Human monoclonal antibody against antigen CT83 and use thereof

Abstract

The present invention relates to a human monoclonal antibody against the antigen CT83 and an anticancer use thereof. The antibody according to the present invention or an antigen-binding fragment thereof specifically binds to the antigen CT83, and has the effect of preventing or treating cancer.

Description

Human monoclonal antibodies against CT83 antigen and uses thereof

[0001] The present invention relates to human monoclonal antibodies against the CT83 antigen and uses thereof.

[0002] The complementarity determining region (CDR) of an antibody is a part of the antibody molecule that forms the antigen binding site, and plays a role in allowing the antibody to interact with, recognize, and bind to a specific antigen.

[0003] CDR is an important factor that determines the diversity of antibodies, and CDR is an important factor in antibody engineering and antibody-based therapeutic development.

[0004] HeLa cells are a cervical cancer cell line derived from Henrietta Lacks in 1951 for the treatment of malaria. This cell line has exceptionally robust growth capacity and can be artificially propagated over long periods of time, making it widely used in cell biology and cancer research. It is also widely utilized for purposes such as studying tumor cell characteristics and evaluating the effectiveness of anticancer treatments.

[0005] Cervical cancer is a cancer that develops in the cervix, the area of ​​a woman's uterus (the organ that carries the placenta). This type of cancer is primarily associated with infection with the human papillomavirus (HPV). HPV is transmitted sexually, and some HPV infections can cause abnormalities in cervical cells, leading to cancer.

[0006] HT-29 cells are a widely used artificial cell line for colorectal cancer. This cell line is derived from human colorectal cancer tissue and is widely used in colorectal cancer research and drug testing.

[0007] Colorectal cancer, a cancer that develops in the colon or rectum, is a type of digestive cancer. It typically develops in the lining of the colon, and as the tumor grows, it can invade and spread to surrounding tissues. Colorectal cancer typically develops through the process of intestinal polyps evolving into cancer. It can present with a variety of symptoms and signs, which vary depending on the location, size, and progression of the tumor. Common symptoms of colorectal cancer include constipation or diarrhea, changes in stool shape or color, abdominal pain, weight loss, and fatigue.

[0008] Accordingly, the present inventors confirmed that overexpression of the CT83 antigen is strongly associated with poor prognosis, and that expression of the CT83 antigen promotes proliferation, metastasis, and EMT of cancer cells, and completed the present invention by developing a recombinant antibody that specifically binds to the CT83 antigen through phage display and biopanning.

[0009] An object of the present invention is to provide a heavy chain variable region comprising CDR 1, CDR 2 and CDR 3; and

[0010] To provide an antibody or antigen-binding fragment thereof that specifically binds to the CT83 antigen, comprising a light chain variable region comprising CDR 1, CDR 2 and CDR 3.

[0011] In addition, another object of the present invention is to provide a gene encoding an antibody or an antigen-binding fragment thereof that specifically binds to the CT83 antigen.

[0012] In addition, it is an object of the present invention to provide a vector containing the above gene.

[0013] In addition, it is an object of the present invention to provide a host cell comprising the vector.

[0014] In addition, an object of the present invention is to provide a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to CT83 protein, including a step of culturing a host cell containing the vector.

[0015] In addition, an object of the present invention is to provide a composition for preventing or treating cancer, comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

[0016] In addition, an object of the present invention is to provide a method for treating cancer using the composition for preventing or treating cancer.

[0017] In addition, it is an object of the present invention to provide an antibody-drug conjugate in which a pharmaceutical composition for preventing or treating cancer is conjugated to an antibody or an antigen-binding fragment thereof that binds to the CT83.

[0018] In addition, it is an object of the present invention to provide a kit for detecting a tumor or cancer, comprising an antibody or an antigen-binding fragment thereof that binds to the CT83.

[0019] In addition, an object of the present invention is to provide a method for detecting a tumor or cancer, comprising the steps of treating a cancer-related sample with an antibody or an antigen-binding fragment thereof that binds to CT83 to form a CT83 antigen-antibody complex; and the step of detecting the CT83 antigen-antibody complex.

[0020] In addition, the purpose of the present invention is to provide a method for providing tumor or cancer diagnostic information, comprising the steps of treating a cancer-related sample with an antibody or an antigen-binding fragment thereof that binds to CT83 to form a CT83 antigen-antibody complex; and the step of detecting the CT83 antigen-antibody complex.

[0021] The present invention relates to a heavy chain variable region comprising CDR 1, CDR 2 and CDR 3; and

[0022] An antibody or antigen-binding fragment thereof that specifically binds to CT83 is provided, comprising a light chain variable region comprising CDR 1, CDR 2 and CDR 3.

[0023] In addition, the present invention provides a gene encoding an antibody or an antigen-binding fragment thereof that specifically binds to the CT83.

[0024] Additionally, the present invention provides a vector comprising the above gene.

[0025] In addition, the present invention provides a host cell comprising the above vector.

[0026] In addition, the present invention provides a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to CT83 protein, comprising the step of culturing a host cell containing the vector.

[0027] In addition, the present invention provides a composition for preventing or treating cancer, comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

[0028] In addition, the present invention provides a method for treating cancer using the composition for preventing or treating cancer.

[0029] In addition, the present invention provides an antibody-drug conjugate in which a pharmaceutical composition for preventing or treating cancer is conjugated to an antibody or an antigen-binding fragment thereof that binds to the CT83.

[0030] In addition, the present invention provides a kit for detecting a tumor or cancer, comprising an antibody or an antigen-binding fragment thereof that binds to the CT83.

[0031] In addition, the present invention provides a method for detecting a tumor or cancer, comprising the steps of treating a cancer-related sample with an antibody or an antigen-binding fragment thereof that binds to CT83 to form a CT83 antigen-antibody complex; and the step of detecting the CT83 antigen-antibody complex.

[0032] In addition, the present invention provides a method for providing tumor or cancer diagnostic information, comprising the steps of treating a cancer-related sample with an antibody or an antigen-binding fragment thereof that binds to CT83 to form a CT83 antigen-antibody complex; and the step of detecting the CT83 antigen-antibody complex.

[0033] The present invention relates to a human monoclonal antibody against the CT83 antigen and its anticancer use, wherein the antibody of the present invention or an antigen-binding fragment thereof specifically binds to the CT83 antigen and has a preventive or therapeutic effect on cancer.

[0034] Figure 1 shows the ELISA results of 400 anti-CT83 single antibody candidates.

[0035] Figure 2a shows the ELISA results of SKAI-5, which was selected as an antibody candidate of the present invention.

[0036] Figure 2b shows the ELISA results of SKAI-33, which was selected as an antibody candidate of the present invention.

[0037] Figure 2c shows the ELISA results of SKAI-48, which was selected as an antibody candidate of the present invention.

[0038] Figure 3 shows the microarray results confirming binding to the antigen for epitope mapping.

[0039] Figure 4 shows the expression level of KK-LC-1 protein in HeLa cells or HT-29 cells.

[0040] Figure 5a shows whether SKAI-5, selected as an antibody candidate of the present invention, specifically binds to HeLa cells or HT-29 cells.

[0041] Figure 5b shows whether SKAI-33, selected as an antibody candidate of the present invention, specifically binds to HeLa cells or HT-29 cells.

[0042] Figure 5c shows whether SKAI-48, selected as an antibody candidate of the present invention, specifically binds to HeLa cells or HT-29 cells.

[0043] Figure 6a illustrates key information about one of the epitopes of the antibody (SKAI-5) of the present invention. The upper part of Figure 6a illustrates information about the sequence, length, mass, isoelectric point, charge, hydrophobicity, and extinction coefficient of the epitope, the middle part of Figure 6a illustrates the structure of the epitope, and the lower part of Figure 6a illustrates the sequence, sequence number, length, and hydrophobicity index (hydropathy_index) for the amino acids constituting the epitope sequence.

[0044] Figure 6b illustrates key information about another epitope of the antibody (SKAI-5) of the present invention. The upper part of Figure 6b illustrates the sequence, length, mass, isoelectric point, charge, hydrophobicity, and extinction coefficient information of the epitope, the middle part of Figure 6b illustrates the structure of the epitope, and the lower part of Figure 6b illustrates the sequence, sequence number, length, and hydrophobicity index (hydropathy_index) for the amino acids constituting the epitope sequence.

[0045] Figure 6c shows key information about one of the epitopes of the antibody (SKAI-33) of the present invention. The upper part of Figure 6c shows information about the sequence, length, mass, isoelectric point, charge, hydrophobicity, and extinction coefficient of the epitope, the middle part of Figure 6c shows the structure of the epitope, and the lower part of Figure 6c shows the sequence, sequence number, length, and hydrophobicity index (hydropathy_index) for the amino acids that make up the epitope sequence.

[0046] Figure 6d shows key information about one of the epitopes of the antibody (SKAI-33) of the present invention. The upper part of Figure 6d shows information about the sequence, length, mass, isoelectric point, charge, hydrophobicity, and extinction coefficient of the epitope, the middle part of Figure 6d shows the structure of the epitope, and the lower part of Figure 6d shows the sequence, sequence number, length, and hydrophobicity index (hydropathy_index) for the amino acids that make up the epitope sequence.

[0047] Figure 6e illustrates key information about one of the epitopes of the antibody (SKAI-48) of the present invention. The upper part of Figure 6e illustrates information about the sequence, length, mass, isoelectric point, charge, hydrophobicity, and extinction coefficient of the epitope, the middle part of Figure 6e illustrates the structure of the epitope, and the lower part of Figure 6e illustrates the sequence, sequence number, length, and hydrophobicity index (hydropathy_index) for the amino acids constituting the epitope sequence.

[0048] Figure 6f illustrates key information about one of the epitopes of the antibody (SKAI-48) of the present invention. The upper part of Figure 6f illustrates information about the sequence, length, mass, isoelectric point, charge, hydrophobicity, and extinction coefficient of the epitope, the middle part of Figure 6f illustrates the structure of the epitope, and the lower part of Figure 6f illustrates the sequence, sequence number, length, and hydrophobicity index (hydropathy_index) for the amino acids constituting the epitope sequence.

[0049] Figure 7a shows the location of one repeat sequence among the epitopes of the antibody (SKAI-5) of the present invention.

[0050] Figure 7b shows the location of a repeat sequence among other epitopes of the antibody (SKAI-5) of the present invention.

[0051] Figure 7c shows the location of one repeat sequence among the epitopes of the antibody (SKAI-33) of the present invention.

[0052] Figure 7d shows the location of one repeat sequence among the epitopes of the antibody (SKAI-48) of the present invention.

[0053] Figure 7e shows the location of a repeat sequence among other epitopes of the antibody (SKAI-48) of the present invention.

[0054]

[0055] Hereinafter, the present invention will be described in detail with reference to the attached drawings and embodiments thereof. However, the following embodiments are provided as examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0056] Furthermore, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the customs of the field to which the present invention pertains. Therefore, the definitions of these terms should be determined based on the contents throughout this specification. Throughout this specification, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless otherwise specifically stated.

[0057] Those skilled in the art will appreciate that variations and modifications can be made without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered illustrative rather than restrictive. The scope of the present invention is defined by the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as encompassed by the present invention.

[0058] “Phage display” is an experimental method for detecting interactions between proteins or between proteins and other target substances.

[0059] By cloning a gene into a bacteriophage and displaying the expressed protein on the phage surface, the presence and degree of interaction can be measured by binding strength with other proteins or other target substances.

[0060] Panning is a method for selecting antibodies or antigen-binding fragments that specifically bind to a specific antigen. Using a phage library, various antibody fragments are expressed on phages. Specific binding to the antigen is confirmed. The genetic information encoded by the phages with specific binding is then identified, enabling antibody production.

[0061] The "bio-panning" of the present invention is a technology for selecting peptides that bind to a specific target substance based on affinity, and it is known that this can be used to identify the sequence of peptides that bind to a specific target and to develop antibodies and pharmaceuticals.

[0062] The amino acid sequence of the antibody of the present invention is shown in Table 2.

[0063] The CT83 antigen is a protein expressed by the CT83 gene and is also called CXorf61, KKLC1, Kita-kyushu lung cancer antigen 1, KK-LC-1, or Cancer / testis antigen 83. It has a molecular weight of approximately 26.8 kDa and is known to be one of the carcinogenic factors in the human body.

[0064] As one embodiment for achieving the above technical task, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CT83, comprising a heavy chain variable region comprising CDR 1, CDR 2, and CDR 3; and a light chain variable region comprising CDR 1, CDR 2, and CDR 3.

[0065] The above antibody or antigen-binding fragment thereof may be characterized by specifically binding to an epitope of SEQ ID NO: 17 or 18.

[0066] The above antibody or antigen-binding fragment thereof may be a scFv that specifically binds to CT83. The scFv may refer to a single-chain fragment antibody and may refer to a linkage of the variable regions of the light and heavy chains of an immunoglobulin.

[0067] As one embodiment for achieving the above technical task, the present invention provides a gene encoding an antibody or an antigen-binding fragment thereof that specifically binds to CT83.

[0068] When the above gene is expressed in a cell, the antibody or antigen-binding fragment thereof can be produced.

[0069] As one embodiment for achieving the above technical task, the present invention provides a vector containing the gene.

[0070] The above vector may refer to, but is not limited to, a plasmid, a cosmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a retroviral vector, an adenoviral vector, a liposome, or an exosome.

[0071] As one embodiment of achieving the above technical task, the present invention provides a host cell comprising the vector. The method of injecting the vector into the host cell may be electroporation, but is not limited thereto.

[0072] As one embodiment for achieving the above technical task, the present invention provides a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to CT83 protein, comprising a step of culturing a host cell containing the vector.

[0073] As one embodiment for achieving the above technical task, the present invention provides a composition for preventing or treating cancer, comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

[0074] As one aspect of achieving the above technical task, the present invention provides a method for treating cancer using the composition for preventing or treating cancer. In the present invention, the method for treating cancer may exclude treatment targeting humans and may target mammals. The mammal may refer to any animal afflicted with cancer, including dogs, cats, giraffes, tigers, whales, sheep, goats, monkeys, camels, cows, pigs, elephants, and horses.

[0075] As one embodiment for achieving the above technical task, the present invention provides an antibody-drug conjugate in which a pharmaceutical composition for preventing or treating cancer is conjugated to an antibody or an antigen-binding fragment thereof that binds to CT83.

[0076] The above-mentioned antibody-drug conjugate for preventing or treating cancer may mean an antibody or an antigen-binding fragment thereof conjugated to a linker or a secondary antibody.

[0077] As one aspect for achieving the above technical task, the present invention also provides a CAR T therapeutic agent in which a T cell is bound to the antibody or an antigen-binding fragment thereof.

[0078] The pharmaceutical composition for preventing or treating cancer may mean at least one selected from the group consisting of a toxin, a chemotherapeutic agent, a cytotoxic anticancer agent, a targeted anticancer agent, an immunotherapy agent, an immune checkpoint inhibitor, an antibiotic, an ADP-ribosyl transferase, a radioisotope, and a nucleolytic enzyme.

[0079] The cancer may include a solid cancer or a blood cancer, and specifically, the solid cancer may include at least one selected from the group consisting of cervical cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, pancreatic cancer, biliary tract cancer, liver cancer, stomach cancer, rectal cancer, oral cancer, pharynx cancer, larynx cancer, colon cancer, endometrial cancer, ovarian cancer, testis cancer, bladder cancer, renal cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer and / or thyroid cancer, and the Blood cancer may include one or more selected from the group consisting of leukemia, Hodgkin's lymphoma, lymphoma, and / or multiple myeloid blood cancer.

[0080] As one embodiment for achieving the above technical task, the present invention provides a kit for detecting a tumor or cancer, comprising an antibody or an antigen-binding fragment thereof that binds to the CT83.

[0081] The above CT83 antigen can act as a biomarker in the above tumor or cancer detection kit, and an antibody or an antigen-binding fragment thereof that binds to the CT83 antigen can be used as a reactant.

[0082] As one aspect for achieving the above technical task, the present invention provides a method for detecting a tumor or cancer, comprising the steps of treating a cancer-related sample with an antibody or an antigen-binding fragment thereof that binds to CT83 to form a CT83 antigen-antibody complex; and the step of detecting the CT83 antigen-antibody complex.

[0083] In addition, as another aspect for achieving the above technical task, the present invention provides a method for providing tumor or cancer diagnostic information, comprising the steps of treating a cancer-related sample with an antibody or an antigen-binding fragment thereof that binds to CT83 to form a CT83 antigen-antibody complex; and the step of detecting the CT83 antigen-antibody complex.

[0084]

[0085] Through examples, it was confirmed that SKAI-5, SKAI-33 or SKAI-48 proteins are antibodies or antigen-binding fragments that specifically bind to the CT83 antigen, and that SKAI-5, SKAI-33 or SKAI-48 proteins can be combined with a drug for preventing or treating cancer to be used as an ADC, or a chimeric antigen receptor T-cell (CAR-T) can be produced to be used for anticancer treatment.

[0086]

[0087] The present invention will be described in more detail below through the following examples. However, these examples are merely illustrative examples intended to easily illustrate the content and scope of the technical concept of the present invention and are not intended to limit or alter the technical scope of the present invention. Furthermore, those skilled in the art will readily recognize that various modifications and variations within the scope of the technical concept of the present invention can be made based on these examples.

[0088]

[0089] Example 1. Library construction and expression

[0090] To identify peptide sequences that can specifically bind to the CT83 protein, an antibody library was constructed and expressed as follows.

[0091] Biopanning to discover antibody candidate sequences is 7.6 X 10 9 The OPAL library (OPAL library, oriented peptide array library, Construction of a Large synthetic human scFv library with six diversified CDRs and high functional diversity., Yang Hym at. al., Molecules and cells, 2009. DOI / 10.1007 / s10059-009-0028-9), which is a form in which an HA tag is linked to nucleotides encoding scFv, was used.

[0092] The antibody expressed by the above OPAL library comprises a human variable heavy chain encoded by the gene Vh3-23 located at V3-23 and a human variable light chain encoded by the gene Vλ1g located at 1g, and CDR-H1, H2, L1, L2, and L3 comprise some CDR loops and CDR sequences combined or combined with human-derived CDR sequences.

[0093] CDR-H3 consists of four sublibraries, AE, BE, CF, and DF (Table 1), and forms an intraloop disulfide bond between cysteines of the DF sublibrary (C-(Xaa)4-C).

[0094] CDR-H3AE(N-ter)-(RK)-(Xaa)4-FDY-(C-ter) SEQ ID NO. 1 BE(N-ter)-(RK)-(DG)-(LPRVAG)-(Xaa)7-FDY-(C-ter) SEQ ID NO. 2 CF(N-ter)-(RK)-(FSYCLPHRVADG)-(LPRITSVAG)-(Xaa)7-(YS)-(YS)-(ADSY)-(YND)-(GA)-MDV-(C-ter) SEQ ID NO. 3 DF(N-ter)-(RK)-(VADG)-(LPRITSVAG)-(LRISVG)-Xaa-C-(Xaa)4-C-(YS)-(YS)(ADSY)-(YND)-(GA)-MDV-(C-ter) SEQ ID NO. 4

[0095] X represents any amino acid (X)n, where n represents n repetitions of that amino acid

[0096]

[0097] The OPAL library was transformed into ER2738 (E. coli, AMID BIOSCIENCES, cat. No. ER-201), and 20 MOI of helper phage (VCSM13) was added to 2xYT medium (BD Difco™, 2xYT powder) containing 50 ug / ml carbenicillin and 70 ug / ml kanamycin, and cultured at 30°C for 14 hours. Afterwards, the bacteriophage was activated (rescued) using 4% Polyethylene glycol 8000 (PEG8000).

[0098]

[0099] Example 2. Selection of candidates that specifically bind to CT83 protein.

[0100] To select candidates that specifically bind to the CT83 protein, biopanning was performed as follows.

[0101]

[0102] Specifically, biopanning was performed to search for substances that specifically bind to the (CT83)-VLPs antigen using recombinant human lung cancer antigen 1 (CT83)-VLPs (KK-LC-1 VLP, CSB-MP711093HU, CUSABIO) (Table 2).

[0103] Protein sequence number CAT# Amino acid sequence Expression cell tag KK-LC-1 (CT83) (NM_001017978) 5TP309391 (N-ter) -MNFYLLLASSILCALIVFWKYRRFQRNTGEMSSNSTALALVRPSSSGLINSNTDNNLAVYDLSRDILNNFPHSIARQKRILVNLSMVENKLVELEHTLLSKGFRGASPHRKST-(C-ter) HEK293T (C-ter) -Myc / DDK recombinant human lung cancer antigen 1(CT83)-VLPs(active)6CSB-MP711093HU(N-ter)-MNFYLLLASSILCALIVFWKYRRFQRNTGEMSNSTALALVRPSSSGLINSNTDNNLAVYDLSRDILNNFPHSIARQKRILVNLSMVENKLVELEHTLLSKGFRGASPHRKST-(C-ter)Mammalian cells(N-ter)- 6xHis-tag

[0104] CT83 antigen was bound to epoxy magnetic beads (M-270 Epoxy) overnight at 4°C, and then incubated with scFv-expressing phage at 37°C for 2 hours. The CT83-bound phage was eluted using 100 mM Triethylamine (TEA) and then infected with new ER2738 cells. The biopanning process of specifically binding the scFv-expressing bacteriophage to the CT83 antigen and then eluted using TEA was repeated three times. By repeating the bio-panning process, substances that nonspecifically bind to the antigen could be removed. Afterwards, to filter out substances that nonspecifically bind to the VLP itself, bio-panning was performed one more time using KK-LC-1 (CT83) (NM_001017978) human recombinant protein (CAT#: TP309391, Origene) (Table 2) to identify antibody candidates.

[0105] A set of 400 monoclonal antibody candidates was randomly selected from independently formed colonies cultured after the four rounds of panning described above (Fig. 1). Each candidate was expressed as an scFv and subjected to ELISA analysis to select 50 antibody candidates that specifically bind to KK-LC-1 (CT-83) versus BSA and KK-LC-1 VLPs versus BSA.

[0106]

[0107] Example 3. NGS analysis of selected candidates and final candidate selection.

[0108] The gene sequences of 50 selected candidates that bind to KK-LC-1 (CT83) and KK-LC-1 VLPs but not to BSA and VLP-(negative) were analyzed through Next Generation Sequencing (NGS) genetic analysis.

[0109] NGS analysis was performed on the above 50 selected antibody candidates, and 46 antibody candidates were selected after excluding overlapping antibodies and antibodies with frame shifts. The scFv of the 46 antibody candidates was reproduced using the gene sequence, and the recombinant KK-LC-1 / BSA and KK-LC-1 VLPs / BSA were analyzed by ELISA to select the final antibody candidates that can specifically bind to the CT83 protein.

[0110] As a result, three final antibody candidates were selected, with an absorbance ratio exceeding 10 and an absorbance of the negative control group below the reference value. The final antibody candidates were named SKAI-5, SKAI-33, and SKAI-48, and the heavy and light chain CDR amino acid sequences of the corresponding antibody candidates are listed in Table 3.

[0111] The ELISA results of SKAI-5 are as shown in Fig. 2a. TMB (3,3',5,5' - tetramethylbenzidine) was added, and the absorbance at 450 nm was measured using a plate reader after 20 minutes.

[0112] The ELISA results of SKAI-33 are as shown in Fig. 2b. TMB (3,3',5,5' - tetramethylbenzidine) was added, and the absorbance at 450 nm was measured using a plate reader after 20 minutes.

[0113] The ELISA results of SKAI-48 are as shown in Fig. 2c. TMB (3,3',5,5' - tetramethylbenzidine) was added, and the absorbance at 450 nm was measured using a plate reader after 20 minutes.

[0114] SKAI-5VHCDR1CDR2CDR3SEQ ID NO: 7SEQ ID NO: 8SEQ ID NO: 9(N-ter)-NYSMS-(C-ter)(N-ter)-SLSSGGGST-(C-ter)(N-ter)-ARRKTRFDY-(C-ter)VLCDR1CDR2CDR3SEQ ID NO: 10SEQ ID NO: 11SEQ ID NO: 12(N-ter)-SGSSSNIGSNTVN-(C-ter)(N-ter)-ADSQ-(C-ter)(N-ter)-GSWDSSLNG-(C-ter)SKAI-33VHCDR1CDR2CDR3SEQ ID NO: 19SEQ ID NO: 20SEQ ID NO: 21(N-ter)-SYSMS-(C-ter)(N-ter)-AISSSGGNK-(C-ter)(N-ter)-ARRKKLFDY-(C-ter)VLCDR1CDR2CDR3SEQ ID NO: 22SEQ ID NO: 23SEQ ID NO: 24(N-ter)-SGSSSNIGTNDVT-(C-ter)(N-ter)-ANNQ-(C-ter)(N-ter)-GAWDASLNG-(C-ter)SKAI-48VHCDR1CDR2CDR3SEQ ID NO: 25SEQ ID NO: 26SEQ ID NO: 27(N-ter)-DYYMS-(C-ter)(N-ter)-AISYDSSST-(C-ter)(N-ter)-ARKKKKFDY-(C-ter)VLCDR1CDR2CDR3SEQ ID NO: 28SEQ ID NO: 29SEQ ID NO: 30(N-ter)-SGSSSNIGSNNVT-(C-ter)(N-ter)-ANSN-(C-ter)(N-ter)-GTWDASLSG-(C-ter)

[0115]

[0116] Example 4. Identification of an epitope sequence binding to CT83

[0117] The final antibody candidate group confirmed in Example 3 was manufactured as an IgG antibody, and the following experiment was performed to clearly identify the amino acid sequence of the site where the antibody binds to the CT83 protein.

[0118] 4-1. IgG antibody production

[0119] The scFv sequence of SKAI-5, SKAI-33, or SKAI-48 that binds to CT83 was inserted into the human IgG vector system (TGEX-HC, Antibody design labs, MX001, and TGEX-LC, Antibody design labs, MX002) by the following method.

[0120] Specifically, a gene cloning vector was constructed to produce IgG using the base sequence encoding scFv by amplifying the antibody gene binding region using forward and reverse primers containing restriction enzyme sites. The base sequences of the primers are listed in Table 4.

[0121]

[0122] Vector designation forward primer (5'->3') reverse primer (5'->3') TGEX-VH SEQ ID NO: 13 SEQ ID NO: 14 (BSSHll) NNGCGCGCACTCCGAGGTGCAGCTGTTG (Bsmbl) NNCGTCTCNATGCTGAGCTCACGGTGACT GEX-VL SEQ ID NO: 15 SEQ ID NO: 16 (BspEl) NNNNNNTCCGGACAGTCTGTGCTGACT (Bsal) NNGGTCTCNTTCGTAGGACCGTCAGCT

[0123] A vector was constructed using the following method to enable expression of the above IgG in mammalian cells. The VH primer was designed to have a restriction site for BSSH11 at the 5' end and for BsmB1 at the 3' end, and the VL primer was designed to have a restriction site for BspE1 at the 5' end and for Bsa1 at the 3' end. The base sequences of the VH primer are the base sequences of SEQ ID NOs: 13 and 14, and the base sequences of the VL primer are the base sequences of SEQ ID NOs: 15 and 16.

[0124] The amplified VH fragment was inserted into the TGEX-HC (Antibody design labs, MX001) vector, and the VL fragment was inserted into the TGEX-LC (Antibody design labs, MX002) vector. The plasmids were amplified using Escherichia coli DH5α-competent cells, and cloning was confirmed by DNA sequencing.

[0125] The cloned heavy and light chain human IgG plasmids were co-transfected into Expi-CHO-s cells using the following method.

[0126] 6 x 10 6 Expi-CHO-s cells were prepared, and 15 µg each of heavy and light chain IgG plasmids were reacted in Opti-pro SFM medium (ThermoFisher scientific, Cat no. 12309019) for 5 minutes. After 5 minutes, the complex was mixed with Expifectamine™ CHO. The complex was transfected into the prepared Expi-CHO-s cells (ThermoFisher scientific, A29127) without exceeding 5 minutes of reaction. The next day, ExpiCHO™ Feed and ExpiFectamine™ CHO Enhancer (ExpiFectamine™ CHO Transfection kit, ThermoFisher scientific, Cat no. A29129) were added to increase nutrient supply and protein production efficiency. Cells were harvested when the cell viability was approximately 75% (approximately 7 days after transfection).

[0127] To purify the produced antibody, a monoclonal antibody was purified using Protein A Resin (Amicogen). The produced antibody (volume 50 ml) and 1 ml of Protein A resin (≥ 40 mg human IgG / ml resin) were mixed and incubated at 4°C for 6 hours. The mixture was then loaded onto an affinity chromatography column and eluted with 0.1 M glycine HCl buffer (pH 2.7). Finally, the purified antibody was concentrated using a 50 kDa cut-off amicon (merkmilipore), and the antibody concentration was measured using a protein assay kit (PierceTM, BCA Protein Assay kit, Thermo Scientific).

[0128]

[0129] 4-2. Epitope mapping

[0130] Epitope mapping was performed using microarray to identify the antigen binding site (epitope) of the selected antibody against the target antigen (CT83).

[0131] Before running the microarray, the SKAI-5, SKAI-33, or SKAI-48 antibodies prepared according to Example 4-1 were biotinylated. Thereafter, the biotinylated antibody samples were subjected to microarray chip analysis for approximately 18,000 CT83 antigen peptide positions to confirm binding to the CT83 antigen (Fig. 3).

[0132] Antigen-antibody binding was confirmed by measuring overlapping biotinylation signals. The sequence, length, mass, isoelectric point, charge, hydrophobicity, and extinction coefficient of the two major epitopes binding to the SKAI-5 antibody are shown in Figures 6a and 6b, and the locations of the repeat sequences (Figures 7a and 7b) were confirmed. The identified epitope sequences are listed in Table 5.

[0133] The sequence, sequence number, length, mass, isoelectric point, charge, hydrophobicity, and extinction coefficient of the major epitope binding to SKAI-33 are shown in Figures 6c and 6d, and the location of the repeat sequence was confirmed (Figure 7c). The confirmed epitope sequences are listed in (Table 5).

[0134] The sequence, sequence number, length, mass, isoelectric point, charge, hydrophobicity, and extinction coefficient of the major epitope binding to SKAI-48 are shown in Figures 6e and 6f, and the locations of the repeat sequences were confirmed (Figures 7d and 7e). The identified epitope sequences are listed in (Table 5).

[0135] SKAI-5Epitope 1 (SEQ ID NO: 17)Epitope 2 (SEQ ID NO: 18)(N-ter)-PHSIARQKR-(C-ter)(N-ter)-FRGASPHRKST-(C-ter)SKAI-33Epitope 1 (SEQ ID NO: 31)(N-ter)-NNLAVYDLLSRDILNNFPHSIARQKR-(C-ter)SKAI-48Epitope 1 (SEQ ID NO: 32)Epitope 2 (SEQ ID NO: 33)(N-ter)-LVELEHT-(C-ter)(N-ter)-FRGASPHRKST-(C-ter)

[0136]

[0137] Example 5. Confirmation of cancer cell-specific binding

[0138] To determine whether SKAI-5, SKAI-33, or SKAI-48 proteins specifically bind to the CT83 (KK-LC-1) antigen expressed in cancer cells, FACS experiments were performed using the following methods.

[0139] The HeLa cells used in this experiment are cancer cells that express the CT83 (KK-LC-1) protein, and HT-29 are cancer cells that do not express the CT83 (KK-LC-1) protein. As shown in Fig. 4, it was confirmed that the HeLa cells express the CT83 (KK-LC-1) antigen, and the HT-29 cells do not express the CT83 (KK-LC-1) antigen.

[0140] For FACS experiments, HeLa and HT-29 cells were harvested by trypsinization, washed with PBS, and centrifuged twice to pellet the cells. The cells were then prepared by incubating with a mouse monoclonal antibody against the CT83 antigen (Invitrogen, Catalog # MA5-24711) diluted 1 / 100 at room temperature for 1 hour. After 1 hour, the secondary antibody was diluted 1 / 100, incubated for 1 hour at room temperature, and analyzed using forward-scattered light (FSC).

[0141] By analyzing the extent to which HeLa cells expressing CT83 (KK-LC-1) protein and HT-29 cells not expressing CT83 (KK-LC-1) bound to SKAI-5 using FACS, we confirmed that SKAI-5 specifically binds to the CT83 (KK-LC-1) antigen. (Fig. 5a)

[0142] By analyzing the extent to which HeLa cells expressing CT83 (KK-LC-1) protein and HT-29 cells not expressing CT83 (KK-LC-1) bound to SKAI-33 using FACS, we confirmed that SKAI-33 specifically binds to the CT83 (KK-LC-1) antigen. (Fig. 5b)

[0143] By analyzing the extent to which HeLa cells expressing CT83 (KK-LC-1) protein and HT-29 cells not expressing CT83 (KK-LC-1) bound to SKAI-48 using FACS, we confirmed that SKAI-48 specifically binds to the CT83 (KK-LC-1) antigen. (Fig. 5c)

[0144] The present invention relates to a human monoclonal antibody against the CT83 antigen and its anticancer use. The antibody of the present invention or its antigen-binding fragment can be used as an anticancer agent by specifically binding to the CT83 antigen, and thus has industrial applicability.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to CT83, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 8, and a heavy chain CDR3 of SEQ ID NO: 9; and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 10, a light chain CDR2 of SEQ ID NO: 11, and a light chain CDR3 of SEQ ID NO: 12; An antibody or antigen-binding fragment thereof.

2. In paragraph 1, An antibody or antigen-binding fragment thereof, characterized in that the antibody or antigen-binding fragment specifically binds to an epitope of SEQ ID NO: 17 or 18.

3. In paragraph 1, An antibody or antigen-binding fragment thereof, characterized in that the antibody or antigen-binding fragment thereof is scFv.

4. A gene encoding an antibody or an antigen-binding fragment thereof that binds to CT83 of paragraph 1.

5. A vector containing the gene of clause 4.

6. A host cell comprising the gene of paragraph 4 or the vector of paragraph 5.

7. In paragraph 6, A host cell characterized in that the gene or vector is transformed.

8. A method for producing an antibody or an antigen-binding fragment thereof that specifically binds to CT83, comprising the step of culturing the host cell of paragraph 6.

9. A composition for preventing or treating cancer, comprising the antibody of paragraph 1 or an antigen-binding fragment thereof as an active ingredient.

10. In paragraph 9, The cancers include cervical cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, pancreatic cancer, biliary tract cancer, liver cancer, stomach cancer, rectal cancer, oral cancer, pharynx cancer, larynx cancer, colon cancer, endometrial cancer, ovarian cancer, testis cancer, bladder cancer, renal cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma, and multiple A composition for preventing or treating cancer, characterized in that it is selected from the group consisting of multiple myeloid blood cancer.

11. A kit for detecting a tumor or cancer, comprising an antibody or an antigen-binding fragment thereof that binds to CT83 of paragraph 1.

12. A method for detecting a tumor or cancer, comprising: a step of treating a cancer-related sample with an antibody or an antigen-binding fragment thereof that binds to CT83 of the first clause to form a CT83 antigen-antibody complex; and a step of detecting the CT83 antigen-antibody complex.

13. A method for providing tumor or cancer diagnostic information, comprising: a step of treating a cancer-related sample with an antibody or an antigen-binding fragment thereof that binds to CT83 of the first clause to form a CT83 antigen-antibody complex; and a step of detecting the CT83 antigen-antibody complex. 14.An antibody or antigen-binding fragment thereof that specifically binds to CT83, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 19, a heavy chain CDR2 of SEQ ID NO: 20, and a heavy chain CDR3 of SEQ ID NO: 21; and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 22, a light chain CDR2 of SEQ ID NO: 23, and a light chain CDR3 of SEQ ID NO: 24; An antibody or antigen-binding fragment thereof.

15. In paragraph 14, An antibody or antigen-binding fragment thereof, characterized in that the antibody or antigen-binding fragment specifically binds to an epitope of SEQ ID NO:

31.

16. In paragraph 14, An antibody or antigen-binding fragment thereof, characterized in that the antibody or antigen-binding fragment thereof is scFv.

17. A gene encoding an antibody or antigen-binding fragment thereof that binds to CT83 of paragraph 14.

18. A vector containing the gene of Article 17.

19. A host cell comprising the gene of paragraph 17 or the vector of paragraph 5.

20. In paragraph 19, A host cell characterized in that the gene or vector is transformed.

21. A method for producing an antibody or an antigen-binding fragment thereof that specifically binds to CT83, comprising a step of culturing the host cell of item 19.

22. A composition for preventing or treating cancer, comprising the antibody of Article 14 or an antigen-binding fragment thereof as an active ingredient.

23. In paragraph 22, The cancers include cervical cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, pancreatic cancer, biliary tract cancer, liver cancer, stomach cancer, rectal cancer, oral cancer, pharynx cancer, larynx cancer, colon cancer, endometrial cancer, ovarian cancer, testis cancer, bladder cancer, renal cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma, and multiple A composition for preventing or treating cancer, characterized in that it is selected from the group consisting of multiple myeloid blood cancer.

24. A kit for detecting a tumor or cancer, comprising an antibody or antigen-binding fragment thereof that binds to CT83 of Article 14.

25. A method for detecting a tumor or cancer, comprising: treating a cancer-related sample with an antibody or antigen-binding fragment thereof that binds to CT83 of claim 14 to form a CT83 antigen-antibody complex; and detecting the CT83 antigen-antibody complex.

26. A method for providing tumor or cancer diagnostic information, comprising: treating a cancer-related sample with an antibody or an antigen-binding fragment thereof that binds to CT83 of claim 14 to form a CT83 antigen-antibody complex; and detecting the CT83 antigen-antibody complex. 27.An antibody or antigen-binding fragment thereof that specifically binds to CT83, A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 25, a heavy chain CDR2 of SEQ ID NO: 26, and a heavy chain CDR3 of SEQ ID NO: 27; and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 28, a light chain CDR2 of SEQ ID NO: 29, and a light chain CDR3 of SEQ ID NO: 30; An antibody or antigen-binding fragment thereof.

28. In paragraph 27, An antibody or antigen-binding fragment thereof, characterized in that the antibody or antigen-binding fragment specifically binds to an epitope of SEQ ID NO: 32 or 33.

29. In paragraph 27, An antibody or antigen-binding fragment thereof, characterized in that the antibody or antigen-binding fragment thereof is scFv.

30. A gene encoding an antibody or antigen-binding fragment thereof that binds to CT83 of item 27.

31. A vector containing the gene of Article 30.

32. A host cell comprising the gene of item 30 or the vector of item 31.

33. In paragraph 32, A host cell characterized in that the gene or vector is transformed.

34. A method for producing an antibody or an antigen-binding fragment thereof that specifically binds to CT83, comprising the step of culturing the host cell of item 32.

35. A composition for preventing or treating cancer, comprising the antibody of Article 27 or an antigen-binding fragment thereof as an active ingredient.

36. In paragraph 35, The cancers include cervical cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, pancreatic cancer, biliary tract cancer, liver cancer, stomach cancer, rectal cancer, oral cancer, pharynx cancer, larynx cancer, colon cancer, endometrial cancer, ovarian cancer, testis cancer, bladder cancer, renal cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's lymphoma, lymphoma, and multiple A composition for preventing or treating cancer, characterized in that it is selected from the group consisting of multiple myeloid blood cancer.

37. A kit for detecting a tumor or cancer, comprising an antibody or antigen-binding fragment thereof that binds to CT83 of Article 27.

38. A method for detecting a tumor or cancer, comprising: treating a cancer-related sample with an antibody or antigen-binding fragment thereof that binds to CT83 of item 27 to form a CT83 antigen-antibody complex; and detecting the CT83 antigen-antibody complex.

39. A method for providing tumor or cancer diagnostic information, comprising: treating a cancer-related sample with an antibody or antigen-binding fragment thereof that binds to CT83 of claim 27 to form a CT83 antigen-antibody complex; and detecting the CT83 antigen-antibody complex.

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