WO2025005704A1 - Human monoclonal antibody against antigen ct83 and use thereof - Google Patents

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 to CT83 antigen and uses thereof

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 allows the antibody to recognize and bind to a specific antigen.

CDR is an important factor that determines antibody diversity, and CDR is an important factor in antibody engineering and antibody-based therapeutic development.

HeLa cells are a cervical cancer cell line that was collected from a woman named Henrietta Lacks in 1951 for the treatment of malaria. This cell line has a very strong growth ability and can be artificially propagated over a long period of time, so it is widely used in cell biology and cancer research, and is also widely utilized for purposes such as studying the characteristics of tumor cells and evaluating the effectiveness of anticancer treatment.

Cervical cancer is a cancer that occurs in the cervix of a woman's uterus (the organ that releases the placenta). This type of cancer is mainly related to infection with the human papillomavirus (HPV). HPV is transmitted through sexual intercourse, 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 as one of the colon cancer cell lines. This cell line is derived from human colon cancer tissue and is widely used in colon cancer research and drug testing.

Colon cancer is a cancer that occurs in the colon or rectum, and is one of the digestive system cancers. Cancer usually occurs in the lining of the colon, and as the tumor grows, it can invade and spread to surrounding tissues. Colon cancer is mainly a process in which intestinal polyps evolve into cancer, and can present various symptoms and signs, which can vary depending on the location, size, and progression of the tumor. Common symptoms of colon cancer include constipation or diarrhea, changes in the shape or color of stool, abdominal pain, weight loss, and fatigue.

Accordingly, the inventors of the present invention 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 bio-panning.

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

Provided is 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 comprising 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, comprising the 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, it is an object of the present invention 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 relates to 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 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 comprising a pharmaceutical composition for preventing or treating cancer, 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 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.

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

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

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

Figure 2c shows the ELISA results of SKAI-48, 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 5a shows whether SKAI-5, selected as an antibody candidate of the present invention, specifically binds to HeLa cells or HT-29 cells.

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

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

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

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

Figure 6c shows key information of one of the epitopes of the antibody (SKAI-33) of the present invention. The upper part of Figure 6c shows information on 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 amino acids constituting the epitope sequence.

Fig. 6d shows key information of one of the epitopes of the antibody (SKAI-33) of the present invention. The upper part of Fig. 6d shows information on the sequence, length, mass, isoelectric point, charge, hydrophobicity, and extinction coefficient of the epitope, the middle part of Fig. 6d shows the structure of the epitope, and the lower part of Fig. 6d shows the sequence, sequence number, length, and hydrophobicity index (hydropathy_index) for amino acids constituting the epitope sequence.

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

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

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

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

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

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

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

Hereinafter, the present invention will be described in detail with reference to the attached drawings as embodiments of the present invention. However, the following embodiments are presented as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description thereof 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.

In addition, the terminology used in this specification is a term used to appropriately express the preferred embodiment of the present invention, and this may vary depending on the intention of the user or operator, or the custom of the field to which the present invention belongs. Therefore, the definition of these terms should be determined based on the contents throughout this specification. Throughout the specification, when a certain part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components can be included, unless specifically stated otherwise.

Those skilled in the art will understand that the present invention can be implemented in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated by the claims, not the foregoing description, and all differences within the scope equivalent thereto should be interpreted as being included in the present invention.

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

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

Panning is a method of selecting antibodies or antigen-binding fragments that bind to a specific antigen. Using a phage library, various antibody fragments are expressed on phages, and then specific binding to the antigen is confirmed. By confirming the genetic information encoded by the phages that have specific binding, antibodies can be produced.

The "bio-panning" of the present invention is a technology for selecting peptides that bind to a specific target substance based on affinity, and through this, it is known that the sequence of a peptide that binds to a specific target can be identified and utilized 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 an 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 mean a single-chain fragment antibody and may mean a linkage of variable regions of light and heavy chains of 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 within a cell, the antibody or an 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 for achieving the above technical task, the present invention provides a host cell comprising the vector. At this time, the method for 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 effective ingredient.

As one aspect for 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 an animal that has developed 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 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 embodiment 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 CT83.

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

As one embodiment 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 diagnosis 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, 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 utilize them as ADCs, or chimeric antigen receptor T-cells (CAR-Ts) can be produced to utilize them for anticancer treatment.

Hereinafter, the present invention will be described in more detail through the following examples. However, the examples are only examples for easily explaining the content and scope of the technical idea of the present invention, and the technical scope of the present invention is not limited or changed thereby. In addition, it can be determined by those skilled in the art that various modifications and changes are possible within the scope of the technical idea of the present invention 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 was performed using 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 library in the form of HA tags linked to nucleotides encoding 7.6 X 10 9 scFvs.

The antibody expressed by the 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 CDR sequences derived from humans.

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

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 (X)n, where n represents n repetitions of that amino acid

ER2738 ( E. coli , AMID BIOSCIENCES, cat. No. ER-201) was transformed with the OPAL library, and 20MOI of helper phage (VCSM13) was added to 2xYT medium (BD DifcoTM, 2xYT powder) containing 50 ug/ml carbenicillin and 70 ug/ml kanamycin, and cultured at 30°C for 14 h. 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 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 Expression
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 bio-panning 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 non-specifically bind to the antigen could be removed. Afterwards, to filter out substances that non-specifically 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 were randomly selected from independently formed colonies cultured after the above four rounds of panning (Fig. 1). ELISA analysis was performed on each candidate expressed in the form of scFv, and 50 antibody candidates that specifically bind to KK-LC-1 (CT-83) versus BSA and KK-LC-1 VLPs versus BSA were selected.

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

For the 50 selected candidates that bind to KK-LC-1 (CT83) and KK-LC-1 VLPs, but not to BSA and VLP-(negative), the gene sequences of each of the 50 candidates were analyzed through next generation sequencing (NGS) genetic analysis.

NGS analysis was performed on the above 50 selected antibody candidates, excluding overlapping antibodies and antibodies with frame shifts, to select 46 antibody candidates, and the scFv of the above 46 antibody candidates was reproduced using the gene sequence, and the final antibody candidates that can specifically bind to the CT83 protein were selected. 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 Fig. 2a. TMB (3,3',5,5' - tetramethylbenzidine) was added, and 20 minutes later, the absorbance at 450 nm was measured using a plate reader.

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

The ELISA results of SKAI-48 are as shown in Fig. 2c. TMB (3,3',5,5' - tetramethylbenzidine) was added, and 20 minutes later, the absorbance at 450 nm was measured 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) SKAI-33 VH CDR1 CDR2 CDR3 Sequence number 19 Sequence number 20 Sequence number 21 (N-ter)-SYSMS-(C-ter) (N-ter)-AISSSGGNK-(C-ter) (N-ter)-ARRKKLFDY-(C-ter) VL CDR1 CDR2 CDR3 Sequence number 22 Sequence number 23 Sequence number 24 (N-ter)-SGSSSNIGTNDVT-(C-ter) (N-ter)-ANNQ-(C-ter) (N-ter)-GAWDASLNG-(C-ter) SKAI-48 VH CDR1 CDR2 CDR3 Sequence number 25 Sequence number 26 Sequence number 27 (N-ter)-DYYMS-(C-ter) (N-ter)-AISYDSSST-(C-ter) (N-ter)-ARKKKKFDY-(C-ter) VL CDR1 CDR2 CDR3 Sequence number 28 Sequence number 29 Sequence number 30 (N-ter)-SGSSSNIGSNNVT-(C-ter) (N-ter)-ANSN-(C-ter) (N-ter)-GTWDASLSG-(C-ter)

Example 4. Identification of epitope sequences 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. Manufacturing of IgG antibodies

The scFv sequences of SKAI-5, SKAI-33, or SKAI-48 that bind to CT83 were inserted into the human IgG vector system (TGEX-HC, Antibody design labs, MX001, and TGEX-LC, Antibody design labs, MX002) by the following method.

Specifically, a gene cloning vector was constructed so that IgG can be produced 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 in the following manner 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.

Cloned heavy and light chain human IgG plasmids were co-transfected into Expi-CHO-s cells as follows.

6 x 10 ⁶ Expi-CHO-s cells were prepared and 15 ug 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, they were mixed with ExpifectamineTM CHO to form a complex. The formed 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) that increase nutrient supply and protein production efficiency were added. 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 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 using microarray was performed to identify the antigen binding site (epitope) of the selected antibodies against the target antigen (CT83).

Before running the microarray, the SKAI-5, SKAI-33, or SKAI-48 antibodies manufactured by 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).

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

The sequence, sequence number, length, mass, isoelectric point, charge, hydrophobicity, and extinction coefficient information 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).

The sequence, sequence number, length, mass, isoelectric point, charge, hydrophobicity, and extinction coefficient information 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 confirmed 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) SKAI-33 Epitope 1 (SEQ ID NO: 31) (N-ter)-NNLAVYDLLSRDILNNFPHSIARQKR-(C-ter) SKAI-48 Epitope 1 (SEQ ID NO: 32) Epitope 2 (SEQ ID NO: 33) (N-ter)- LVELEHT-(C-ter) (N-ter)-FRGASPHRKST-(C-ter)

Example 5. Confirmation of cancer cell-specific binding

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.

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. Through 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 collected by trypsinization, washed with PBS, and centrifuged to precipitate the cells twice. The cells were prepared by diluting 1/100 with mouse monoclonal antibody against CT83 antigen (Invitrogen, Catalog # MA5-24711) and incubating for 1 hour at room temperature. After 1 hour, the secondary antibody was diluted 1/100 and incubated for 1 hour at room temperature, and analyzed by 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. 5a)

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-33 using FACS, we confirmed that SKAI-33 specifically binds to the CT83 (KK-LC-1) antigen. (Fig. 5b)

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-48 through FACS, we confirmed that SKAI-48 specifically binds to the CT83 (KK-LC-1) antigen. (Fig. 5c)

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

Claims (39)

An antibody or an 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 an antigen-binding fragment thereof. In the first paragraph, 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. 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 Article 6, A host cell characterized in that the gene or vector is transformed. 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 claim 6. A composition for preventing or treating cancer, comprising the antibody of claim 1 or an antigen-binding fragment thereof as an active ingredient. In Article 9, The above 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. A kit for detecting a tumor or cancer, comprising an antibody or an antigen-binding fragment thereof that binds to CT83 of claim 1. 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. 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 claim 1 to form a CT83 antigen-antibody complex; and a step of detecting the CT83 antigen-antibody complex. An antibody or an 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 an antigen-binding fragment thereof. In Article 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. In Article 14, 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 clause 14. A vector containing the gene of claim 17. A host cell comprising the gene of claim 17 or the vector of claim 5. In Article 19, A host cell characterized in that the gene or vector is transformed. A method for producing an antibody or an antigen-binding fragment thereof that specifically binds to CT83, comprising the step of culturing a host cell of claim 19. A composition for preventing or treating cancer, comprising the antibody of claim 14 or an antigen-binding fragment thereof as an active ingredient. In Article 22, The above 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. A kit for detecting a tumor or cancer, comprising an antibody or an antigen-binding fragment thereof that binds to CT83 of claim 14. A method for detecting a tumor or cancer, 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. 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 claim 14 to form a CT83 antigen-antibody complex; and a step of detecting the CT83 antigen-antibody complex. An antibody or an 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 an antigen-binding fragment thereof. In Article 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. In Article 27, 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 clause 27. A vector containing the gene of claim 30. A host cell comprising the gene of claim 30 or the vector of claim 31. In Article 32, A host cell characterized in that the gene or vector is transformed. A method for producing an antibody or an antigen-binding fragment thereof that specifically binds to CT83, comprising the step of culturing a host cell of claim 32. A composition for preventing or treating cancer, comprising the antibody of claim 27 or an antigen-binding fragment thereof as an active ingredient. In Article 35, The above 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. A kit for detecting a tumor or cancer, comprising an antibody or an antigen-binding fragment thereof that binds to CT83 of claim 27. A method for detecting a tumor or cancer, comprising: treating a cancer-related sample with an antibody or an 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. 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 27 to form a CT83 antigen-antibody complex; and detecting the CT83 antigen-antibody complex.

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