34 and 45; A heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 37, and 46; A heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 38 to 41, 47, and 48; and A heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 42 to 44.

2. An antibody or antigen-binding fragment thereof that specifically binds to prostate-specific membrane antigen (PSMA) comprising a light chain variable region represented by any one sequence selected from the group consisting of SEQ ID NOs: 12, 14, 16, 18, 20, 21, and 53 in the first paragraph.

3. An antibody or antigen-binding fragment thereof that specifically binds to prostate-specific membrane antigen (PSMA) comprising a heavy chain variable region represented by any one sequence selected from the group consisting of SEQ ID NOs: 13, 15, 17, 19, 22, and 54 to 56 in the first paragraph.

4. An antibody or antigen-binding fragment thereof that specifically binds to prostate-specific membrane antigen (PSMA), characterized in that it comprises at least one combination of a light chain variable region and a heavy chain variable region comprising the following: i) a light chain variable region represented by SEQ ID NO: 12 and a heavy chain variable region represented by SEQ ID NO: 13; ii) a light chain variable region represented by SEQ ID NO: 14 and a heavy chain variable region represented by SEQ ID NO: 15; iii) a light chain variable region represented by SEQ ID NO: 16 and a heavy chain variable region represented by SEQ ID NO: 17; iv) a light chain variable region represented by SEQ ID NO: 18 and a heavy chain variable region represented by SEQ ID NO: 19; v) a light chain variable region represented by SEQ ID NO: 20 and a heavy chain variable region represented by SEQ ID NO: 22; vi) a light chain variable region represented by SEQ ID NO: 21 and a heavy chain variable region represented by SEQ ID NO: 22; vii) a light chain variable region represented by SEQ ID NO: 53 and a heavy chain variable region represented by SEQ ID NO: 54; viii) a light chain variable region represented by SEQ ID NO: 53 and a heavy chain variable region represented by SEQ ID NO: 55; and ix) A light chain variable region represented by SEQ ID NO: 53 and a heavy chain variable region represented by SEQ ID NO:

56.

5. A nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4.

6. A recombinant expression vector containing the nucleic acid of Article 5.

7. A recombinant cell in which the nucleic acid of paragraph 5 or a recombinant expression vector containing the same has been introduced into a host cell.

8. A method for producing an antibody or an antigen-binding fragment thereof that specifically binds to prostate-specific membrane antigen (PSMA), comprising: a step of culturing the recombinant cell of paragraph 7 to produce an antibody; and a step of isolating and purifying the produced antibody.

9. A chimeric antigen receptor (CAR) comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4.

10. An immune cell expressing the chimeric antigen receptor of clause 9 on its surface.

11. A pharmaceutical composition for preventing or treating cancer, comprising the immune cells of Article 10.

12. An antibody-drug conjugate (ADC) in which an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4 is bound to a drug.

13. A composition for diagnosing, preventing or treating cancer, comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4, or an antibody-drug conjugate comprising the same.

14. A composition for diagnosing, preventing or treating cancer, characterized in that the cancer in claim 13 is selected from the group consisting of prostate cancer, breast cancer, pancreatic cancer, lung cancer, thyroid cancer, stomach cancer, ovarian cancer, colon cancer, liver cancer, gallbladder cancer, kidney cancer, cervical cancer, bladder cancer, acute myeloid leukemia and multiple myeloma.

15. A bispecific or multispecific antibody comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

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