Viral vector-derived target proteins for anti-cancer therapy and binding molecules or fragments thereof that specifically bind to them.

Abstract

The present invention relates to a target protein for anti-cancer therapy based on a viral vector, and a binding molecule or fragment thereof that specifically binds to the target protein. More specifically, the present invention relates to a structural epitope of protein A56, and a binding molecule or fragment thereof that specifically binds to the structural epitope. The A56 binding molecule or fragment thereof that binds to the structural epitope of A56 according to the present invention forms a specific structural bond with A56 and shows high affinity for A56. The present inventors found that A56 is expressed on the surface of cancer cells in various carcinomas when an oncolytic virus based on vaccinia virus is administered. The A56 binding molecule or fragment thereof according to the present invention effectively targets A56 specifically expressed on the surface of cancer cells, enabling targeted therapy for cancer cells that survive even infection with an oncolytic virus, thereby providing an effective anti-cancer therapy.

Description

Technical Field

[0001] The present invention relates to a target protein for anti-cancer treatment based on a viral vector, and a binding molecule or a fragment thereof that specifically binds to the target protein. More specifically, the present invention relates to a structural epitope of protein A56, and a binding molecule or a fragment thereof that specifically binds to the structural epitope.

Background Art

[0002] Cancer, also called a tumor, refers to cells that have abnormally grown due to the autonomous overgrowth of body tissues. The cancer incidence rate continues to increase in modern society due to the aging population, the increase in the smoking population, the increase in alcohol consumption, westernized eating habits, and environmental pollution.

[0003] Methods for treating cancer include surgery, radiotherapy, chemotherapy, etc. Specifically, surgery is a treatment method for removing cancerous tissue from the body and is very effective for early-stage cancer or cancer limited to a specific location with a lesion. However, it is difficult to remove cancer that has invaded the surrounding tissues or metastasized to lymph nodes, and such cancer has a high possibility of recurrence. In this case, radiotherapy or chemotherapy is used in combination with surgery. Radiotherapy or chemotherapy is mainly used for the treatment of progressive or terminal cancer; however, this treatment also affects normal cells and therefore causes serious adverse effects.

[0004] [[ID=十九]] On the other hand, clinical studies using oncolytic viruses with increased tumor selectivity and anti-cancer efficacy have been initiated with the full use of genetic recombination technology. The first recombinant oncolytic virus reported in the literature was the herpes simplex virus. Since then, research on tumor lysis using other viruses has been actively conducted

[0005] Among these, research has been conducted on thymidine kinase (TK) gene - deficient vaccinia virus obtained by using vaccinia virus; however, despite its clinical utility, the virus has limitations in maximizing its clinical effects due to its narrow therapeutic concentration range. For TK - deficient vaccinia virus, a narrow therapeutic concentration range means that a high virus dose has high clinical efficacy but may be accompanied by a clinical risk due to virus toxicity. To overcome this problem, studies have been carried out in such viruses to cause an increase in therapeutic effect and a decrease in toxicity by deleting, along with the deletion of the TK gene, the gene region encoding non - essential proteins such as protein A56 (Izmailyan R, Chang W Journal of virology 2008 Oct;82(20):10079~87).

[0006] Recently, immune cell therapy has been actively studied as a cancer treatment method. Immune cell therapy is different from existing treatment methods in that it uses the patient's immune cells to kill cancer cells. Specifically, immune cell therapy is a method in which immune cells are obtained from a patient, activated to specifically attack proliferative cells or cancer cells, and then returned to the patient's body; and this method maximizes the anti - cancer effect while minimizing drug - induced adverse effects.

[0007] Over the past five years, immune checkpoint inhibitors have been actively studied as an immunotherapy for treating cancer. In particular, inhibitors against immune checkpoints such as cytotoxic T - lymphocyte - associated protein 4 (CTLA - 4), programmed cell death protein 1 (PD - 1), and PD - L1 have been studied. Immune checkpoint inhibitors such as ipilimumab (anti - CTLA - 4), nivolumab (anti - PD - 1), and pembrolizumab (anti - PD - 1) have been approved by regulatory authorities for the treatment of some types of cancer. However, immune checkpoint inhibitors are used limitedly as a treatment for patients with certain types of cancer such as melanoma, lung cancer, head and neck cancer, kidney cancer, and bladder cancer.

[0008] In addition, chimeric antigen receptor T cells (CAR-T cells) have shown excellent therapeutic effects against various types of hematological malignancies. However, there have been very few reported cases of clinical success using CAR-T cells for solid tumors. Reasons for this include difficulties in the transport and invasion of CAR-T cells into tumors, problems with the persistence and proliferation of CAR-T cells, and the limitations of CAR-T cells in that their therapeutic effects are not exerted due to the unsuitable tumor microenvironment. However, the biggest limitation is the serious safety problem that arises due to the lack of appropriate target antigens that are specifically expressed in tumors. In fact, most of the target antigens used in the clinical development of CAR-T cells for solid tumors are overexpressed in tumors and also expressed at low levels in normal cells. For this reason, there have been reports of CAR-T cells binding to normal cells as well, causing serious toxicity problems. To overcome this on-target-off-tumor toxicity problem, target antigens that meet the conditions of tumor specificity, high and uniform applicability, and expression stability are needed. However, there are currently very few solid tumor target antigens that meet all three conditions. Therefore, there is a need for continued research and development of cancer cell targeting methods that have the ability to target solid tumors in a safe and effective manner. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Izmailyan R, Chang W Journal of virology 2008 Oct;82(20):10079~87 [Overview of the project] [Problems that the invention aims to solve]

[0010] Therefore, the inventors have been studying to develop a method to safely and effectively target or treat cancer, particularly solid tumors. As a result, the inventors have found that when cancer cells are treated with a vector containing nucleic acids encoding vaccinia virus protein A56 or a fragment thereof for various carcinomas, protein A56 is expressed on the surface of cancer cells in a tumor-specific and stable manner. In addition, the inventors have analyzed the binding of several antibodies that bind to protein A56 or a fragment thereof to A56. As a result, the inventors have found that the antibodies cross-inhibit the binding to A56, even though their epitope sequences are not completely identical and are unique. Subsequently, the inventors have identified structural epitopes that determine binding to A56 and have found that binding molecules that bind to such structural epitopes are highly effective in reducing the burden on cancer cells, thereby completing the present invention. [Means for solving the problem]

[0011] In an embodiment of the present invention, a binding molecule or fragment thereof is provided that specifically binds to a structural epitope or fragment thereof of A56, wherein the structural epitope comprises a basic or nucleophilic amino acid located in the region from amino acid 60 to amino acid 63 in the amino acid sequence of protein A56 represented by Sequence ID No. 1, and further comprises (i) a nucleophilic amino acid located in the region from amino acid 44 to amino acid 50 in the amino acid sequence of protein A56, (ii) a nucleophilic amino acid located in the region from amino acid 53 to amino acid 59 in the amino acid sequence of protein A56, (iii) a nucleophilic amino acid located in the region from amino acid 85 to amino acid 90 in the amino acid sequence of protein A56, or (iv) a basic or nucleophilic amino acid located in the region from amino acid 91 to amino acid 94 in the amino acid sequence of protein A56.

[0012] In another embodiment of the present invention, a method is provided for reducing the burden on cancer cells in a subject, comprising the step of administering a binding molecule or a fragment thereof to the subject.

[0013] In yet another embodiment of the present invention, a pharmaceutical composition for preventing or treating cancer is provided, comprising a binding molecule or a fragment thereof.

[0014] In yet another embodiment of the present invention, a kit for preventing or treating cancer is provided, comprising a binding molecule or a fragment thereof and an oncolytic virus.

[0015] In yet another embodiment of the present invention, the use of a binding molecule or fragment thereof for the treatment of cancer is provided. [Effects of the Invention]

[0016] The A56-binding molecule or fragment thereof, which binds to the structural epitope of A56 according to the present invention, forms a specific structural bond with A56 and exhibits high affinity for A56. The inventors have found that when oncolytic viruses based on vaccinia virus are administered, A56 is expressed on the surface of cancer cells in various cancers. The A56-binding molecule or fragment thereof according to the present invention effectively targets A56 specifically expressed on the surface of cancer cells, enabling targeted therapy for cancer cells that have survived even infection with oncolytic viruses, thereby providing effective anti-cancer therapy. For example, the A56-binding molecule according to the present invention itself enables effective targeting of cancer cells that have survived even infection with oncolytic viruses. In addition, when used in cancer immunotherapy such as CAR-T cell therapy, the A56-binding molecule specifically shows increased activation and proliferative capacity against cancer cells expressing A56 and exerts excellent cytotoxic effects on those cancer cells. Therefore, the A56-binding molecule can provide effective anti-cancer therapy against cancer cells expressing A56. Cancer immunotherapy using the A56-binding molecule according to the present invention is preferably used in combination with an oncolytic virus, and may additionally be used in combination with a drug capable of enhancing the anticancer effect of the oncolytic virus (e.g., hydroxyurea, chemotherapeutic agents for controlling lymphocyte removal (e.g., cyclophosphamide and fludarabine), or immunotherapeutic agents). Where used herein, unless otherwise indicated, the expression “in combination with” includes both administration of the therapeutic agent / active substance in question carried out at time intervals in any order, and administration of the therapeutic agent / active substance carried out simultaneously. [Brief explanation of the drawing]

[0017] [Figure 1] This figure shows the results obtained by infecting human lung cancer cell lines (A549), human colorectal cancer cell lines (HCT-116), or human melanoma cell lines (SK-MEL-5) with an oncolytic virus containing nucleic acid encoding protein A56, and then identifying the presence or absence of protein A56 expression on the cell surface of each cell line by immunofluorescence staining. [Figure 2]This figure shows the results obtained by infecting a human colorectal cancer cell line (HCT-116) with an oncolytic virus containing nucleic acid encoding protein A56, and then identifying the presence or absence of protein A56 expression on the cell surface of the infected HCT-116 cell line by immunofluorescence staining. [Figure 3] This figure shows the results obtained by subjecting mice transplanted with the human colorectal cancer cell line (HCT-116) to intraperitoneal administration of an oncolytic virus containing nucleic acid encoding protein A56, and then identifying the expression of protein A56 on the surface of each tissue. [Figure 4] This figure shows the results obtained by intravascular administration of an oncolytic virus containing nucleic acid encoding protein A56 to normal rabbits, followed by identification of protein A56 expression on the surface of various tissues. [Figure 5] This figure shows the results obtained by administering an oncolytic virus containing nucleic acid encoding protein A56 to mice transplanted with the mouse renal cell carcinoma cell line (Renca) via intratumoral administration, and then identifying the expression of protein A56 on the tumor tissue surface on days 7, 10, and 14. [Figure 6] This figure shows the results obtained by administering intratumoral administration of a hydroxyurea and an oncolytic virus containing nucleic acid encoding protein A56 to mice transplanted with the mouse renal cell carcinoma cell line (Renca), and then identifying the expression of protein A56 on the tumor tissue surface on days 7, 10, and 14. [Figure 7] This figure shows the results obtained by administering a second dose of oncolytic virus containing hydroxyurea and nucleic acid encoding protein A56 to mice transplanted with the mouse renal cell carcinoma cell line (Renca), and then identifying the expression of protein A56 on the tumor tissue surface on days 21, 24, and 28. [Figure 8] This figure shows embodiments of protein A56 and its fragments. [Figure 9]This figure shows the results obtained by identifying whether protein A56 or its fragments are expressed intracellularly and on the cell surface. [Figure 10] This figure shows the results obtained by identifying the expression level of the Fc-fusion A56 fusion protein. [Figure 11] This figure shows the results obtained by measuring the affinity between the anti-A56 antibody prepared according to the embodiment of the present invention and A56. [Figure 12] This figure shows the results obtained by measuring the affinity between the anti-A56 antibody prepared according to the embodiment of the present invention and A56. [Figure 13] This figure shows the results obtained by measuring the affinity between the anti-A56 antibody prepared according to the embodiment of the present invention and A56. [Figure 14] This figure shows the results obtained by measuring the affinity between the anti-A56 antibody prepared according to the embodiment of the present invention and A56. [Figure 15] This figure shows the results obtained by measuring the affinity between the anti-A56 antibody prepared according to the embodiment of the present invention and A56. [Figure 16] This figure shows the results obtained by measuring the affinity between the anti-A56 antibody prepared according to the embodiment of the present invention and A56. [Figure 17A] This figure shows the results obtained by identifying the productivity of antibodies A56-01A02 to A56-02B06. [Figure 17B] This graph shows the productivity of antibodies A56-01A02 to A56-02B06. [Figure 17C] This figure shows the results obtained by identifying the productivity of antibodies A56-02D04 to A56-59E12. [Figure 18] This figure shows the results obtained by purifying the anti-A56 antibodies prepared in the embodiments of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 19]This figure shows the results obtained by purifying the anti-A56 antibodies prepared in the embodiments of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 20] This figure shows the results obtained by purifying the anti-A56 antibodies prepared in the embodiments of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 21] This figure shows the results obtained by purifying the anti-A56 antibodies prepared in the embodiments of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 22] This figure shows the results obtained by purifying the anti-A56 antibodies prepared in the embodiments of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 23] This figure shows the results obtained by purifying the anti-A56 antibodies prepared in the embodiments of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 24] This figure shows the results obtained by purifying the anti-A56 antibodies prepared in the embodiments of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 25] This figure shows the results obtained by purifying the anti-A56 antibodies prepared in the embodiments of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 26] This figure shows the results obtained by purifying the anti-A56 antibodies prepared in the embodiments of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 27] This figure shows the results obtained by purifying the anti-A56 antibodies prepared in the embodiments of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 28] This figure shows the results obtained by purifying the anti-A56 antibodies prepared in the embodiments of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 29]This figure shows the results obtained by purifying the anti-A56 antibodies prepared in the embodiments of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 30] This figure shows the results obtained by purifying the anti-A56 antibodies prepared in the embodiments of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 31] This figure shows the results obtained by purifying the anti-A56 antibodies prepared in the embodiments of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 32] This figure shows the results obtained by purifying the anti-A56 antibodies prepared in the embodiments of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 33] This figure shows the results obtained by purifying the anti-A56 antibodies prepared in the embodiments of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 34] This figure shows the results obtained by purifying the anti-A56 antibodies prepared in the embodiments of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 35] This figure shows the results obtained by comparing the amino acid sequence homology of protein A56, which is dependent on the vaccinia virus strain. [Figure 36] This figure shows the results obtained by measuring the binding ability of 10 anti-A56 antibodies (Ab18, Ab19, Ab01, Ab13, Ab14, Ab08, Ab03, Ab51, Ab55, Ab16) to protein A56. [Figure 37] This figure shows a photograph taken after reacting protein A56 with a commercially available anti-A56 antibody and then performing Western blotting. The antibody was used as a control to determine whether the anti-A56 antibody possesses a structural epitope. [Figure 38]This figure shows photographs taken after reacting protein A56 with each of 10 anti-A56 antibodies (Ab18, Ab19, Ab01, Ab13, Ab14, Ab08, Ab03, Ab51, Ab55, Ab16), followed by Western blotting. [Figure 39] This figure shows the results obtained by analyzing the antigen-antibody conjugate (A56-C-His / Ab13) using high-mass MALDI. [Figure 40] This figure shows the results obtained by analyzing the antigen-antibody conjugate (A56-C-His / Ab16) using high-mass MALDI. [Figure 41] This figure shows the results obtained by analyzing the antigen-antibody conjugate (A56-C-His / Ab18) using high-mass MALDI. [Figure 42] This figure shows the results obtained by analyzing the antigen-antibody conjugate (A56-C-His / Ab01) using high-mass MALDI. [Figure 43] This figure shows the results obtained by analyzing the antigen-antibody conjugate (A56-C-His / Ab19) using high-mass MALDI. [Figure 44] This figure shows the results obtained by treating protein A56 with five proteolytic enzymes (trypsin, chymotrypsin, ASP-N, elastase, and thermolysin) and then performing LTQ-Orbitrap MS (mass spectrometry) analysis. [Figure 45] This figure shows the epitope site on A56-C-His to which the anti-A56 antibody (Ab13) binds, and a model structure of the protein A56-C-His. The amino acid numbering in A56 shown here is based on the sequence obtained by removing N-terminal amino acids 1-16 from the amino acid sequence represented by SEQ ID NO: 1 (for example, the serine at position 30 corresponds to the serine at position 46 in the amino acid sequence represented by SEQ ID NO: 1). [Figure 46]This figure shows the epitope site on A56-C-His to which the anti-A56 antibody (Ab16) binds, and a model structure of the protein A56-C-His. The amino acid numbering in A56 shown here is based on the sequence obtained by removing N-terminal amino acids 1-16 from the amino acid sequence represented by SEQ ID NO: 1 (for example, the serine at position 30 corresponds to the serine at position 46 in the amino acid sequence represented by SEQ ID NO: 1). [Figure 47] This figure shows the epitope site on A56-C-His to which the anti-A56 antibody (Ab18) binds, and a model structure of the protein A56-C-His. The amino acid numbering in A56 shown here is based on the sequence obtained by removing N-terminal amino acids 1-16 from the amino acid sequence represented by SEQ ID NO: 1 (for example, the serine at position 30 corresponds to the serine at position 46 in the amino acid sequence represented by SEQ ID NO: 1). [Figure 48] This figure shows the epitope site on A56-C-His to which the anti-A56 antibody (Ab01) binds, and a model structure of the protein A56-C-His. The amino acid numbering in A56 shown here is based on the sequence obtained by removing N-terminal amino acids 1-16 from the amino acid sequence represented by SEQ ID NO: 1 (for example, the serine at position 30 corresponds to the serine at position 46 in the amino acid sequence represented by SEQ ID NO: 1). [Figure 49] This figure shows the epitope site on A56-C-His to which the anti-A56 antibody (Ab19) binds, and a model structure of the protein A56-C-His. The amino acid numbering in A56 shown here is based on the sequence obtained by removing N-terminal amino acids 1-16 from the amino acid sequence represented by SEQ ID NO: 1 (for example, the serine at position 40 corresponds to the serine at position 56 in the amino acid sequence represented by SEQ ID NO: 1). [Figure 50]This figure was obtained by summarizing the results of epitope mapping of five antibodies (Ab13, Ab16, Ab18, Ab01, Ab19) to protein A56. The amino acid numbering in A56 shown here is based on the sequence obtained by removing N-terminal amino acids 1-16 from the amino acid sequence represented by SEQ ID NO: 1 (for example, the serine at position 30 corresponds to the serine at position 46 in the amino acid sequence represented by SEQ ID NO: 1). [Figure 51] This figure was obtained by modeling protein A56 into a 3D protein structure and then analyzing that structure using iCn3D. [Figure 52] This figure shows the paratope position on the anti-A56 antibody (Ab13) and the model structure formed by the binding of that antibody to the protein A56-C-His. [Figure 53] This figure shows the paratope position on the anti-A56 antibody (Ab16) and the model structure formed by the binding of that antibody to the protein A56-C-His. [Figure 54] This figure shows the paratope position on the anti-A56 antibody (Ab18) and the model structure formed by the binding of that antibody to the protein A56-C-His. [Figure 55] This figure shows the paratope position on the anti-A56 antibody (Ab01) and the model structure formed by the binding of that antibody to the protein A56-C-His. [Figure 56] This figure shows the paratope position on the anti-A56 antibody (Ab19) and the model structure formed by the binding of that antibody to the protein A56-C-His. [Figure 57A] This figure shows the formation of hydrogen bonds between K91 in protein A56 and T52 in the heavy chain of Ab13, and the measured bond distance. [Figure 57B] This figure shows the formation of a hydrogen bond between S62 in protein A56 and K57 in the heavy chain of Ab13, and the measured bond distance. [Figure 58A]This figure shows the results obtained by binding primary antibodies (SA2038, Ab13, A56-02A02) to an A56-C-His antigen-immobilized biosensor (NTA) to a saturated state, treating it with nine antibodies, and then analyzing whether additional binding of such antibodies to the biosensor occurred using surface plasmon resonance (SPR). [Figure 58B] This figure shows the results obtained by measuring the binding affinity (KD) between proteins A56 and Ab13 using OCTET(SPR) and ELISA. [Figure 59] This figure shows the binding between proteins A56 and Ab16 and the measured distance between them. [Figure 60A] This figure shows the results obtained by binding primary antibodies (SA2041, Ab16, A56-02B08) to an A56-C-His antigen-immobilized biosensor (NTA) to a saturated state, treating it with nine antibodies, and then analyzing whether additional binding of such antibodies to the biosensor occurred using the SPR method. [Figure 60B] This figure shows the results obtained by measuring the binding affinity (KD) between proteins A56 and Ab16 using OCTET(SPR) and ELISA. [Figure 61A] This figure shows the formation of a hydrogen bond between S62 in protein A56 and R98 in the heavy chain of Ab18, and the measured bond distance. [Figure 61B] This figure shows the formation of a hydrogen bond between K91 in protein A56 and Y32 in the light chain of Ab18, and the measured bond distance. [Figure 62A] This figure shows the results obtained by binding primary antibodies (SA2043, Ab18, A56-02C06) to an A56-C-His antigen-immobilized biosensor (NTA) to a saturated state, treating it with nine antibodies, and then analyzing whether additional binding of such antibodies to the biosensor occurred using the SPR method. [Figure 62B]This figure shows the results obtained by measuring the binding affinity (KD) between proteins A56 and Ab18 using OCTET(SPR) and ELISA. [Figure 63] This figure shows the binding between proteins A56 and Ab01, and the measured distance between them. [Figure 64A] This figure shows the results obtained by binding primary antibodies (SA2026, Ab01, A56-01A02) to an A56-C-His antigen-immobilized biosensor (NTA) to a saturated state, treating it with nine antibodies, and then analyzing whether additional binding of such antibodies to the biosensor occurred using the SPR method. [Figure 64B] This figure shows the results obtained by measuring the binding affinity (KD) between protein A56 and Ab01 using OCTET(SPR) and ELISA. [Figure 65] This figure shows the binding between proteins A56 and Ab19, and the measured distance between them. [Figure 66A] This figure shows the results obtained by binding primary antibodies (SA2044, Ab19, A56-02C07) to an A56-C-His antigen-immobilized biosensor (NTA) to a saturated state, treating it with nine antibodies, and then analyzing whether additional binding of such antibodies to the biosensor occurred using the SPR method. [Figure 66B] This figure shows the results obtained by measuring the binding affinity (KD) between proteins A56 and Ab19 using OCTET(SPR) and ELISA. [Figure 67] This is a schematic diagram of the structure of a chimeric antigen receptor constructed in an embodiment of the present invention. [Figure 68] This figure shows the results obtained by comparing the cytotoxicity observed when HeLa cell lines were administered either alone or in combination with five types of CAR-T cells (OTS-412). [Figure 69]This figure shows the results obtained by comparing the cytotoxicity observed when the NCI-H522 cell line was administered with five types of CAR-T cells, either alone or in combination with an oncolytic virus (OTS-412). [Figure 70] This figure shows the results obtained by comparing the cytotoxicity observed when the HCT-116 cell line was administered alone or in combination with five types of CAR-T cells (OTS-412). [Figure 71] This figure shows the results obtained by measuring the transduction efficiency of five types of CAR-T cells using flow cytometry (FACS). [Figure 72] This figure shows the results of cytotoxicity observed when the A549 cell line or HCT-116 cell line was administered with UTD, Ab16 CAR-T cells, or Ab18 CAR-T cells, either alone or in combination with an oncolytic virus (OTS-412). [Figure 73] This figure shows a photograph taken after administering UTD and five types of CAR-T cells (Ab13 CAR-T, Ab16 CAR-T, Ab18 CAR-T, Ab01 CAR-T, Ab19 CAR-T) to an HCT-116 cell line infected with the oncolytic virus (OTS-412), followed by staining of dead cells. [Figure 74] This figure shows a photograph taken after administering UTD and five types of CAR-T cells (Ab13 CAR-T, Ab16 CAR-T, Ab18 CAR-T, Ab01 CAR-T, Ab19 CAR-T) to an HCT-116 cell line infected with the oncolytic virus (WOTS-418), followed by staining of dead cells. [Modes for carrying out the invention]

[0018] The present invention is described in detail below.

[0019] In an embodiment of the present invention, a binding molecule or fragment thereof is provided that specifically binds to a structural epitope or fragment thereof of A56, wherein the structural epitope comprises a basic or nucleophilic amino acid located in the region from amino acid 60 to amino acid 63 in the amino acid sequence of protein A56 represented by Sequence ID No. 1, and further comprises (i) a nucleophilic amino acid located in the region from amino acid 44 to amino acid 50 in the amino acid sequence of protein A56, (ii) a nucleophilic amino acid located in the region from amino acid 53 to amino acid 59 in the amino acid sequence of protein A56, (iii) a nucleophilic amino acid located in the region from amino acid 85 to amino acid 90 in the amino acid sequence of protein A56, or (iv) a basic or nucleophilic amino acid located in the region from amino acid 91 to amino acid 94 in the amino acid sequence of protein A56.

[0020] As used herein, the term "A56" refers to a protein translated from the gene represented by A56, A56R, or HA (e.g., gene ID: 3707652) encoded in the gene of a poxviridae virus after the virus has infected a host cell. Protein A56 or a fragment thereof may contain any one amino acid sequence selected from SEQ ID NOs: 1 to 15. The nucleic acid encoding protein A56 or a fragment thereof may contain any one nucleotide sequence selected from SEQ ID NOs: 16 to 31. In the present invention, protein A56 may be used interchangeably with "UTTA".

[0021] More specifically, protein A56 may be wild-type protein A56 or a variant thereof. Wild-type protein A56 may have the amino acid sequence represented by SEQ ID NO: 1 or 12. In addition, the nucleic acid encoding wild-type protein A56 may be the nucleotide sequence represented by SEQ ID NO: 16 or 28.

[0022] In addition, protein A56 variants may have one or more amino acids substituted, deleted, or added, as long as they can be located on the surface of cancer cells like protein A56. The nucleotide sequence encoding a protein A56 variant may be a nucleotide sequence encoding an amino acid sequence having at least 60%, at least 70%, at least 80%, or at least 90% sequence homology with the amino acid sequence represented by SEQ ID NO: 1 or 15, and most preferably a nucleotide sequence encoding an amino acid sequence having at least 95% sequence homology with the amino acid sequence represented by SEQ ID NO: 1 or 15. In particular, protein A56 variants may include the amino acid sequence represented by SEQ ID NO: 13 or 15. The nucleic acid encoding a protein A56 variant may include the nucleotide sequence represented by SEQ ID NO: 29 or 31.

[0023] The protein A56 fragment may be a polypeptide containing any one amino acid sequence selected from SEQ ID NOs: 2-14. In addition, the nucleic acid encoding the fragment may be a nucleotide sequence encoding a polypeptide containing any one amino acid sequence selected from SEQ ID NOs: 2-14. More specifically, the nucleotide sequences encoding a polypeptide containing any one amino acid sequence selected from SEQ ID NOs: 2-14 may be represented by SEQ ID NOs: 17-31, in the order described.

[0024] In addition, the base sequence encoding the protein A56 fragment may be a base sequence encoding an amino acid sequence having at least 60%, at least 70%, at least 80%, or at least 90% sequence homology with any one amino acid sequence selected from SEQ ID NOs: 2 to 14, and most preferably, a base sequence encoding an amino acid sequence having at least 95% sequence homology with any one amino acid sequence selected from SEQ ID NOs: 2 to 14.

[0025] The binding molecule according to the present invention refers to a biological molecule that has the ability to specifically bind to A56, and typical examples include antibodies or chimeric antigen receptors. More specifically, the binding molecule according to the present invention can specifically bind to A56 exposed on the surface of cancer cells, and this ability enables effective targeting of cancer cells in anticancer treatment, particularly cancer cells that have survived even infection with oncolytic viruses as a second-line anticancer treatment. In addition, when the binding molecule is used in cancer immunotherapy such as CAR-T cell therapy, it itself exhibits excellent cytotoxic effects, and therefore enables effective treatment of cancer.

[0026] Structural epitopes may contain adjacent amino acids, or amino acids that are not adjacent due to the three-dimensional folding of the protein. More specifically, structural epitopes may contain at least 2, 5, 8, 10, or 11 amino acids in a three-dimensional structure in an independent space.

[0027] Several amino acid residues of an antigen involved in antigen / antibody binding are called epitopes, and the specific portion of an antibody that recognizes an epitope is called a paratope. Depending on how the paratope binds, epitopes are broadly classified into linear epitopes, which consist of a sequence of approximately 4 to 12 consecutive amino acids, and structural epitopes, which consist of a sequence of discontinuous amino acids. In the case of structural epitopes, antigen / antibody binding occurs three-dimensionally in the folding structure. Therefore, structural epitopes have a relatively broad and complex range of amino acid residues involved in binding. Consequently, even if the structure of the antigen or a partial sequence of its components is known, it is difficult to predict or mimic a monoclonal antibody that binds to the antigen three-dimensionally. Whether an antigen's epitope is linear or structural can be identified through Western blot analysis by denaturing the antigen and then binding the antibody to it. In the case of linear epitopes, the antibody can bind to the epitope even if the sample (antigen) is denatured. However, in the case of structural epitopes, if the antigen is denatured, it is difficult or impossible for the antibody to bind to the epitope three-dimensionally, and therefore, the band corresponding to the expected molecular weight is not detected.

[0028] The structural epitope may include basic or nucleophilic amino acids located in the region from amino acid 60 to amino acid 63 in the amino acid sequence of protein A56 represented by Sequence ID No. 1, and may further include (i) nucleophilic amino acids located in the region from amino acid 44 to amino acid 50 in the amino acid sequence of protein A56, (ii) nucleophilic amino acids located in the region from amino acid 53 to amino acid 59 in the amino acid sequence of protein A56, (iii) nucleophilic amino acids located in the region from amino acid 85 to amino acid 90 in the amino acid sequence of protein A56, or (iv) basic or nucleophilic amino acids located in the region from amino acid 91 to amino acid 94 in the amino acid sequence of protein A56.

[0029] Protein A56, represented by Sequence ID No. 1, may preferably have the following secondary structural configuration consisting of a total of eight sheets (green), four helices (red), and seven loops (blue) (however, the structural configuration does not have to be this): The region from amino acid position 41 to amino acid position 43: Helix 1 and 2 The region from amino acid position 44 to amino acid position 50: Sheet 3 The region from amino acid position 51 to amino acid position 52: Loop The region from amino acid position 53 to amino acid position 59: Sheet 4 The region from amino acid position 60 to amino acid position 63: Loop The region from amino acid position 64 to amino acid position 66: Sheet 5 The region from amino acid position 67 to amino acid position 74: Loop The region from amino acid position 75 to amino acid position 78: Sheet 6 The region from amino acid position 79 to amino acid position 80: Loop The region from amino acid position 81 to amino acid position 83: Helix 3 Amino acid ranked 84th: Loop The region from amino acid position 85 to amino acid position 90: Sheet 7 The region from amino acid position 91 to amino acid position 94: Loop The region from amino acid position 95 to amino acid position 97: Helix 4 98th ranked amino acid: Loop The region from amino acid position 99 to amino acid position 105: Sheet 8 (Sheets 1 and 2 are not shown)

[0030] Nucleophilic amino acids are amino acids that have a nucleophilic side chain that readily undergoes covalent reactions with an electrophilic side chain, and may include, for example, cysteine ​​(C), lysine (K), serine (S), threonine (T), or tyrosine (Y). Basic amino acids are amino acids whose side chains are basic and whose side chains have a (+) charge when the amino acid dissociates in the neutral pH range, and may include, for example, histidine (H), arginine (R), or lysine (K).

[0031] The structural epitope of A56 according to the present invention may include (iii) a nucleophilic amino acid located in the region from the 60th to the 63rd amino acid in the amino acid sequence of protein A56 represented by Sequence ID No. 1, and (iv) a nucleophilic amino acid located in the region from the 85th to the 90th amino acid in the amino acid sequence of protein A56, and (iv) a basic or nucleophilic amino acid located in the region from the 91st to the 94th amino acid in the amino acid sequence of protein A56.

[0032] More specifically, the structural epitope may include the nucleophilic amino acids serine at position 62 (S62), tyrosine at position 66 (Y66), threonine at position 87 (T87), lysine at position 91 (K91), and serine at position 92 (S92) in the amino acid sequence of protein A56 represented by Sequence ID No. 1. More specifically, the structural epitope may include the amino acids at positions 54 (S54), 62 (S62), 66 (Y66), 86 (T86), 87 (T87), 91 (K91), 92 (S92), 94 (T94), 96 (arginine, R96), 100 (T100), and 101 (Y101) in the amino acid sequence of protein A56 represented by Sequence ID No. 1. In addition, the structural epitopes may include the amino acids at positions 62 (S62), 66 (Y66), 71 (T71), 72 (K72), 76 (S76), 87 (T87), 91 (K91), and 92 (S92) in the amino acid sequence of protein A56 represented by Sequence ID 1.

[0033] In addition, the structural epitope of A56 according to the present invention may include basic or nucleophilic amino acids located in the region from the 60th to the 63rd amino acid in the amino acid sequence of protein A56 represented by Sequence ID No. 1, and may include (i) nucleophilic amino acids located in the region from the 44th to the 50th amino acid in the amino acid sequence of protein A56, and (ii) nucleophilic amino acids located in the region from the 53rd to the 59th amino acid in the amino acid sequence of protein A56.

[0034] More specifically, the structural epitope may include the nucleophilic amino acids at positions 46 (S46), 54 (S54), and 61 (K61) in the amino acid sequence of protein A56 represented by SEQ ID NO: 1. More specifically, the structural epitope may include the amino acids at positions 46 (S46), 54 (S54), 61 (K61), 91 (K91), and 100 (T100) in the amino acid sequence of protein A56 represented by SEQ ID NO: 1. In addition, the structural epitope may include the amino acids at positions 46 (S46), 49 (Y49), 54 (S54), 61 (K61), 62 (K62), and 71 (T71) in the amino acid sequence of protein A56 represented by SEQ ID NO: 1.

[0035] In addition, the structural epitope may include the amino acids at positions 61 (K61) and 62 (S62) of the amino acid sequence of protein A56, represented by Sequence ID No. 1.

[0036] An analysis was conducted on the binding properties of several different antibodies that bind to A56 with high affinity. As a result, it was found that the basic amino acids at position 61 or 91 of A56 (K61, K91) commonly play a crucial role in binding. Specifically, K61 or K91 forms a strong hydrogen bond with the nucleophilic amino acid present in the paratope of each A56 antibody. As such binding proceeds, the heavy and light chains of the antibody fold, causing contraction folding, which leads to the remaining antigen-antibody binding. Separately, K61 or K91 may also contribute to antigen-antibody binding by influencing the serine (S62, S92) nucleophilic amino acid located immediately next to it. Specifically, K61 or K91 excites the hydroxyl group of the adjacent S62 or S92, generating an electrostatic force, and therefore, S62 or S92 is also thought to actively contribute to strong binding with the antibody.

[0037] The binding molecule or fragment thereof may compete for binding to A56 by preventing one or more of the following antigen-binding molecules from binding to A56. Such competitive binding means that they share a binding site (epitope) for A56. The binding molecule or fragment thereof may compete for binding to one or more of the following antibodies: An antibody (e.g., Ab13) comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 32, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 33, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 34, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 35, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 36, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 37; An antibody (e.g., Ab16) comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 38, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 39, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 40, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 41, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 42, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 43; An antibody (e.g., Ab18) comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 44, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 45, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 46, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 47, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 48, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 49; An antibody (e.g., Ab01) comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 50, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 51, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 52, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 53, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 54, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 55; An antibody (e.g., Ab19) comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 56, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 57, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 58, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 59, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 60, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 61; An antibody (e.g., Ab03) comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 62, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 64, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 65, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 66, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 67; An antibody (e.g., Ab08) comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 68, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 69, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 70, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 71, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 72, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 73; An antibody (e.g., Ab14) comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 74, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 75, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 76, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 77, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 78, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 79; An antibody (e.g., Ab51) comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 80, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 81, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 82, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 83, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 84, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 85; and An antibody (e.g., Ab55) comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 86, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 87, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 88, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 89, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 90, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 91.

[0038] "Competing for binding" means that an antibody or other antigen-binding moiety has the ability to interfere with the binding of another antibody or antigen-binding moiety to a specific antigen in a standard competitive binding assay. The ability or degree to which an antibody or other antigen-binding moiety can interfere with the binding of another antibody or antigen-binding moiety to a specific antigen, and therefore whether cross-competition can be said to exist according to the present invention, can be determined using a standard competitive binding assay. One suitable assay is the Biacore technique, in which the level of interaction can be measured using surface plasmon resonance technology. Another assay for measuring cross-competition uses an ELISA-based approach. A high-throughput method for "epitope binning" antibodies based on cross-competition is described in International Publication No. 2003 / 48731.

[0039] It was identified that A56 antibodies cross-inhibit binding to A56, even though their epitope sequences are not completely identical. This suggests that a specific higher-order structure formed between the A56-binding molecule and A56, i.e., the structural epitopes of A56, is important for binding to A56. In particular, antibody Ab19 inhibits binding to A56 to a relatively lower degree than other A56 antibodies. However, antibody Ab19 still significantly inhibited the binding of other A56 antibodies to A56. Analysis of the binding between antibody Ab19 and A56 showed strong antigen-antibody binding only at K61 and S62 in A56. This suggests that the amino acid sequences of K61 and S62 (or K91 and K92, which exhibit similar electrostatic properties) in A56 are important for specific binding to A56, and that other additional structural epitopes contribute to stronger binding to A56.

[0040] As used herein, the term “antibody” refers to an immunoprotein that binds to an antigen and interferes with or removes the antigen. There are five types of antibodies: IgM, IgD, IgG, IgA, and IgE, each containing a heavy chain produced from the heavy chain constant region gene μ, δ, γ, α, or ε. In antibody technology, IgG is the most commonly used. IgG comprises four isotypes: IgG1, IgG2, IgG3, and IgG4, each of which can have different structural and functional properties.

[0041] IgG forms a highly stable Y-shaped structure (molecular weight: approximately 150 kDa) composed of two heavy chain (approximately 50 kDa) proteins and two light chain (approximately 25 kDa) proteins. Antibodies have both light and heavy chains, and each chain is divided into a variable region with an amino acid sequence that differs among antibodies and a constant region with an amino acid sequence that is the same among antibodies. The heavy chain constant region contains CH1, H (hinge), CH2, and CH3 domains. Each of these domains consists of two β-sheets, and these domains are linked by intramolecular disulfide bonds. The two variable regions in the heavy and light chains associate together to form an antigen-binding site. This site is located on each of the two arms of the Y-shape. In the Y-shape, the portion that can bind to the antigen is called the antibody-binding fragment (Fab), and the portion that does not bind to the antigen is called the crystalline fragment (Fc). The Fab and Fc are connected by a movable hinge region.

[0042] As used herein, the term "CDR" refers to the antigen-binding site, which is a hypervariable region located in the heavy chain and light chain variable regions of the antibody, and whose amino acid sequence differs depending on the antibody. In the three-dimensional structure of the antibody, the CDR takes the form of a loop on the antibody surface. Below this loop is a framework region (FR) that structurally supports the CDR. Each of the heavy and light chains has three loop structures, and these six loop region structures associate with each other to make direct contact with the antigen. For convenience, the antigen-binding sites on the six loop region structures are referred to as CDR1, CDR2, CDR3, CDR4, CDR5, and CDR6, respectively.

[0043] In addition, the antibody fragment may be one selected from the group consisting of Fab, scFv, F(ab)2, and Fv. The antibody fragment refers to the antigen-binding domain, excluding the crystalline region (Fc region) which has effector function in transmitting antigen-binding stimuli to cells, complement, etc., and may include single-domain antibodies or third-generation antibody fragments such as minibodies.

[0044] In addition, antibody fragments have the following advantages: antibody fragments are smaller in size than complete IgG, resulting in improved penetration into tissues or tumors; and antibody fragments can be produced in bacteria, which reduces manufacturing costs. Furthermore, antibody fragments lack Fc and are therefore used when the function of transmitting antigen-binding stimuli to cells, complement, etc., is not desired. Antibody fragments have a short half-life in the human body and are therefore suitable for in vivo diagnostics. However, substitution of some basic, acidic, or neutral amino acids in the amino acids that make up the antibody can alter the antibody's intrinsic isoelectric point (pI). Such alterations in the isoelectric point of an antibody can induce changes such as reducing the antibody's in vivo toxic side effects or increasing its water solubility. Therefore, for therapeutic antibodies, complete IgG can be used, taking into account affinity or structural form.

[0045] Antibodies can be readily produced using known monoclonal antibody production techniques. Methods for producing monoclonal antibodies may include preparing hybridomas using B lymphocytes obtained from immunized animals, or using phage display technology. However, the present invention is not limited to these methods.

[0046] Antibodies or fragments thereof can be used as part of a chimeric antigen receptor (CAR). Specifically, a chimeric antigen receptor comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, and antibodies or fragments thereof can be used as the antigen-binding domain.

[0047] The antigen-binding domain refers to the region of the antibody that binds to the antigen. The antigen-binding domain may be the antibody or its antigen-binding fragment. Preferably, the antigen-binding domain may be an antigen-binding fragment. In addition, the antigen-binding fragment may be a fragment having one antigen-binding site, formed by linking one heavy chain and one light chain within the antibody by a disulfide bond. The antigen-binding fragment may be any one selected from the group consisting of scFv, Fab, and Fab'. Preferably, the antigen-binding fragment may be scFv. That is, the antigen-binding domain may be scFv.

[0048] A transmembrane domain (SDM) is a region in the structure of a protein located on the cell membrane that connects the antigen-binding domain with the intracellular signaling domain, and extends across the cell membrane. The SDM can tether other proteins located on the cell membrane. The SDM can originate from any one of the following: T cell receptors, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PD-1. In particular, the SDM may originate from CD8α.

[0049] The intracellular signaling domain refers to the region on the cell surface that transmits signals to the cell when an antigen receptor (antigen-binding domain) recognizes an extracellular antigen, inducing responses such as cell activation, release of cytotoxic factors, cytokine production, and proliferation. In addition, generally, a signal transmitted through only one antigen receptor (antigen-binding domain) is insufficient for cell activation, and therefore, secondary or co-stimulatory signals are required. Thus, the intracellular signaling domain may include a primary signaling domain, a secondary signaling domain, and / or a co-stimulatory domain. In particular, the intracellular signaling domain may include a co-stimulatory domain and a primary signaling domain.

[0050] The co-stimulatory domain may originate from at least one molecule selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70. In particular, the co-stimulatory domain may originate from CD137 (4-1BB).

[0051] The primary signaling domain can originate from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, or CD66d. In particular, for T cells, signals are transmitted to the cell via the CD3 chain γ, δ, ε, or ζ. When creating chimeric antigen receptor T cells (CAR-T cells), the CD3 chain γ, δ, ε, or ζ can be used as the primary signaling domain. In particular, the primary signaling domain can originate from CD3ζ.

[0052] CAR-T cells can be produced using vectors containing polynucleotides encoding chimeric antigen receptors. These polynucleotides can be prepared, manipulated, expressed, and delivered using any of the various established techniques known and available in the art. To express a desired chimeric antigen receptor on the T cell surface, the polynucleotide encoding the chimeric antigen receptor can be inserted into a suitable vector.

[0053] Regarding vectors, various vectors known in the art can be used. Depending on the type of host cell intended to produce the antigen receptor, expression regulatory sequences such as promoters, terminators, and enhancers, and sequences for membrane targeting or secretion, can be appropriately selected and combined in various ways according to the purpose. In particular, vectors include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. A suitable vector includes expression regulatory elements such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and can be constructed in various ways according to the purpose.

[0054] Methods known in the art can be used to introduce vectors into immune cells. For example, vectors can be introduced into cells by transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, or by using a gene gun, or by other known methods for introducing nucleic acids into cells (Wu et al., J. Bio. Chem., 267:963~967, 1992; Wu and Wu, J. Bio. Chem., 263:14621~14624, 1988). However, the present invention is not limited thereto.

[0055] Transduced or transfected immune cells are proliferated after vector introduction. In some embodiments, transfected immune cells can be proliferated by incubation for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, preferably 12 to 14 days.

[0056] Methods for identifying whether a vector has been successfully introduced into immune cells include, for example, molecular biology assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; and biochemical assays, such as detection of the presence or absence of specific peptides by immunological methods (e.g., ELISA and Western blotting).

[0057] The A56 binding molecule or fragment thereof according to the present invention can bind to protein A56 with a binding affinity of less than about 1.0 nM (1×10 D , -10 , -9 ) as measured by surface plasmon resonance. Specifically, the binding molecule or fragment thereof can have a binding affinity for protein A56 of less than 1×10 D , less than 9×10 -9 , or less than 8×10 -10 of K -10 (M). In an embodiment of the present invention, the binding molecule in that embodiment was determined to have a binding affinity of 7.39×10 D , 2.28×10 -10 , 1.96×10 -10 , 4.92×10 -10 , and 4.38×10 -10 , and 4.38×10 -10 of K D (M).

[0058] In the present invention, in order to identify a novel structural epitope of protein A56, the structure of the complex formed by the binding of the anti-A56 antibody to protein A56 in an embodiment was crystallized at a resolution of 1.8 Å (Figs. 61A - 65). As a result, a novel structural epitope of protein A56 and the paratope of the anti-A56 antibody in the embodiment were identified and are shown in Table 1.

[0059]

Table 1

[0060] The binding molecule or fragment thereof may bind to protein A56 within an intermolecular distance of 6.5 Å. The binding molecule or fragment thereof may form van der Waals bonds, hydrophobic bonds, or electrostatic bonds with protein A56.

[0061] The binding molecule or fragment thereof can be used to reduce the burden on cancer cells when administered together with an oncolytic virus. The burden on cancer cells may refer to the weight, volume, or number of cancer cells. Cancer cells that survive even infection with an oncolytic virus express the protein A56 on their cell surface. Therefore, the binding molecule or fragment thereof that specifically binds to such A56 according to the present invention enables secondary anticancer therapy, or targeting for secondary anticancer therapy.

[0062] The oncolytic virus may be a vaccinia virus. The vaccinia virus may be one of the following vaccinia virus strains, but is not limited to: Western Reserve (WR), New York Vaccinia Virus (NYVAC), Wyeth (The New York City Board of Health; NYCBOH), LC16m8, Lister, Copenhagen, Tian Tan, USSR, Tashkent, Evans, International Health Division-J (IHD-J), and International Health Division-White (IHD-W).

[0063] Oncolytic viruses may be viruses in which the thymidine kinase (TK) gene is deleted. In particular, oncolytic viruses may be recombinant vaccinia viruses in which the thymidine kinase gene is deleted.

[0064] As used herein, the term “thymidine kinase (TK)” refers to an enzyme called thymidine kinase that is involved in nucleotide biosynthesis. TK is an enzyme used in nucleotide biosynthesis in both cells and viruses. In the case of cells, normal cells no longer divide and therefore do not contain TK. Even in rapidly dividing cells such as hair follicle cells, TK is not present in sufficient quantities for a virus to utilize. From these perspectives, by deleting the TK gene, a virus can proliferate only in the presence of cancer cells in which TK is present, and as a result, cancer cells can be selectively killed.

[0065] The oncolytic virus may contain nucleic acids encoding protein A56 or a fragment thereof. Protein A56 or a fragment thereof is as described above. Protein A56 may be wild-type protein A56 or a variant thereof. The nucleic acid encoding protein A56 or a fragment thereof may be wild-type protein A56 or a variant thereof. The oncolytic virus may be administered simultaneously with hydroxyurea, sequentially, or in the reverse order.

[0066] In another embodiment of the present invention, a method is provided for reducing the burden on cancer cells in a subject, comprising the step of administering a binding molecule or a fragment thereof to the subject. The binding molecule or fragment thereof is as described above.

[0067] The subjects may be mammals, including humans, or non-human animals. The term "non-human animals" refers to any vertebrate, and may include mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, and reptiles. In addition, the subjects are those suffering from cancer or a disease that can be mitigated, inhibited, or treated by the administration of an oncolytic virus.

[0068] The dosage of the binding molecule or its fragments will vary depending on the patient's condition and weight, the severity of the disease, the type of drug, the route of administration, and the duration of administration, and can be appropriately selected by those skilled in the art.

[0069] The binding molecule or fragment thereof can be administered parenterally, and such administration can be carried out by any suitable method, such as intratumoral, intraperitoneal, subcutaneous, intradermal, intranodal, or intravenous administration. Among these, intratumoral, intraperitoneal, or intravenous administration may be preferred.

[0070] With regard to the route of administration, dosage, and frequency of administration, the conjugated molecule or its fragments can be administered to a subject in various ways and in various amounts, depending on the subject's condition and the presence or absence of side effects; and the optimal route of administration, dosage, and frequency of administration can be appropriately selected by those skilled in the art. In addition, the conjugated molecule or its fragments may be administered in combination with another drug or physiologically active substance known to have a therapeutic effect on the disease to be treated, or may be formulated in the form of a compound preparation with other drugs. In particular, the conjugated molecule or its fragments can be provided in the form of an injectable solution.

[0071] In yet another embodiment of the present invention, a pharmaceutical composition for preventing or treating cancer is provided, comprising a binding molecule or a fragment thereof. The binding molecule or fragment thereof is as described above. The pharmaceutical composition may further contain hydroxyurea.

[0072] The pharmaceutical composition may further contain a physiologically acceptable carrier. In addition, the pharmaceutical composition may further contain suitable excipients and diluents commonly used in the preparation of pharmaceutical compositions. Furthermore, the pharmaceutical composition may be formulated and used in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, as well as aerosols, topical preparations, suppositories, or injections, according to conventional methods. In particular, anticancer agents may take the form of injections. Suitable formulations known in the art may be those disclosed in the literature (Remington's Pharmaceutical Science, 1985).

[0073] The pharmaceutical composition may be intended to prevent or treat cancers selected from the group consisting of lung cancer, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymic cancer, gastric cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, lymphoma, acute leukemia, multiple myeloma, and combinations thereof.

[0074] In addition, examples of carriers, excipients, and diluents for pharmaceutical compositions include sodium chloride, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, crystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When anticancer drugs are formulated, the preparations can be made using commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and diluents or excipients such as surfactants.

[0075] Regarding pharmaceutical compositions, preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injection esters such as ethyl oleate. For suppository bases, Witepsol, macrogol, Tween 61, cocoa butter, lauric acid butter, and glycerol gelatin can be used.

[0076] Cancer can be a solid tumor or a hematological cancer. More specifically, a solid tumor may be any one selected from the group consisting of lung cancer, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymic cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, and combinations thereof. In addition, a hematological cancer may be any one selected from the group consisting of lymphoma, acute leukemia, multiple myeloma, and combinations thereof.

[0077] In yet another embodiment of the present invention, a kit for preventing or treating cancer is provided, comprising a binding molecule or fragment thereof and an oncolytic virus. The binding molecule or fragment thereof and the oncolytic virus are as described above. The kit may further comprise a hydroxyurea.

[0078] The dosage of oncolytic viruses varies depending on the patient's condition and weight, the severity of the disease, the type of drug, the route of administration, and the duration of administration, and can be appropriately selected by those skilled in the art. The dosage is 1 × 10⁻⁶ for the patient. 5 ~1 × 10 18 Virus particles, infectious virus units (TCID) 50 ), or an amount that allows the oncolytic virus to be received in plaque-forming units (PFUs). Specifically, the dosage may be such that the patient receives 1 × 10⁶ doses. 5 , 2×10 5 , 5×10 5 , 1 x 10 6 , 2×10 6 , 5×10 6 , 1 x 10 7 , 2×10 7 , 5×10 7 , 1 x 10 8 , 2×10 8 , 5×10 8 , 1 x 10 9 , 2×10 9 , 5×10 9 , 1 x 10 10 , 5×10 10 , 1 x 10 11 , 5×10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14, 1 x 10 15 , 1 x 10 16 , 1 x 10 17 This can be an amount that receives , or more viral particles, infectious viral units, or oncolytic viruses in plaque-forming units, and various numerical values ​​and ranges between the numbers mentioned above may also be included. Preferably, the oncolytic virus is 1 × 10⁻⁶ 5 ~1 × 10 10 It can be administered in doses of pfu. More preferably, the oncolytic virus is 1 × 10⁻⁶ 5 PFU or higher and 1 × 10 9 It can be administered in doses less than pfu.

[0079] Oncolytic viruses can be administered parenterally, and such administration can be carried out by any suitable method, such as intratumoral, intraperitoneal, subcutaneous, intradermal, intranodal, or intravenous administration. Of these, intratumoral, intraperitoneal, or intravenous administration may be preferred.

[0080] With regard to the route of administration, dosage, and frequency of administration, oncolytic viruses can be administered to a subject in various ways and in various amounts, depending on the subject's condition and the presence or absence of side effects; and the optimal route of administration, dosage, and frequency of administration can be appropriately selected by those skilled in the art. In addition, oncolytic viruses may be administered in combination with another drug or physiologically active substance known to have a therapeutic effect on the disease to be treated, or may be formulated in the form of a compound preparation with other drugs. In particular, oncolytic viruses can be provided in the form of an injectable solution.

[0081] In yet another embodiment of the present invention, the use of a binding molecule or fragment thereof for the treatment of cancer is provided. [Examples]

[0082] The present invention will be described in more detail below using the following embodiments. However, the following embodiments are for illustrative purposes only, and the scope of the present invention is not limited to these embodiments.

[0083] I. Construction of a vector containing nucleic acids encoding protein A56 or a fragment thereof, and identification of protein A56 expression on the surface of tumor cells. Preparation 1.1. Construction of a vector containing nucleic acids encoding protein A56 or a fragment thereof. To construct plasmids encoding wild-type protein A56 and its fragments, duplicate PCR was performed by preparing primers to remove specific regions and forming regions that overlapped with GFP.

[0084] Preparation 1.2. Preparation of oncolytic vaccinia virus containing nucleic acids encoding protein A56 or a fragment thereof. To produce oncolytic vaccinia virus containing protein A56, the HeLaS3 (ATCC) cell line was placed in a 6-well plate with 4 × 10⁶ cells per well. 5 Cells were seeded individually and then prepared in EMEM medium containing 10% fetal bovine serum. Treatment with wild-type vaccinia virus strains Weiss and Western Reserve strain was carried out at a MOI of 0.05. After 2 hours, the medium was replaced with EMEM medium containing 2% fetal bovine serum, and then the cells were transfected with a vector containing the reporter gene and insertion gene using Xfect reagent buffer. Culture was carried out for 4 hours. Subsequently, the medium was replaced with EMEM medium containing 2% fetal bovine serum, and the cells were cultured for a further 72 hours. Finally, infected cells were collected and then frozen and thawed three times. The cells were lysed by sonication, and free oncolytic vaccinia virus (OTS-412, WOTS-418) containing nucleic acids encoding protein A56 or a fragment thereof was obtained using the sucrose cushion method.

[0085] Reference Example 1. Comparison of the protein A56 sequences for each vaccinia virus strain. We commissioned Macrogen to sequence the membrane protein A56 in the Weiss strain OTS-412 and the Western Reserve strain WOTS-418, both vaccinia viruses lacking a TK region. Alignment of the A56 sequences in OTS-412 and WOTS-418 was performed via NCBI Blast and Uniprot. No 100% identical sequences were found; in particular, a deletion of amino acids between positions 245 and 250 was identified. Comparisons were made with four other strains exhibiting high sequence homology.

[0086] Experimental Example 1. Expression of protein A56 on the surface of cancer cells Using oncolytic vaccinia virus containing nucleic acids encoding protein A56 or a fragment thereof, prepared in Preparation Example 1.2, human lung cancer cell lines (A549), human colorectal cancer cell lines (HCT-116), or human melanoma cell lines (SK-MEL-5) were infected to identify whether protein A56 was expressed on the surface of the cancer cells.

[0087] Specifically, A549 (lung carcinoma, ATCC, USA), HCT-116 (colorectal carcinoma, Korea Cell Line Bank), or SK-MEL-5 (human melanoma, Korea Cell Line Bank) cell lines were placed on the coverslip of a 12-well plate at a rate of 3.5 × 10⁶ cells per well. 4Cells were seeded individually. Subsequently, the cells were infected with oncolytic vaccinia virus at an MOI of 0.1, and then incubated for 30 hours at 37°C and 5% CO2. Each incubated cell line was collected. Subsequently, treatment was carried out with 4% (v / v) paraformaldehyde (PFA), 1% (v / v) BSA, a 1:500 dilution of anti-A56 primary antibody (catalog number ABIN1606294, Antibodies-Online), and a 1:200 dilution of secondary antibody (Alexa 594, catalog number A21205, Invitrogen). DAPI staining was performed. After that, samples from each cell line were placed on glass slides and observed using a confocal microscope (Olympus, FV1000).

[0088] As a result, protein A56 was identified as being expressed on the cell surface of A549 and HCT-116 cell lines infected with oncolytic vaccinia virus (Figures 1 and 2).

[0089] Experimental Example 2. Identification of protein A56 expression on the surface of mouse tumor tissue (I) A cancer-inducible mouse model was subjected to intraperitoneal administration of oncolytic vaccinia virus containing nucleic acids encoding protein A56 or a fragment thereof, prepared in Preparation Example 1.2, to identify whether protein A56 was expressed on the tissue surface.

[0090] In detail, BALB / c nude mice were inoculated with 6.3 × 10⁶ HCT-116 colorectal cancer cell line (Korea Cell Line Bank, KCLB). 6 Individual cells were subcutaneously transplanted to induce cancer. The average tumor volume was 150 mm². 3 ~200mm 3 When this is reached, the mouse is subjected to 2 × 10⁶ of the oncolytic vaccinia virus prepared in Preparation Example 1.2. 7 The mice were administered intraperitoneally at doses of pfu. Subsequently, on day 4, the mice were sacrificed, and tumor tissue, as well as brain, heart, lung, muscle, kidney, liver, and spleen tissue, were collected from the mice.

[0091] Immunofluorescence staining for protein A56 was performed using the same method as in Experimental Example 1. DAPI staining was also performed. Subsequently, each tissue sample was placed on a glass slide and observed using a fluorescence microscope.

[0092] As a result, it was identified that protein A56 is expressed on the surface of tumor tissue in mice administered with oncolytic vaccinia virus. On the other hand, it was identified that protein A56 is not expressed in brain, heart, lung, muscle, kidney, liver, and spleen tissue (Figure 3). These results indicate that if a binding molecule targeting protein A56 expressed on the surface of tumor tissue is produced after administration of oncolytic vaccinia virus, this antibody can be used as an anti-cancer immunotherapy.

[0093] Experimental Example 3. Identification of protein A56 expression on rabbit tissue surface. Normal rabbits were subjected to intravenous administration of oncolytic vaccinia virus containing nucleic acids encoding protein A56 or a fragment thereof, prepared in Preparation Example 1.2, to identify whether protein A56 was expressed on the tissue surface. The toxicity risk was then analyzed.

[0094] In detail, New Zealand rabbits were subjected to 1 × 10⁶ of the oncolytic vaccinia virus prepared in Preparation Example 1.2. 8 PFU or 1 × 10 9 The rabbits were administered intravascularly at doses of pfu. Subsequently, at 3 or 8 weeks, the rabbits were sacrificed, and brain, heart, lung, muscle, kidney, liver, and spleen tissues were collected from them.

[0095] Immunofluorescence staining for protein A56 was performed using the same method as in Experimental Example 1. DAPI staining was also performed. Subsequently, each tissue sample was placed on a glass slide and observed using a fluorescence microscope.

[0096] As a result, it was identified that protein A56 was not expressed in the heart, lung, muscle, kidney, liver, and spleen tissues of rabbits administered with oncolytic vaccinia virus.

[0097] On the other hand, a fluorescence reaction was detected in the brain tissue of rabbits administered with oncolytic vaccinia virus. To determine whether the detected fluorescence reaction was a nonspecific reaction of the anti-A56 antibody, brain tissue from normal rabbits that had not been administered oncolytic vaccinia virus was subjected to immunofluorescence staining using the same method as above, and the fluorescence reaction in the brain tissue was then checked using a fluorescence microscope.

[0098] As a result, the fluorescence was detected even in the brain tissue of rabbits that were not administered oncolytic vaccinia virus. This result identified such fluorescence as a nonspecific reaction (Figure 4).

[0099] In addition, when brain tissue from normal humans who had not been administered oncolytic vaccinia virus (Pusan ​​National University Yangsan Hospital, Korea) was subjected to immunofluorescence staining using the same method as above, a weak fluorescence reaction was detected using a fluorescence microscope. However, from the perspective that the antibody does not cross the blood-brain barrier, it is determined that the anti-A56 antibody will not cause a nonspecific reaction unless the antibody is directly administered into the ventricles of the brain.

[0100] Experimental Example 4. Identification of protein A56 expression on the surface of mouse tumor tissue (II) A cancer-inducible mouse model was subjected to simultaneous administration of oncolytic vaccinia virus and hydroxyurea containing nucleic acids encoding protein A56 or a fragment thereof, prepared in Preparation Example 1.2, to identify whether protein A56 was expressed on the tissue surface.

[0101] In detail, BALB / c nude mice, 6.3 × 10 6Numerous Renca cancer cell lines (Korea Cell Line Bank) were subcutaneously transplanted to induce cancer. The average tumor volume was 150 mm². 3 ~200mm 3 When this is reached, the mouse is subjected to 2 × 10⁶ of the oncolytic vaccinia virus prepared in Preparation Example 1.2. 7 The subjects were subjected to intratumoral administration of pfu at a dose and hydroxyurea at a dose of 30 mg / kg.

[0102] The renal cancer cell transplanted mice were divided into three groups (n=4). The group receiving intratumoral administration of saline was set as the control group, and oncolytic vaccinia virus (1 × 10⁻¹⁶) was administered to the mice. 7 The group receiving pfu (1x10) monotherapy, and the group receiving oncolytic vaccinia virus (1x10) 7 A group receiving simultaneous administration of pfu and hydroxyurea (30 mg / kg) was designated as the experimental group. Oncolytic vaccinia virus was administered intratumorally twice, on days 0 and 14. Hydroxyurea was administered intraperitoneally six times a week, from one day before administration of oncolytic vaccinia virus until 21 days after administration of oncolytic vaccinia virus, excluding the days on which oncolytic vaccinia virus was administered.

[0103] Mice were sacrificed on days 7, 10, and 14 after the first administration of oncolytic vaccinia virus, and tumor tissue was collected from these mice. Mice were also sacrificed on days 21, 24, and 28 after the second administration of oncolytic vaccinia virus, and tumor tissue was collected from these mice. Immunofluorescence staining for protein A56 was performed using the same method as in Experimental Example 1. DAPI staining was performed. Subsequently, each tissue sample was placed on a glass slide and observed using a fluorescence microscope.

[0104] As a result, protein A56 was identified as being clearly expressed on the tumor surface of mice that received only oncolytic vaccinia virus and those that received co-administration of oncolytic vaccinia virus and hydroxyurea by days 7, 10, and 14 after the first administration of oncolytic vaccinia virus (Figures 5 and 6).

[0105] Experimental Example 5. Expression of protein A56 or its fragments on the cell surface. HeLa cell lines were treated with plasmids encoding protein A56 or a fragment thereof to identify whether protein A56 was expressed on the cell surface. For the protein A56 fragment, primers were prepared to remove specific regions from wild-type protein A56, and overlapping PCR was performed to form a region overlapping with GFP.

[0106] In detail, the HeLa (cervical cancer cell, ATCC, USA) cell line was placed on a 12-well plate cover slip at a rate of 3.5 × 10⁴ per well. 4 Cells were seeded individually. Subsequently, plasmids containing the A56 fragment, diluted by mixing with Xfect reaction buffer and Xfect polymer, were incubated with HeLa cell lines at 37°C and 5% CO2 for 30 hours. Each incubated cell line was collected. Treatment was then carried out with 4% (v / v) paraformaldehyde (PFA), 1% (v / v) BSA, anti-A56 antibody diluted at a ratio of 1:500 (catalog number ABIN1606294, Antibodies-Online), and secondary antibody diluted at a ratio of 1:200 (Alexa 594, catalog number A21205, Invitrogen). The nuclei were stained with DAPI, and the Golgi apparatus was stained with a fluorescent dye. Cell line samples were then placed on glass slides and observed using a confocal fluorescence microscope (Olympus, FV1000). The results are shown in Figures 8 and 9 and Table 2.

[0107] [Table 2]

[0108] Wild-type A56 (A56-G) and fragments of A56 obtained by partial shortening of six regions of A56 were expressed on the cell surface. As a result, it was identified that only wild-type A56 (A56-G) and A56-121, in which the IgV-like domain is shortened, reached the cell surface and were expressed there. Among the A56 variants in which the IgV-like domain is excluded, it was identified that variants in which the signal peptide, transmembrane domain, stalk region, and tandem repeat region are shortened individually or in combination were not expressed on the cell membrane. In addition, it was identified that even variants containing only the transmembrane domain were not expressed on the cell membrane when the IgV-like domain was present. In other words, it was identified that variants in which the IgV-like domain is excluded and which contain five regions are expressed on the cell membrane.

[0109] II. Preparation of antibodies that bind to protein A56 or its fragments Preparation Example 2. Purification and Production of Protein A56 The vector DNA (N293F-A56-C-HIS) was amplified and introduced into HEK293F cells, where it was overexpressed. Subsequently, primary purification was performed by affinity chromatography (Ni-NTA), followed by secondary purification by cation exchange chromatography (CEX). In this way, the A56-C-HIS protein was finally produced (Figure 10).

[0110] Preparation Example 3. Preparation of antibodies that bind to protein A56 or its fragments (I) Ybiologics was commissioned to produce anti-A56 antibodies that specifically bind to protein A56 or its variants or fragments. Sixty-one antibodies were produced using phage library technology, and their CDRs were analyzed. The phage library was added to tubes coated with A56 antigen, and biopanning was performed to identify binding hits. Phages exhibiting specific binding were selected by performing an average of three washes. Three panning steps were performed. Subsequently, affinity testing was conducted, and small samples of colonies showing high affinity were collected to identify whether the colonies exhibited affinity for the actual antigen. Sets with relatively high hit counts were selected, and an automated system was used for collection and hit selection.

[0111] The affinity between each of the anti-A56 antibodies thus produced and protein A56 was measured. The results are shown in Figures 11-16. In addition, the productivity of each anti-A56 antibody is shown in Figures 17A-17C. Furthermore, Figures 18-34 show the results obtained by identifying each of the produced anti-A56 antibodies via SDS-PAGE.

[0112] Experimental Example 6. Affinity Measurement of Anti-A56 Antibody Each well of the immunotube was coated with protein A56, followed by a blocking step. After the blocking step, each well was reacted with antibody, which had been subjected to 3-fold serial dilutions starting from 100 nM, at room temperature for a predetermined time. Subsequently, washing with PBS was performed three times, followed by treatment with secondary antibody, also at room temperature for a predetermined time. Subsequently, the affinity of each anti-A56 antibody for protein A56 was measured at their respective concentrations.

[0113] Experimental Example 7. Comparison of amino acid sequence homology among protein A56 for each vaccinia virus strain. A comparison of amino acid sequence homology among vaccinia virus strains, namely Copenhagen, Ankara, Western Reserve (WR), International Health Division-J (IHD-J), Tiantan, and Weiss (New York City Department of Health; NYCBOH). As a result, as shown in Figure 35, it was identified that the amino acids from position 30 to 90 of the amino acid sequence of protein A56 in all strains were identical.

[0114] III. Identification of the structural epitope of protein A56 Example 1. Distinction between linear and structural epitopes on protein A56. To distinguish between linear and structural epitopes, Western blot analysis was performed on 10 anti-A56 antibodies (Ab18, Ab19, Ab01, Ab13, Ab14, Ab08, Ab03, Ab51, Ab55, Ab16) that bind to A56. Ten anti-A56 antibodies were obtained by selecting the top 10 antibodies based on binding affinity from 61 antibodies selected as representative antibodies from a human phage library that specifically bind to protein A56 (Figure 36).

[0115] In detail, HeLa cell lines were infected with OTS-412; after 24 hours, the HeLa cells were collected, lysed, and total protein was extracted. The protein was subjected to a denaturation process and then loaded onto an SDS-PAGE gel for electrophoresis. After electrophoresis, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane and reacted with 10 primary anti-A56 antibodies. Subsequently, washing with PBST (PBS: Donginbiotech Co., Ltd, Tween 20: Sigma) was performed. Subsequently, the protein was reacted with a secondary antibody (goat anti-human IgG Fc cross-adsorbed, HRP (Abcam, catalog number ab98624)). Washing with PBST was performed again. After that, treatment with a luminescent reagent (ECL, Amersham Biosciences) was performed, and an imaging system (chemiluminescence imaging system) was used for identification. Here, recombinant protein A56 (A56-C-His, 1.56 mg / mL) and a commercially available anti-A56 antibody (Immune Technology Corp., catalog number IT-012-006M1) were used as positive control groups.

[0116] As a result, first, as shown in Figure 37, it was found that in the positive control group, protein A56 had a molecular weight corresponding to the 85kD–100kD band, while most of the 10 anti-A56 antibodies had weaker band sizes or intensities than the positive control group (Figure 38). In particular, many weak bands with small sizes were observed, or no bands were observed, for Ab03, Ab08, Ab13, Ab19, Ab51, Ab55, and Ab16. The absence of bands, or bands with small size and intensity, indicates that the epitope is formed three-dimensionally.

[0117] Furthermore, for epitope mapping of seven anti-A56 antibodies, Ab18, Ab13, and Ab16 were preferentially mapped, and then epitopes for two highly binding antibodies (Ab01 and Ab19) were identified.

[0118] Example 2. Analysis of intact proteins of protein A56 and anti-A56 antibody, and characterization of the binding complex. Prior to epitope mapping, each sample (A56-C-His, three antibodies (Ab13, Ab16, Ab18) and antigen-antibody conjugates (A56-C-His / Ab13, A56-C-His / Ab16, A56-C-His / Ab18) were characterized to analyze the integrity of the sample and the degree of aggregation of the conjugates.

[0119] In detail, for intact mass spectrometry of the control group, 5 μl each of protein A56 and anti-A56 antibodies (Ab13, Ab16, Ab18) were mixed. Subsequently, 1 μl of the 10 μl mixture was mixed with acetonitrile / water in a 1:1 ratio and 0.1% TFA (K200 MALDI Kit) in a recrystallized sinapic acid matrix (10 mg / ml) placed on a MALDI plate (SCOUT 384), and crystallized at room temperature. After that, molecular weight measurements were performed three times by MALDI-MS (mass spectrometry).

[0120] Here, the complex (A56-C-His / anti-A56 antibody) was subjected to crosslinked high-mass matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS, Autoflex II MALDI ToF mass spectrometer, Bruker). 9 μl of the antigen-antibody conjugate remaining after intact mass spectrometry was mixed with 1 μl of crosslinking reagent (K200 stabilizer, 2 mg / ml) and reacted at room temperature for 180 minutes. Subsequently, MALDI ToF MS was performed using a standard nitrogen laser (MS: linear and positive modes, ion source 1: 20 kV, 2: 17 kV, lens: 12 kV, pulsed ion extraction: 400 ns; HM4: gain voltage: 3.14 kV, acceleration voltage: 20 kV).

[0121] As a result, molecular weights very similar to the peak values ​​of the control group were detected in the samples identified by crosslinking (Table 3), and no non-covalent complexes were detected. Therefore, it was identified that the samples used for analysis were not clumped or damaged.

[0122] [Table 3]

[0123] Furthermore, antigen-antibody binding complexes (A56-C-His / Ab13, A56-C-His / Ab16, A56-C-His / Ab18, A56-C-His / Ab01, A56-C-His / Ab19) were stabilized by treatment with a dedicated reagent for crosslinking non-covalent protein complexes, and then high-mass MALDI analysis was performed. As a result, two types of peaks were observed. These data were evaluated using complex tracker software to identify the presence of A56·Ab complexes and 2A56·Ab complexes (Figures 39-43). In addition, the molecular weights of each sample and complex are shown in Table 4.

[0124] [Table 4]

[0125] Example 3. Epitope mapping of protein A56 against 5 anti-A56 antibodies The amino acid sequence numbering in A56, as shown in Examples 3.2 to 3.7 and the drawings referred thereto, is based on the sequence obtained by removing N-terminal amino acids 1 to 16 from the amino acid sequence represented by SEQ ID NO: 1 (for example, the serine at position 30 corresponds to the serine at position 46 in the amino acid sequence represented by SEQ ID NO: 1).

[0126] Example 3.1. Sequencing of protein A56 For epitope mapping of protein A56, the sequence of protein A56 was first identified. Protein A56 was then fragmented into peptides by treatment with five proteolytic enzymes (trypsin, chymotrypsin, ASP-N, elastase, and thermolysin). Subsequently, mass fingerprint information was obtained from the digested peptides by LTQ-Orbitrap MS (mass spectrometry), and a comparative analysis was performed to determine whether this information matched existing sequence information for protein A56.

[0127] As a result, as shown in Figure 44, amino acid fragments obtained by digestion of protein A56 with trypsin had 38.76% sequence coverage; amino acid fragments obtained by digestion of protein A56 with chymotrypsin had 66.28% sequence coverage; amino acid fragments obtained by digestion of protein A56 with ASP-N had 43.80% sequence coverage; amino acid fragments obtained by digestion of protein A56 with elastase had 94.57% sequence coverage; and amino acid fragments obtained by digestion of protein A56 with thermolysin had 83.33% sequence coverage. Considering all sequences identified by the five proteolytic enzymes, these sequences were identified as 99.61% identical to existing protein A56 in terms of sequence information.

[0128] Example 3.2. Epitope mapping of protein A56 against anti-A56 antibody (Ab13). Samples of the antigen-antibody conjugate (A56-C-His / Ab13) were subjected to deuterated crosslinking and combined treatment with various proteolytic enzymes, resulting in alkylation and reduction of the samples. Subsequently, molecular weight data was obtained from these samples using high-mass MALDI MS and nLC-LTQ-Orbitrap MS, and then analyzed using software (XQuest, Stavrox).

[0129] As a result, the sequences of the sites where protein A56 and each anti-A56 antibody (Ab13) are in direct contact with each other were identified using crosslinking. Based on this, the epitopes and paratopes shown in Table 5 were identified as follows.

[0130] [Table 5]

[0131] As shown in Table 5, the epitopes on the binding sites of A56-C-His and the anti-A56 antibody (Ab13) were identified as being at positions 38, 46, 50, 70, 71, 75, 76, 78, 80, 84, and 85 in the amino acid sequence of A56-C-His.

[0132] In detail, as shown in Figure 45, the epitope positions of amino acid residues from position 30 to 90 in the amino acid sequence of A56-C-His are shown in red (positions 38, 46, 50, 70, 71, 75, 76, 78, 80, 84, and 85); and when the protein structure of A56-C-His (at positions 2 to 148 in the amino acid sequence) is represented in silico using Swiss-Model software, the binding sites of Ab13 and A56-C-His are shown in blue. Within the amino acid sequence of A56-C-His, the identified binding sites were found to correspond to positions 38-50 (SIILLAAKSDVLY) and 70-85 (TTITIKSLTARDAGTY).

[0133] Example 3.3. Epitope mapping of protein A56 against anti-A56 antibody (Ab16). Samples of the antigen-antibody conjugate (A56-C-His / Ab16) were subjected to deuterated crosslinking and combined treatment with various proteolytic enzymes, resulting in alkylation and reduction of the samples. Subsequently, molecular weight data was obtained from these samples using high-mass MALDI MS and nLC-LTQ-Orbitrap MS, and then analyzed using software (XQuest, Stavrox).

[0134] The sequences of the sites where protein A56 and each anti-A56 antibody (Ab16) are in direct contact with each other were identified using cross-linking. Based on this, the epitopes and paratopes shown in Table 6 were identified as follows.

[0135] [Table 6]

[0136] As shown in Table 6, the epitopes on the binding sites of A56-C-His and the anti-A56 antibody (Ab16) were identified as being at positions 30, 33, 38, 45, 46, 55, 56, 58, and 60 in the amino acid sequence of A56-C-His.

[0137] In detail, as shown in Figure 46, the epitope positions of amino acid residues from position 30 to 90 in the amino acid sequence of A56-C-His are shown in red (positions 30, 33, 38, 45, 46, 55, 56, 58, and 60); and when the protein structure of A56-C-His (at positions 2-148 in the amino acid sequence) is represented in silico using Swiss-Model software, the binding sites of Ab16 and A56-C-His are shown in blue. Within the amino acid sequence of A56-C-His, the binding sites were identified as corresponding to positions 30-46 (SAWYKEPNSIILLAAKS) and 55-60 (TKDKIS).

[0138] Example 3.4. Epitope mapping of protein A56 against anti-A56 antibody (Ab18). Samples of the antigen-antibody conjugate (A56-C-His / Ab18) were subjected to deuterated crosslinking and combined treatment with various proteolytic enzymes, resulting in alkylation and reduction of the samples. Subsequently, molecular weight data was obtained from these samples using high-mass MALDI MS and nLC-LTQ-Orbitrap MS, and then analyzed using software (XQuest, Stavrox).

[0139] The sequences of the sites where protein A56 and each anti-A56 antibody (Ab18) are in direct contact with each other were identified using cross-linking. Based on this, the epitopes and paratopes shown in Table 7 were identified as follows.

[0140] [Table 7]

[0141] As shown in Table 7, the epitopes on the binding sites of A56-C-His and the anti-A56 antibody (Ab18) were identified as being at positions 46, 50, 55, 56, 60, 71, 75, and 76 in the amino acid sequence of A56-C-His.

[0142] In detail, as shown in Figure 47, the epitope positions of amino acid residues from position 30 to 90 in the amino acid sequence of A56-C-His are shown in red (positions 46, 50, 55, 56, 60; 71, 75, 76); and when the protein structure of A56-C-His (at positions 2-148 in the amino acid sequence) is represented in silico using Swiss-Model software, the binding sites of Ab18 and A56-C-His are shown in blue. Within the amino acid sequence of A56-C-His, the identified binding sites were found to correspond to positions 46-60 (SDVLYTKDKIS) and 71-76 (TITIKS).

[0143] Example 3.5. Epitope mapping of protein A56 against anti-A56 antibody (Ab01). Samples of the antigen-antibody conjugate (A56-C-His / Ab01) were subjected to deuterated crosslinking and combined treatment with various proteolytic enzymes, resulting in alkylation and reduction of the samples. Subsequently, molecular weight data was obtained from these samples using high-mass MALDI MS and nLC-LTQ-Orbitrap MS, and then analyzed using software (XQuest, Stavrox).

[0144] The sequences of the sites where protein A56 and each anti-A56 antibody (Ab01) are in direct contact with each other were identified using cross-linking. Based on this, the epitopes and paratopes shown in Table 8 were identified as follows.

[0145] [Table 8]

[0146] As shown in Table 8, the epitopes on the binding sites of A56-C-His and the anti-A56 antibody (Ab01) were identified as being at positions 30, 38, 45, 75, and 84 in the amino acid sequence of A56-C-His.

[0147] In detail, as shown in Figure 48, the epitope positions of amino acid residues from position 30 to 90 in the amino acid sequence of A56-C-His are shown in red (positions 30, 38, 45, 75, and 84); and when the protein structure of A56-C-His (from position 2 to 148 in the amino acid sequence) is represented in silico using Swiss-Model software, the binding sites of Ab01 and A56-C-His are shown in blue. Within the amino acid sequence of A56-C-His, the identified binding sites were found to correspond to positions 30-45 (SAWYKEPNSIILLAAK) and 75-84 (KSLTARDAGT).

[0148] Example 3.6. Epitope mapping of protein A56 against anti-A56 antibody (Ab19). Samples of the antigen-antibody conjugate (A56-C-His / Ab19) were subjected to deuterated crosslinking and combined treatment with various proteolytic enzymes, resulting in alkylation and reduction of the samples. Subsequently, molecular weight data was obtained from these samples using high-mass MALDI MS and nLC-LTQ-Orbitrap MS, and then analyzed using software (XQuest, Stavrox).

[0149] The sequences of the sites where protein A56 and each anti-A56 antibody (Ab19) are in direct contact with each other were identified using cross-linking. Based on this, the epitopes and paratopes shown in Table 9 were identified as follows.

[0150] [Table 9]

[0151] As shown in Table 9, the epitopes on the binding site between A56-C-His and the anti-A56 antibody (Ab19) were identified as being at positions 45 and 46 in the amino acid sequence of A56-C-His.

[0152] In detail, as shown in Figure 49, the epitope positions of amino acid residues 30 through 90 in the amino acid sequence of A56-C-His are shown in red (positions 45 and 46); and when the protein structure of UTTA-C-His (positions 2 through 148 in the amino acid sequence) is represented in silico using Swiss-Model software, the binding sites of Ab19 and A56-C-His are shown in blue. Within the amino acid sequence of A56-C-His, the identified binding sites were identified to correspond to positions 45 and 46 (KS).

[0153] Example 3.7. Comparison of epitope mapping of protein A56 against anti-A56 antibodies (Abl3, Ab16, Ab18, Ab01, Ab19) The results obtained by epitope mapping in Examples 3.2 to 3.6 were compared and are shown in Figures 50 and 51. As a result, it was identified that the epitope in the amino acid sequence of protein A56 maps to positions 30 to 85, which correspond to the IgG-like domain region.

[0154] In addition, the IgG-like domain of protein A56 was modeled in primary 3D protein structure using SWISS-MODEL and structurally analyzed using iCn3D. As a result, it was identified that the domain consists of a total of eight sheets (green), four helices (red), and seven loops (blue). In particular, it was identified that helices 1 and 2 are structurally folded immediately next to helix 3.

[0155] Structural analysis of the A56 epitope involved in binding to anti-A56 antibodies (Ab13, Ab16, Ab18, Ab01, Ab19) has shown that the epitope consists of positively charged basic amino acid residues lysine (K / Lys) and arginine (R / Arg), nucleophilic amino acid residues serine (S / Ser) and threonine (T / Thr), and the aromatic tyrosine (Y / Tyr). Basic amino acids readily form hydrogen bonds due to their positively charged side chains, while nucleophilic and aromatic amino acids can form hydrogen bonds by being partially negatively charged by adjacent amino acids. The main physicochemical properties of the amino acid residues that crosslink protein A56 and anti-A56 antibodies are summarized.

[0156] Example 4.5 Paratope analysis of protein A56 for anti-A56 antibodies. The amino acid sequence numbering in A56, as shown in Examples 4.1 to 4.5 and the drawings referred thereto, is based on the sequence obtained by removing N-terminal amino acids 1 to 16 from the amino acid sequence represented by SEQ ID NO: 1 (for example, the serine at position 30 corresponds to the serine at position 46 in the amino acid sequence represented by SEQ ID NO: 1).

[0157] Example 4.1. Paratope analysis of protein A56 using anti-A56 antibody (Ab13) The primary 3D protein structure of Ab13 was modeled using SWISS-MODEL software, and the structural analysis of the amino acids involved in binding was performed using iCn3D. As a result, it was analyzed that the nucleophilic amino acids serine (S / Ser) and threonine (T / Thr), and the aromatic tyrosine (Y / Tyr) are distributed in the terminal exposed portion of the heavy chain, and therefore have a strong binding affinity to the strongly positively charged lysine (K91) and arginine (R96) in protein A56; and that the strongly positively charged lysine (K26) is located in CDR1 of the light chain, lysine (K57) is located in the loop between sheet 5 (S5) and sheet 6 (S6), and that the nucleophilic amino acids serine (S) and tyrosine (Y / Tyr) in protein A56 have a binding affinity to lysine (K26, K57) (Figure 52).

[0158] Example 4.2. Paratope analysis of protein A56 using anti-A56 antibody (Ab16) Ab16 was modeled as a primary 3D protein structure using SWISS-MODEL software, and the structure of the amino acids involved in binding was analyzed using iCn3D. As a result, it was analyzed and predicted that arginine (R56) and lysine (K58) in CDR2, located in the loop between sheet 5 (S5) and sheet 6 (S6), which are the terminal exposed portions of the heavy chain, have a stronger positive charge (blue mash in Figure 58) due to the nucleophilic amino acid serine (S57) located between these two amino acids, which is advantageous in that binding to the nucleophilic amino acids serine (S46, S54) in protein A56 proceeds preferentially; and that this antibody has a high level of binding affinity to the nucleophilic amino acids serine (S62) and lysine (K61) in protein A56 due to the strongly positively charged lysine (K60) and nucleophilic amino acid serine (S) located in sheet 6 (S6) of FR3 of the light chain (Figure 53).

[0159] Example 4.3. Paratope analysis of protein A56 using anti-A56 antibody (Ab18) The primary 3D protein structure of Ab18 was modeled using SWISS-MODEL software, and the structural analysis of amino acids involved in binding was performed using iCn3D. As a result, the nucleophilic amino acids serine (S / Ser) and threonine (S / Ser), as well as the aromatic tyrosine (Y / Tyr), are distributed in the exposed portion of the CDR1 helix of the heavy chain. Therefore, this portion is predicted to have a relatively negative charge and thus bind to the strongly positively charged lysine (K72) in protein A56; and the strongly positively charged arginine (R98) was identified as being located in CDR3. In addition, it was analyzed that the nucleophilic amino acid serine (S30) in the CDR1 of the light chain binds to the strongly positively charged lysine (K91) in protein A56, and that lysine (K50) located between sheet 5 (S5) and the loop binds to tyrosine (Y / Tyr, T71) in protein A56 (Figure 54).

[0160] Example 4.4. Paratope analysis of protein A56 using anti-A56 antibody (Ab01) The primary 3D protein structure of Ab01 was modeled using SWISS-MODEL software, and the structural analysis of the amino acids involved in binding was performed using iCn3D. As a result, it was analyzed that the nucleophilic amino acids serine (S / Ser) and threonine (T / Thr), and the aromatic tyrosine (Y106) are distributed in the terminal exposed portion of the heavy chain, and therefore have a binding affinity to the strongly positively charged lysine (K91) in protein A56; and the nucleophilic amino acids serine (S) and threonine (T102) are mainly located in the CDR of the light chain, and in particular, threonine (T102) has a binding affinity to the strongly positively charged lysine (K61) in protein A56 (Figure 55).

[0161] Example 4.5. Paratope analysis of protein A56 using anti-A56 antibody (Ab19) Ab19 was modeled as a primary 3D protein structure using SWISS-MODEL software, and the structure of the amino acids involved in binding was analyzed using iCn3D. As a result, it was analyzed that this antibody has a relatively low binding affinity due to simple hydrogen bonding caused by serine (S93, S94), and that this antibody cannot overlap with other anti-A56 antibodies in terms of epitopes due to the binding site (K61, S62) located in the loop between sheet 4 (S4) and sheet 5 (S5) of protein A56 (Figure 56).

[0162] Example 5. Analysis of the binding of anti-A56 antibody to protein A56. The amino acid sequence numbering in A56, as shown in Examples 5.1.1 to 5.5.3 and the drawings referred thereto, is based on the sequence obtained by removing N-terminal amino acids 1 to 16 from the amino acid sequence represented by SEQ ID NO: 1 (for example, the serine at position 30 corresponds to the serine at position 46 in the amino acid sequence represented by SEQ ID NO: 1).

[0163] Example 5.1.1. Analysis of electrostatic force binding between anti-A56 antibody (Ab13) and protein A56. To analyze the electrostatic force of the binding of the anti-A56 antibody (Ab13) to protein A56, we used the DelPhi potential, a scientific application for calculating the electrostatic potential and corresponding electrostatic energy in and around macromolecules. The DelPhi potential is a commonly used analytical method for visualizing electrostatic changes along the surface of proteins or other macromolecules and for calculating electrostatic components of various energies. In the DelPhi potential, the effect of ion intensity-mediated screening is incorporated by evaluating the Poisson-Boltzmann equation at a finite number of points in a three-dimensional lattice box. Here, blue and red represent positive and negative potentials, respectively, and the force lines indicate the direction and intensity of the electrostatic force surrounding the protein.

[0164] In detail, in protein A56, the positively charged basic amino acid lysine (K91), which has a strong DelPhi surface electrostatic potential, excited the hydroxyl group of the serine (S92) residue immediately adjacent to lysine. As a result, an electrostatic force was generated, and the relatively low level of the DelPhi surface electrostatic potential became strongly positively charged. At that time, for K26 and K57 of the light chain in Ab13, the negatively charged D25 and D55 surrounding them formed a relatively strongly positively charged DelPhi surface electrostatic potential.

[0165] In addition, T52 and T58 of the heavy chain in Ab13 are highly exposed to the outside due to the adjacent hydrophobic residue I51 (isoleucine) and the aromatic and hydrophilic-free F54 (phenylalanine), and the high levels of DelPhi surface electrostatic potential of T52 and T58 are strongly negatively charged due to the negatively charged D56.

[0166] As a result, the electrostatic properties shown in Table 10 were identified.

[0167] [Table 10]

[0168] In this table, the letters (H or L) following the numbers of the amino acid positions shown in the rightmost column indicate the heavy chain (H) or light chain (L).

[0169] Example 5.1.2. Analysis of the binding distance between anti-A56 antibody (Ab13) and protein A56. To analyze the binding distance between the anti-A56 antibody (Ab13) and protein A56, the structural alignment of protein A56 with Ab13 was estimated using Swiss-PdbViewer (4.1.0), and the interatomic H bonds, angles, and distances were predicted by comparing the active site or other relevant regions.

[0170] As a result, the NH residue, specifically the strongly positive terminal functional group K91 in protein A56, formed a hydrogen bond with the O- of T52 in the heavy chain of Ab13, which is negatively charged at an appropriate level, and the distance between them was measured to be 4.88 Å (Figure 57A). In addition, the NH residue, specifically the strongly positive terminal functional group K57 in the heavy chain of Ab13, formed a hydrogen bond with the O- of S62 in protein A56, which is negatively charged at an appropriate level, and the distance between them was measured to be 3.73 Å (Figure 57B).

[0171] As the two hydrogen bonds progressed, it was predicted that the negatively charged O- of Y66 in protein A56 would bind to the positively charged K26 on the light chain at Ab13. In addition, as the two hydrogen bonds progressed, the heavy and light chains were predicted to undergo contraction folding, resulting in the electrostatic formation of the remaining bonds (S54(A56)⇔(heavy chain in antibody) and R96(A56)⇔(heavy chain in antibody)).

[0172] Example 5.1.3. Competitive assay for binding of anti-A56 antibody (Ab13) to protein A56: Binning test To determine whether 10 anti-A56 antibodies (Ab18, Ab19, Ab01, Ab13, Ab14, Ab08, Ab03, Ab51, Ab55, Ab16) have the same or different epitopes for protein A56, analysis was performed using surface plasmon resonance (SPR) with an Octet instrument.

[0173] In detail, the A56-C-His antigen was immobilized on a biosensor (NTA), primary antibodies (SA2038, Ab13, A56-02A02) were bound to the antigen to a saturated state, and the remaining nine antibodies were further bound to the antigen as secondary antibodies. Here, if the antibodies have the same epitope, competition occurs and additional binding is difficult to achieve; if the antibodies have different epitopes, additional binding will occur. In addition, the results were checked again by performing additional experiments in which the binding order of the secondary and primary antibodies to the A56-C-His antigen was reversed.

[0174] As a result, as shown in Figure 58A, it was identified that no additional binding occurred, indicating that the antibodies possessed the same epitope.

[0175] In addition, Western blotting was performed using polyacrylamide gel electrophoresis (PAGE) to check the binding affinity in the two-dimensional structure, and it was identified that no binding occurred to the linear protein A56. From these results, it was identified that the anti-A56 antibody (Ab13) recognizes and binds only to the three-dimensional structure of protein A56. Furthermore, the binding affinity (K) calculated by OCTET (SPR) and ELISA was also determined. D The values ​​are shown in Figure 58B.

[0176] Example 5.2.1. Analysis of electrostatic force binding between anti-A56 antibody (Ab16) and protein A56. The DelPhi potential was used to analyze the electrostatic force of the binding between the anti-A56 antibody (Ab16) and protein A56. Specifically, the basic amino acid lysine (K61), which has a strong positive charge in the DelPhi surface electrostatic potential of protein A56, excited the hydroxyl group of serine (S62), the residue immediately adjacent to lysine. As a result, an electrostatic force was generated, and the relatively low level of the DelPhi surface electrostatic potential became strongly positively charged. Here, the positively charged DelPhi surface electrostatic potential of S59 and K60 in the light chain of Ab16 was formed due to the physicochemical properties of protein A56.

[0177] In addition, arginine (R56) and lysine (K58) in CDR2 located in the loop between sheet 5 (S5) and sheet 6 (S6), which is the terminal-exposed part of the heavy chain in Ab16, have a stronger positive charge due to the nucleophilic amino acid serine (S57) located between the two amino acids. As a result, it was predicted that the binding with serine (S46, S54), which is a nucleophilic amino acid in protein A56, would preferentially proceed; the electrostatic properties as shown in Table 11 were identified.

[0178]

Table 11

[0179] In this table, the letter (H or L) following the number of the amino acid position shown in the rightmost column means the heavy chain (H) or the light chain (L).

[0180] Example 5.2.2. Analysis of the binding distance between anti-A56 antibody (Ab16) and protein A56 To analyze the binding distance between anti-A56 antibody (Ab16) and protein A56, the structural alignment of protein A56 with Ab16 was inferred using Swiss-PdbViewer (4.1.0), and the active site or other related parts were compared to predict the H-bonds, angles, and distances between atoms.

[0181] As a result, as shown in Figure 59, the NH residue, that is, the terminal functional group of K61, which is positively charged with a strong positive charge in protein A56, forms a hydrogen bond with the O- of S59, which is negatively charged with an appropriate level of negative charge in the light chain of Ab16, and the distance between them was measured to be 4.78 Å. At the same time, the NH residue, that is, the terminal functional group of K60, which is positively charged with a strong positive charge in the light chain of Ab16, forms a hydrogen bond with the O- of S62, which is negatively charged with an appropriate level of negative charge in protein A56, and the distance between them was measured to be 5.07 Å.

[0182] As the two hydrogen bonds progressed, it was predicted that the negatively charged O- of S46 / S54 in protein A56 would bind to the positively charged R56 / K58 in Ab16, which is strongly positively charged due to the heavy chain's strong positive charge. In addition, as the binding progressed, the heavy and light chains were predicted to undergo contraction folding, resulting in the remaining bond being strongly electrostatically formed.

[0183] Example 5.2.3. Competitive assay for binding of anti-A56 antibody (Ab16) to protein A56: Binning test To determine whether 10 anti-A56 antibodies (Ab18, Ab19, Ab01, Ab13, Ab14, Ab08, Ab03, Ab51, Ab55, Ab16) have the same or different epitopes for protein A56, analysis was performed using surface plasmon resonance (SPR) with an Octet instrument.

[0184] In detail, the A56-C-His antigen was immobilized on a biosensor (NTA), primary antibodies (SA2041, Ab16, A56-02B08) were bound to the antigen to a saturated state, and the remaining nine antibodies were further bound to the antigen as secondary antibodies. Here, if the antibodies have the same epitope, competition occurs and additional binding is difficult to achieve; if the antibodies have different epitopes, additional binding will occur. In addition, the results were checked again by performing additional experiments in which the binding order of the secondary and primary antibodies to the A56-C-His antigen was reversed.

[0185] As a result, as shown in Figure 60A, it was identified that no additional binding occurred, indicating that the antibodies possessed the same epitope.

[0186] In addition, Western blotting was performed using PAGE to check the binding affinity in the two-dimensional structure, and multiple linear bands were identified. This indicates that there was no specific one-to-one binding to the simple linear amino acid sequence in the two-dimensional structure of protein A56, and therefore it was not identified. Furthermore, the binding affinity (K) calculated by OCTET(SPR) and ELISA was also performed. D The values ​​are shown in Figure 60B.

[0187] Example 5.3.1. Analysis of electrostatic force binding between anti-A56 antibody (Ab18) and protein A56. The DelPhi potential was used to analyze the electrostatic force of the binding between the anti-A56 antibody (Ab18) and protein A56. Specifically, the basic amino acid lysine (K61), which has a strong positive charge and a strong DelPhi surface electrostatic potential in protein A56, excited the hydroxyl group of serine (S62), the residue immediately adjacent to lysine. As a result, an electrostatic force was generated, and the relatively low level of the DelPhi surface electrostatic potential became strongly positively charged. Here, R98 in the heavy chain of Ab18 was highly exposed to the outside due to the adjacent A97 and the short methyl group (CH3) of W99 (tryptophan), which is aromatic and does not contain hydrophilic reactive groups, and the high level of the DelPhi surface electrostatic potential of R98 became strongly positively charged. In addition, for K50 in the light chain of Ab18, the negatively charged Y49 and S52 around K50 formed a relatively strongly positively charged DelPhi surface electrostatic potential.

[0188] As a result, the electrostatic properties shown in Table 12 were identified.

[0189] [Table 12]

[0190] In this table, the letters (H, L, or U) following the numbers of the amino acid positions shown in the rightmost column represent the heavy chain (H), light chain (L), or UTTA (or A56) (U).

[0191] Example 5.3.2. Analysis of the binding distance between anti-A56 antibody (Ab18) and protein A56. To analyze the binding distance between the anti-A56 antibody (Ab18) and protein A56, the structural alignment of protein A56 with Ab18 was estimated using Swiss-PdbViewer (4.1.0), and the interatomic H bonds, angles, and distances were predicted by comparing the active site or other relevant regions.

[0192] As a result, the NH residue, i.e., the terminal functional group K91 of protein A56 which is strongly positive, formed a hydrogen bond with the Y32L of the light chain in Ab18 which is negatively charged at an appropriate level, and the distance between them was measured to be 3.46 Å (Figure 61B). In addition, it was predicted that the structure would fold by electrostatically pulling the S30 of the light chain in Ab18 which is negatively charged at an appropriate level. Furthermore, the NH residue, i.e., the terminal functional group R98 of the heavy chain in Ab18 which is strongly positive, formed a hydrogen bond with the O- of S62 of protein A56 which is negatively charged at an appropriate level, and the distance between them was measured to be 3.77 Å (Figure 61A).

[0193] As the two hydrogen bonds progressed, it was predicted that the strongly positively charged K50 on the light chain of Ab18 would fold and bind to the negatively charged O- of S30 in protein A56.

[0194] Example 5.3.3. Competitive assay for binding of anti-A56 antibody (Ab18) to protein A56: Binning test To determine whether 10 anti-A56 antibodies (Ab18, Ab19, Ab01, Ab13, Ab14, Ab08, Ab03, Ab51, Ab55, Ab16) have the same or different epitopes for protein A56, analysis was performed using the SPR method with an Octet instrument.

[0195] Specifically, the A56-C-His antigen was immobilized on a biosensor (NTA), and a primary antibody (SA2043, Ab18, A56-02C06) was bound to the antigen until saturation, and the remaining nine antibodies were further bound to the antigen as secondary antibodies. Here, if these antibodies have the same epitope, competition will occur and additional binding will be difficult; if these antibodies have different epitopes, additional binding will occur. In addition, the results were checked again by conducting additional experiments in which the binding order of the secondary antibody and the primary antibody to the A56-C-His antigen was reversed.

[0196] As a result, as shown in Figure 62A, it was identified that no additional binding occurred, indicating that these antibodies have the same epitope.

[0197] In addition, Western blotting was performed to check the binding affinity in the two-dimensional structure using PAGE, and multiple linear bands were identified. From these results, it was identified that this antibody recognizes and binds not only to the amino acid sequence of the two-dimensional structure in protein A56 but also to the three-dimensional motif in protein A56. In addition, the binding affinity (K D ) values calculated by OCTET (SPR) and ELISA are shown in Figure 62B.

[0198] Example 5.4.1. Analysis of the electrostatic force of the binding between anti-A56 antibody (Ab01) and protein A56 To analyze the electrostatic force of the binding between anti-A56 antibody (Ab01) and protein A56, the DelPhi potential was used. Specifically, in protein A56, the basic amino acid lysine (K61) with a strong positive charge of the DelPhi surface electrostatic potential excited the hydroxyl group of serine (S62), which is the residue immediately adjacent to that lysine, resulting in the generation of an electrostatic force and a relatively low level of the DelPhi surface electrostatic potential being strongly positively charged. At that time, for S32 and T102 of the light chain in Ab01, their DelPhi surface electrostatic potentials were relatively negatively charged at an appropriate level.

[0199] In addition, the heavy chains S103 and Y106 in Ab01 are highly exposed to the outside due to the adjacent hydrophobic residue L107 and the aromatic and hydrophilic reactive group-free F101, and its high level of DelPhi surface electrostatic potential is strongly negatively charged due to the nucleophilic amino acid S103.

[0200] As a result, the electrostatic properties shown in Table 13 were identified.

[0201] [Table 13]

[0202] In this table, the letters (H or L) following the numbers of the amino acid positions shown in the rightmost column indicate the heavy chain (H) or light chain (L).

[0203] Example 5.4.2. Analysis of the binding distance between anti-A56 antibody (Ab01) and protein A56. To analyze the binding distance between the anti-A56 antibody (Ab01) and protein A56, the structural alignment of protein A56 with Ab01 was estimated using Swiss-PdbViewer (4.1.0), and the interatomic H bonds, angles, and distances were predicted by comparing the active site or other relevant regions.

[0204] As a result, as shown in Figure 63, the NH residue, i.e., the strongly positively charged terminal functional group K61 in protein A56, formed a hydrogen bond with the O- of T102 in the light chain of Ab01, which is negatively charged at an appropriate level, and the distance between them was measured to be 3.69 Å. In addition, the NH residue, i.e., the strongly positively charged terminal functional group K91 in protein A56, formed a hydrogen bond with the O- of Y106 in the heavy chain of Ab01, which is negatively charged at an appropriate level, and the distance between them was measured to be 3.12 Å.

[0205] As the two hydrogen bonds progressed, it was predicted that S54 in protein A56 would bind to T57, T100, and S103 in the heavy chain and S32 in the light chain of Ab01. In addition, as the two hydrogen bonds progressed, it was predicted that the heavy and light chains would undergo contraction folding, resulting in the formation of the remaining bonds electrostatically.

[0206] Example 5.4.3. Competitive assay for binding of anti-A56 antibody (Ab01) to protein A56: Binning test To determine whether 10 anti-A56 antibodies (Ab18, Ab19, Ab01, Ab13, Ab14, Ab08, Ab03, Ab51, Ab55, Ab16) have the same or different epitopes for protein A56, analysis was performed using the SPR method with an Octet instrument.

[0207] In detail, the A56-C-His antigen was immobilized on a biosensor (NTA), primary antibodies (SA2026, Ab01, A56-01A02) were bound to the antigen to a saturated state, and the remaining nine antibodies were further bound to the antigen as secondary antibodies. Here, if the antibodies have the same epitope, competition occurs and additional binding is difficult to achieve; if the antibodies have different epitopes, additional binding will occur. In addition, the results were checked again by performing additional experiments in which the binding order of the secondary and primary antibodies to the A56-C-His antigen was reversed.

[0208] As a result, as shown in Figure 64A, it was identified that no additional binding occurred, indicating that the antibodies possessed the same epitope.

[0209] In addition, Western blotting was performed using PAGE to check the binding affinity in the two-dimensional structure, and a linear single band was identified. Furthermore, the binding affinity (K) was calculated by OCTET(SPR) and ELISA. D The values ​​are shown in Figure 64B.

[0210] Example 5.5.1. Analysis of electrostatic force binding between anti-A56 antibody (Ab19) and protein A56. The DelPhi potential was used to analyze the electrostatic force of the binding between the anti-A56 antibody (Ab19) and protein A56. Specifically, the basic amino acid lysine (K61), which has a strong positive charge in the DelPhi surface electrostatic potential of protein A56, excited the hydroxyl group of the serine (S62) residue immediately adjacent to lysine, resulting in the generation of an electrostatic force and a strongly positive charge of the relatively low level of the DelPhi surface electrostatic potential. Here, among the amino acids from position 90 to 95 (DSSSD) of the light chain in Ab19, the serine (S93, S94) at positions 93 and 94 had their DelPhi surface electrostatic potentials relatively strongly negatively charged due to the strong negative charge of the acidic amino acid aspartic acid residues (D90 and D95).

[0211] In addition, the heavy chain Y32 in Ab19 was highly exposed to the outside due to the hydrophobic amino acids V (valine) and L (leucine) immediately adjacent to it, as well as the aromatic and hydrophilic non-reactive F (phenylalanine), and its low level DelPhi surface electrostatic potential was negatively charged at a low level due to the nucleophilic amino acid S (serine).

[0212] As a result, the electrostatic properties shown in Table 14 were identified.

[0213] [Table 14]

[0214] Example 5.5.2. Analysis of the binding distance between anti-A56 antibody (Ab19) and protein A56. To analyze the binding distance between the anti-A56 antibody (Ab19) and protein A56, the structural alignment of protein A56 with Ab19 was estimated using Swiss-PdbViewer (4.1.0), and the interatomic H bonds, angles, and distances were predicted by comparing the active site or other relevant regions.

[0215] As a result, as shown in Figure 65, the terminal functional groups K61 and S62 in protein A56 were ionized to form NH+(K61) and O-(S62), and the light chain S93 and S94 in Ab19 had a strong negative charge due to the adjacent aspartic acid residues (D91 and D95), giving the O-(S93, S94) an electrostatic surface DelPhi potential, which was predicted to result in the formation of a bond. The NH2+(K61) and O-(S62) in protein A56, separated by 9.9 Å, were located between the light chain O-(S93) and the heavy chain Y32 O- in Ab19, separated by 19.5 Å. As a result, K61 in protein A56 bound to S93 in Ab19, and the binding distance was measured to be 4.59 Å (indicated by the pink circle). The binding distance between Y32 in Ab19 and S62 in protein A56 was measured to be 6.08 Å (indicated by the white circle).

[0216] Furthermore, the binding of protein A56 to Ab19 indicates that the two molecules bind horizontally. Therefore, it was predicted that if another antibody with a specific paratope other than the binding site has a complementary electrostatic surface DelPhi potential, multiple bindings would be possible due to the presence of different epitopes on protein A56.

[0217] Example 5.5.3. Competitive assay for binding of anti-A56 antibody (Ab19) to protein A56: Binning test To determine whether 10 anti-A56 antibodies (Ab18, Ab19, Ab01, Ab13, Ab14, Ab08, Ab03, Ab51, Ab55, Ab16) have the same or different epitopes for protein A56, analysis was performed using the SPR method with an Octet instrument.

[0218] In detail, the A56-C-His antigen was immobilized on a biosensor (NTA), primary antibodies (SA2044, Ab19, A56-02C07) were bound to the antigen to a saturated state, and the remaining nine antibodies were further bound to the antigen as secondary antibodies. Here, if the antibodies have the same epitope, competition occurs and additional binding is difficult; if the antibodies have different epitopes, additional binding will occur. In addition, the results were checked again by performing additional experiments in which the binding order of the secondary and primary antibodies to the A56-C-His antigen was reversed.

[0219] As a result, as shown in Figure 66A, it was analyzed that a small amount of additional binding occurred to two antibodies (SA2041(Ab16) and SA2043(Ab18)) during the secondary antibody binding step. These results identified that these two antibodies (SA2041(Ab16) and SA2043(Ab18)) possess epitopes other than the Ab19 epitope.

[0220] In addition, Western blotting was performed using PAGE to check the binding affinity in the two-dimensional structure, and a linear single band was identified. Furthermore, the binding affinity (K) was calculated by OCTET(SPR) and ELISA. D The values ​​are shown in Figure 66B.

[0221] Experimental Example 8. Production of CAR-T cells using the antigen-binding domain of anti-A56 antibody. Experimental Example 8.1. Structural Design of Chimeric Antigen Receptors The structure of the chimeric antigen receptor was designed to include a signal peptide, an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. For example, as shown in Figure 67, the chimeric antigen receptor was designed to include an antigen-binding domain (anti-UTTA scFv), a CD8 transmembrane domain (H+TM), and intracellular signaling domains (4-1BB and CD3Z).

[0222] Experimental Example 8.2. Construction of a vector encoding a chimeric antigen receptor The pLVX-EF1α-IRES-mCherry vector (Spel / Notl) was used as the vector. A gene encoding a chimeric antigen receptor, including a signal peptide (sp), a single-stranded variable fragment that specifically binds to A56 (anti-UTTA scFv), the transmembrane domain of human CD8 (H+TM), and intracellular signaling domains (4-1BB and CD3ζ), was inserted into this vector.

[0223] The amino acid and nucleotide sequences of the chimeric antigen receptor used in this experiment are shown in Table 15.

[0224] [Table 15] JPEG0007842773000016.jpg216149 JPEG0007842773000017.jpg168149 JPEG0007842773000018.jpg215149 JPEG0007842773000019.jpg203149

[0225] Experimental Example 8.3. Production of CAR-T Cells Following approval by the IRB under human-derived materials research, CAR-T cells were generated by isolating CD4+ / CD8+ cells from blood provided by Pusan ​​National University Yangsan Hospital (Korea) using the MACS cell isolation system. Subsequently, CD3+ T cells (≥97%) were isolated using MACS Pan T cell Ab. Subsequently, 1 × 10⁻⁶ cells were isolated. 6 T cell activation was induced by culturing individual CD3+ T cells in a medium containing 20 IU / ml of rhIL-2 and TransAct (CD3 / CD28 agonist) for 24 hours.

[0226] Activated T cells were treated with each of the two types of lentivirus cloned into the vector constructed in Experimental Example 8.2 at a MOI of 50 and cultured for 48 hours. Subsequently, the T cells were placed in a medium containing 20 IU / ml of rhIL-2 at a dose of 1 × 10⁶. 6 The cells were resuspended at a concentration of 10 cells / ml. The cells were counted every 2 or 3 days, with the medium being replaced with fresh medium containing rhIL-2 for cell proliferation. Here, CAR-T cells generated using a lentivirus targeting the antigen-binding domain of Ab16 were named "16 CAR-T," and CAR-T cells generated using a lentivirus targeting the antigen-binding domain of Ab18 were named "18 CAR-T." In addition, T cells that were not transduced by any CAR (UTD, untransduced) were used as a control group.

[0227] Experimental Example 9. Identification of the anticancer effects of oncolytic vaccinia virus (OTS-412) and CAR-T cells. The anticancer effects of UTD cells and five types of CAR-T cells were analyzed by measuring cell death in real time using a fluorescence-based cell imaging system (Incucyte® live cell analysis system, Sartorius).

[0228] In detail, three cancer cell lines HeLa (3 × 10 3 (Individual cells / well), NCI-H522 (6×10) 3 (individual cells / well), and HCT-116 (9 × 10 3 The cells were seeded into a 96-well plate. Subsequently, the cells were incubated at 37°C while being prepared in OTS-412 (1.55 × 10⁶) at an MOI of 0.05. 8The cells were subjected to infection with pfu / ml, resulting in the expression of protein A56 on the surface of the cancer cells. After 2 hours, the culture medium was replaced with a medium containing 2% FBS. After 4 hours, UTD cells and five types of CAR-T cells (Ab13 CAR-T, Ab16 CAR-T, Ab18 CAR-T, Ab01 CAR-T, Ab19 CAR-T) were administered in a T cell:cancer cell ratio of 3:1. The T cells were administered with a medium containing red fluorescent staining reagent (10% FBS). Subsequently, cell death image data was acquired at 30-minute intervals for 5 days using the Incucyte system and analyzed through software.

[0229] As a result, when comparing HeLa cell lines infected with OTS-412 with the group treated with UTD cells, each of the five types of CAR-T cells showed a statistically significant specific cytotoxic effect (Figure 68; * indicates p<0.033 compared to the UTD group, ** indicates p<0.002, and *** indicates p<0.001, and ## indicates p<0.002 compared to treatment with T cells alone in the same scFv Car-T group). In particular, among these, Ab16 CAR-T and Ab18 CAR-T cells showed a statistically significant difference in protein A56-specific cytotoxicity compared to the cytotoxicity of the group not infected with OTS-412.

[0230] When NCI-H522 cell lines infected with OTS-412 were compared with a group treated with UTD cells, groups treated with Ab01 CAR-T, Ab13 CAR-T, Ab16 CAR-T, and Ab19 CAR-T cells, among the five types of CAR-T cells, showed statistically significant specific cytotoxic effects (Figure 69; * indicates p<0.033 compared to the UTD group, ** indicates p<0.002, and *** indicates p<0.001, and ## indicates p<0.002 compared to treatment with T cells alone in the same scFv Car-T group). In particular, among these, Ab16 CAR-T cells showed a statistically significant difference in protein A56-specific cytotoxicity compared to the cytotoxicity of the group not infected with OTS-412.

[0231] When HCT-116 cell lines infected with OTS-412 were treated with UTD cells, the groups treated with each of the five types of CAR-T cells showed statistically significant CAR-T cell-specific cytotoxic effects compared to the group treated with UTD cells (Figure 70; * indicates p<0.033 compared to the UTD group, ** indicates p<0.002, and *** indicates p<0.001, and ## indicates p<0.002 compared to treatment with T cells alone in the same scFv Car-T group). In particular, among these, Ab01 CAR-T, Ab16 CAR-T, Ab18 CAR-T, and Ab19 CAR-T cells showed statistically significant differences in protein A56-specific cytotoxicity compared to the cytotoxicity of the group not infected with OTS-412.

[0232] Furthermore, when the transduction efficiency of the five types of CAR-T cells was measured by flow cytometry (FACS), different peaks were observed for Ab13 CAR-T, Ab16 CAR-T, and Ab19 CAR-T cells; on the other hand, for Ab01 CAR and Ab18 CAR-T cells, a peak distinct from the negative peak was not clearly observed due to the low intensity of CAR expressed on the T cell surface, and their transduction efficiency was also thought to be low (Figure 71). The differences in transduction efficiency among the five CAR-T cells were similar to those of blood derived from subjects of different ages and sexes. Transduction efficiency was consistently higher in Ab13 CAR-T, Ab16 CAR-T, and Ab19 CAR-T than in Ab01 CAR-T and Ab18 CAR-T; among these, Ab16 CAR-T consistently showed the highest transduction efficiency.

[0233] Experimental Example 10. Identification of the anticancer effects of oncolytic vaccinia virus (OTS-412) and CAR-T cells. The anticancer effects of UTD cells and five types of CAR-T cells were analyzed by measuring cell death in real time using a fluorescence-based cell imaging system (Incucyte® live cell analysis system, Sartorius).

[0234] In detail, four cancer cell lines A549 (1 × 10) 4 (individual cells) and HCT-116 (3 × 10) 4 The cells were seeded into a 96-well plate. Subsequently, the cells were incubated at 37°C while being prepared in OTS-412 (1.55 × 10⁶) at an MOI of 0.05. 8 The cells were subjected to infection with pfu / ml, resulting in the expression of protein A56 on the surface of the cancer cells. After 2 hours, the culture medium was replaced with a medium containing 2% FBS. After 4 hours, UTD cells, Ab16 CAR-T cells, and Ab18 CAR-T cells were administered in a T cell:cancer cell ratio of 3:1. The T cells were administered with a medium containing red fluorescent staining reagent (10% FBS). Subsequently, cell death image data was acquired at 30-minute intervals for 5 days using the Incucyte system and analyzed through the software.

[0235] As a result, as shown in Figure 73, it was identified that for A549 and HCT-116 cell lines infected with OTS-412, more dead cells were stained in the groups treated with Ab16 CAR-T or Ab18 CAR-T cells, respectively, compared to the group treated with UTD cells. In addition, for A549 and HCT-116 cell lines infected with OTS-412, both groups treated with Ab16 CAR-T and Ab18 CAR-T cells, respectively, showed statistically significant CAR-T cell-specific cytotoxic effects compared to the group treated with UTD cells (Figure 72).

[0236] Experimental Example 11. Identification of the anticancer effects of oncolytic vaccinia viruses (OTS-412, WOTS-418) and CAR-T cells. The anticancer effects of CAR-T cells were analyzed by measuring cell death in real time using a fluorescence-based cell imaging system (Incucyte® live cell analysis system, Sartorius).

[0237] In detail, cancer cell line HCT-116 (1 × 10 4Cells were seeded in a 96-well plate. Subsequently, while incubating at 37°C, the cells were subjected to infection with OTS-412 and WOTS-418, respectively, at a MOI of 0.05, resulting in the expression of protein A56 on the surface of the cancer cells. After 2 hours, the medium was replaced with a medium containing 2% FBS. After 4 hours, UTD cells and five types of CAR-T cells (Ab13 CAR-T, Ab16 CAR-T, Ab18 CAR-T, Ab01 CAR-T, Ab19 CAR-T) were administered in a T cell:cancer cell ratio of 3:1. The T cells were administered with a medium containing red fluorescent staining reagent (10% FBS). Subsequently, cell death image data was acquired at 30-minute intervals for 5 days using the Incucyte system and analyzed through the software.

[0238] As a result, it was identified that in HCT-116 cell lines infected with OTS-412 or WOTS-418, more dead cells were stained in the groups treated with each of the five types of CAR-T cells (Ab13 CAR-T, Ab16 CAR-T, Ab18 CAR-T, Ab01 CAR-T, Ab19 CAR-T) compared to the group treated with UTD cells (Figures 73 and 74).

Claims

1. An antibody or its antigen-binding fragment that specifically binds to protein A56, (i) Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 32, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 33, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 34, light chain CDR1 having the amino acid sequence of SEQ ID NO: 35, light chain CDR2 having the amino acid sequence of SEQ ID NO: 36, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 37; (ii) Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 38, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 39, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 40, light chain CDR1 having the amino acid sequence of SEQ ID NO: 41, light chain CDR2 having the amino acid sequence of SEQ ID NO: 42, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 43; (iii) Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 44, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 45, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 46, light chain CDR1 having the amino acid sequence of SEQ ID NO: 47, light chain CDR2 having the amino acid sequence of SEQ ID NO: 48, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 49; (iv) Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 50, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 51, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 52, light chain CDR1 having the amino acid sequence of SEQ ID NO: 53, light chain CDR2 having the amino acid sequence of SEQ ID NO: 54, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 55; or (v) Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 56, heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 57, heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 58, light chain CDR1 having the amino acid sequence of SEQ ID NO: 59, light chain CDR2 having the amino acid sequence of SEQ ID NO: 60, and light chain CDR3 having the amino acid sequence of SEQ ID NO: 61 An antibody or its antigen-binding fragment, including the above.

2. Regarding binding to A56, the antibody or antigen-binding fragment according to claim 1 competes with one or more of the following antibodies: An antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 32, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 33, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 34, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 35, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 36, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 37; An antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 38, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 39, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 40, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 41, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 42, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 43; An antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 44, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 45, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 46, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 47, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 48, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 49; An antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 50, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 51, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 52, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 53, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 54, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 55; An antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 56, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 57, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 58, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 59, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 60, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 61; An antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 62, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 63, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 64, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 65, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 66, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 67; An antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 68, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 69, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 70, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 71, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 72, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 73; An antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 74, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 75, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 76, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 77, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 78, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 79; An antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 80, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 81, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 82, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 83, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 84, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 85; An antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 86, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 87, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 88, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 89, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 90, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

91.

3. Regarding binding to A56, the antibody or antigen-binding fragment according to claim 1 or 2 competes with one or more of the following antibodies: An antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 32, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 33, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 34, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 35, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 36, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 37; An antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 38, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 39, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 40, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 41, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 42, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 43; An antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 44, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 45, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 46, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 47, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 48, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 49; An antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 50, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 51, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 52, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 53, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 54, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 55; An antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 56, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 57, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 58, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 59, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 60, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

61.

4. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, used to reduce the burden on cancer cells when administered together with an oncolytic virus.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein the oncolytic virus is vaccinia virus.

6. Vaccinia virus is used by the Western Reserve (WR), New York Vaccinia Virus (NYVAC), Wyeth (The New York City Board of Health; NYCBOH), LC16m8, Lister, Copenhagen, Tian Tan, USSR, Tashkent, Evans, International Health Division-J (IHD-J), or International Health Division-White. The antibody or antigen-binding fragment thereof according to claim 5, wherein it is Division-White (IHD-W).

7. A composition for preventing or treating cancer, comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; and Oncolytic virus A kit for preventing or treating cancer, including [mention specific ingredients / methods].

9. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 for the manufacture of a pharmaceutical product for preventing or treating cancer.

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