KR102846817B1 - Human monoclonal antibodies against CT83 antigen and their use - Google Patents

Abstract

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.

Description

Human monoclonal antibodies against CT83 antigen and their use

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

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.

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.

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.

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, which can lead to cancer.

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.

Colon cancer, a cancer that occurs in the large intestine 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. Colon 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 colon cancer include constipation or diarrhea, changes in stool shape or color, abdominal pain, weight loss, and fatigue.

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.

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

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.

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.

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

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

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.

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.

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

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.

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.

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.

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.

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

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.

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

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

Additionally, the present invention provides a host cell comprising the vector.

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.

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.

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

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.

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.

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.

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.

The antibody or antigen-binding fragment of the present invention can specifically bind to the CT83 antigen, and the antibody or antigen-binding fragment can be usefully used in fields related to diagnosis and treatment of tumors or cancers related to the CT83 antigen.

Figure 1 shows the ELISA results of 400 anti-CT83 single antibody candidates.
Figure 2 shows the ELISA results of SKAI-5, which was selected as an antibody candidate of the present invention.
Figure 3 shows the microarray results confirming binding to the antigen for epitope mapping.
Figure 4 shows the expression level of KK-LC-1 protein in HeLa cells or HT-29 cells.
Figure 5 shows whether SKAI-5, selected as an antibody candidate of the present invention, specifically binds to HeLa cells or HT-29 cells.
Figure 6a illustrates key information about one of the epitopes of the antibody 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.
Figure 6b illustrates key information about another epitope of the antibody 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.
Figure 7a shows the location of one of the repeat sequences among the epitopes of the antibody of the present invention.
Figure 7b shows the location of a repeat sequence among other epitopes of the antibody of the present invention.

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.

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.

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.

"Phage display" is an experimental method for detecting protein-protein interactions or interactions between proteins and other target substances. By cloning genes into bacteriophages, the expressed proteins are displayed on the phage surface. The presence and extent of interaction can then be measured by binding to other proteins or target substances.

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"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, allowing antibodies to be produced.

"Biopanning" is a technology that selects 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, which can be used for antibody development and pharmaceutical development.

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

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.

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.

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

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.

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.

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

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Through examples, it was confirmed that SKAI-5 is an antibody or antigen-binding fragment that specifically binds to the CT83 antigen, and that a pharmaceutical composition including SKAI-5 can be used for anticancer treatment.

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.

Example 1. Library construction and expression

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

Biopanning to discover antibody candidate sequences used 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 HA tags are linked to nucleotides encoding 7.6 X 10 9 scFvs.

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.

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).

CDR-H3 AE (N-ter)-(RK)-(Xaa)4-F-D-Y-(C-ter) Sequence number 1 BE (N-ter)-(RK)-(DG)-(LPRVAG)-(Xaa)7-F-D-Y-(C-ter) Sequence number 2 CF (N-ter)-(RK)-(FSYCLPHRVADG)-(LPRITSVAG)-(Xaa)7-(YS)-(YS)-(ADSY)-(YND)-(GA)-M-D-V-(C-ter) Sequence number 3 DF (N-ter)-(RK)-(VADG)-(LPRITSVAG)-(LRISVG)-Xaa-C-(Xaa)4-C-(YS)-(YS)(ADSY)-(YND)-(GA)-M-D-V-(C-ter) Sequence number 4

X represents any amino acid

In (X)n, n means that the corresponding amino acid is repeated n times.

The OPAL library was transformed into ER2738 ( E. coli , AMID BIOSCIENCES, cat. No. ER-201), and 20MOI 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).

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

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

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).

protein Sequence number CAT# amino acid sequence Manifestation
cell Tags KK-LC-1(CT83)
(NM_001017978) 5 TP309391 (N-ter)-MNFYLLLASSILCALIVFWKYRRFQRNTGEMSSNSTALALVRPSSSGLINSNTDNNLAVYDLSRDILNNFPHSIARQKRILVNLSMVENKLVELEHTLLSKGFRGASPHRKST-(C-ter) HEK293T (C-ter)-Myc/DDK Recombinant human lung cancer antigen 1
(CT83)-VLPs
(Active) 6 CSB-MP711093HU (N-ter)-MNFYLLLASSILCALIVFWKYRRFQRNTGEMSSNSTALALVRPSSSGLINSNTDNNLAVYDLSRDILNNFPHSIARQKRILVNLSMVENKLVELEHTLLSKGFRGASPHRKST-(C-ter) Breastfeeding
cell (N-ter)- 6xHis-tag

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. Afterwards, the CT83-bound phage was eluted using 100 mM Triethylamine (TEA) and 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 biopanning 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 additionally performed once more using KK-LC-1 (CT83) (NM_001017978) human recombinant protein (CAT#: TP309391, Origene) (Table 2) to discover antibody candidates.

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.

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

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.

NGS analysis was performed on the above 50 selected antibody candidates, and 46 antibody candidates were selected by excluding overlapping antibodies and antibodies with frame shifts. In order to reproduce the scFv of the above 46 antibody candidates using the gene sequences and select the final antibody candidates that can specifically bind to the CT83 protein, Recombinant KK-LC-1/BSA and KK-LC-1 VLPs/BSA were analyzed by ELISA.

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.

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

SKAI-5 VH CDR1 CDR2 CDR3 Sequence number 7 Sequence number 8 Sequence number 9 (N-ter)-NYSMS-(C-ter) (N-ter)-SLSSGGGST-(C-ter) (N-ter)-ARRKTRFDY-(C-ter) VL CDR1 CDR2 CDR3 Sequence number 10 Sequence number 11 Sequence number 12 (N-ter)-SGSSSNIGSNTVN-(C-ter) (N-ter)-ADSQ-(C-ter) (N-ter)-GSWDSSLNG-(C-ter)

Example 4. Identification of an epitope sequence binding to CT83

The final antibody candidates confirmed in Example 3 To manufacture an IgG antibody and clearly identify the amino acid sequence of the site where the antibody binds to the CT83 protein, the following experiments were performed.

4-1. IgG antibody production

The scFv sequence of SKAI-5 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) using the following method.

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.

Vector name Forward primer (5'->3') Reverse primer (5'->3') TGEX-VH Sequence number 13 Sequence number 14 (BSSHll)
NNGCGCGCACTCCGAGGTGCAGCTGTTG (Bsmbl)
NNCGTCTCNATGCTGAGCTCACGGTGAC TGEX-VL Sequence number 15 Sequence number 16 (BspEl)
NNNNNNTCCGGACAGTCTGTGCTGACT (Bsal)
NNGGTCTCNTTCGTAGGACCGTCAGCT

A vector was constructed using the following method so that the above IgG can be expressed in mammalian cells.

The VH primer was designed to have a restriction site for BSSHll at the 5' end and for BsmBl 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.

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.

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

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 cells were mixed with ExpifectamineTM CHO to form a complex. The prepared complex was transfected into the prepared Expi-CHO-s cells (ThermoFisher scientific, A29127) without exceeding 5 minutes of reaction. The next day, ExpiCHOTM Feed and ExpiFectamineTM CHO Enhancer (ExpiFectamineTM 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).

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).

4-2. Epitope mapping

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

Before running the microarray, the SKAI-5 antibody prepared according to Example 4-1 was biotinylated. Thereafter, the biotinylated antibody sample was subjected to microarray chip analysis for approximately 18,000 CT83 antigen peptide positions to confirm binding to the CT83 antigen (Fig. 3).

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.

SKAI-5 Epitope 1 (SEQ ID NO: 17) Epitope 2 (SEQ ID NO: 18) (N-ter)-PHSIARQKR-(C-ter) (N-ter)-FRGASPHRKST-(C-ter)

Example 5. Confirmation of cancer cell-specific binding

To determine whether the SKAI-5 protein specifically binds to the CT83 (KK-LC-1) antigen expressed in cancer cells, a FACS experiment was conducted using the following method.

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.

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).

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

Attach an electronic file of the sequence list

Claims (13)

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.
In the first paragraph,
An antibody or antigen-binding fragment thereof, characterized in that the antibody or antigen-binding fragment thereof specifically binds to an epitope of SEQ ID NO: 17 or 18.
In the first paragraph,
An antibody or antigen-binding fragment thereof, characterized in that the antibody or antigen-binding fragment thereof is scFv.
A gene encoding an antibody or an antigen-binding fragment thereof that binds to CT83 of the first clause.
A vector containing the gene of claim 4.
A host cell comprising the gene of claim 4 or the vector of claim 5.
In paragraph 6,
A host cell characterized in that the gene or vector is transformed.
A method for producing an antibody or antigen-binding fragment thereof that specifically binds to CT83, comprising the step of culturing the host cell of claim 6.
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