Chimeric antigen receptors targeting oncolytic virus-derived proteins, immune cells expressing the same, and use of both

Abstract

The present invention relates to a chimeric antigen receptor targeting an oncolytic virus-derived protein, an immune cell expressing the same, and uses thereof. In particular, the present invention relates to a chimeric antigen receptor targeting a protein A56 expressed on the surface of a cancer cell infected with a vaccinia virus, an immune cell expressing the same, and uses thereof. The chimeric antigen receptor-expressing immune cell of the present invention can effectively target the protein A56 specifically expressed on the surface of a cancer cell, enabling targeted treatment of cancer cells that survive even infection with an oncolytic virus, thereby providing an effective anti-cancer treatment. The chimeric antigen receptor-expressing immune cell of the present invention can cause an increase in activation and proliferation ability specifically against the protein A56, and can exhibit excellent cytotoxicity effects, thereby providing an effective anti-cancer treatment against the protein A56-expressing cancer cells. The immune cell is preferably used in combination with an oncolytic virus, and may additionally be used in combination with a drug capable of enhancing the anti-cancer effect of the oncolytic virus (e.g., hydroxyurea, a chemotherapeutic agent for controlling lymphocyte removal (e.g., cyclophosphamide and fludarabine), or an immunotherapeutic agent).

Description

[Technical Field]

[0001] The present invention relates to a chimeric antigen receptor that targets an oncolytic virus-derived protein, an immune cell expressing the same, and uses thereof. Specifically, the present invention relates to a chimeric antigen receptor that targets the protein A56 expressed on the surface of vaccinia virus-infected cancer cells, an immune cell expressing the same, and uses thereof. [Background technology]

[0002] Cancer, also known as tumor, refers to the abnormal growth of cells due to the autonomous overgrowth of body tissues. The incidence of cancer continues to increase in modern society due to an aging population, an increasing number of smokers, increased alcohol consumption, Westernized dietary habits, and environmental pollution.

[0003] Methods for treating cancer include surgery, radiation therapy, chemotherapy, etc. In particular, surgery is a treatment method for removing cancerous tissue from the body, and is very effective for early cancer or cancer with a specific lesion. However, it is difficult to remove cancer that has infiltrated the tissue around the lesion or metastasized to the lymph nodes, and such cancer has a high possibility of recurrence. In this case, radiation therapy or chemotherapy is used in combination with surgery. Radiation therapy or chemotherapy is mainly used to treat advanced or terminal cancer; however, this treatment also affects normal cells and therefore causes serious adverse effects.

[0004] Meanwhile, with the widespread use of genetic recombination technology, clinical studies have begun using oncolytic viruses with increased tumor selectivity and anticancer efficacy. The first recombinant oncolytic virus reported in the literature was herpes simplex virus. Since then, research into oncolysis using other viruses has been actively conducted.

[0005] Among these, research has been conducted on thymidine kinase (TK) gene-deficient vaccinia viruses obtained by using vaccinia viruses; however, despite their clinical usefulness, these viruses have limitations in maximizing their clinical efficacy due to their narrow therapeutic window. The narrow therapeutic window of TK-deficient vaccinia viruses means that high viral doses have high clinical efficacy but may be accompanied by clinical risks due to viral toxicity. To overcome this problem, research has been conducted on such viruses by deleting gene regions encoding non-essential proteins, such as protein A56, in addition to deleting the TK gene, thereby increasing therapeutic efficacy and reducing toxicity (Izmailyan R, Chang W Journal of Virology 2008 Oct;82(20):10079-87).

[0006] Recently, immune cell therapy has been actively researched as a cancer treatment method. Immune cell therapy differs 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 the patient, activated to specifically attack proliferating cells or cancer cells, and then returned to the patient's body; this method maximizes anti-cancer effects while minimizing drug-induced adverse effects.

[0007] Over the past five years, immune checkpoint inhibitors have been actively investigated as immunotherapies for treating cancer. In particular, inhibitors of immune checkpoints such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), and PD-L1 have been investigated. 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 several types of cancer. However, immune checkpoint inhibitors are only used to treat 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-expressing T cells (CAR-T cells) have demonstrated excellent therapeutic efficacy against various types of hematological cancers. However, few clinically successful cases using CAR-T cells have been reported for solid tumors. Related reasons include the difficulty of CAR-T cell delivery and infiltration into tumors, problems with CAR-T cell persistence and proliferation, and the limited therapeutic efficacy of CAR-T cells due to an unfavorable tumor microenvironment. However, the biggest limitation is the serious safety issue caused by the lack of appropriate target antigens specifically expressed in tumors. In fact, most target antigens used in the clinical development of CAR-T cells for solid tumors are overexpressed in tumors and expressed at low levels in normal cells. For this reason, CAR-T cells have been reported to bind to normal cells, causing serious toxicity problems. To overcome this on-target / off-tumor toxicity issue, target antigens that meet the requirements of tumor specificity, high and uniform coverage, and stable expression are needed. However, currently, few solid tumor target antigens that meet all three requirements exist. Therefore, there is a need for continued research and development into cancer cell targeting methods capable of targeting 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 [Non-patent document 2] Anthony K. Park et al., Science Translational Medicine. 2020 Sep;12(559) Summary of the Invention [Problem to be solved by the invention]

[0010] The present inventors have been researching to develop a method for safely and effectively targeting or treating solid tumors using CAR-T therapeutic agents. As a result, the inventors attempted to induce anti-cancer effects by using tumor-selective oncolytic viruses (vectors) to express oncolytic virus-derived proteins that can be targeted by CAR-T on the surface of solid tumor cells, followed by injection of CAR-T therapeutic agents that bind to the proteins. Prior to the present invention, an immunotherapy approach was proposed in which the CD19 gene was inserted into an oncolytic virus so that the CD19 protein was expressed on the surface of solid tumor cells, followed by administration of CD19-specific CAR-T cells (Anthony K. Park et al., Science Translational Medicine. 2020 Sep;12(559)). However, no method for targeting solid tumors using proteins naturally found in oncolytic viruses had previously been proposed. Proteins naturally found in the human body, such as CD19, are also distributed in other organs or normal cells. However, as noted above, when a protein naturally found in an oncolytic virus is used to target the virus, the risk of the CAR-T binding to an antigen in normal cells is reduced because the oncolytic virus has been engineered to grow in a tumor-selective manner.

[0011] As a result of intensive research, the present inventors have found that when cancer cells of various carcinomas are treated by injection of a vector containing a nucleic acid encoding the vaccinia virus protein A56 or a fragment thereof, the protein A56 is expressed on the surface of cancer cells in a tumor-specific and stable manner. Additionally, the present inventors analyzed the binding of several antibodies that bind to the protein A56 or a fragment thereof to A56. As a result, the present inventors found that the antibodies cross-blocked the binding to A56, even though their epitope sequences were not completely identical but unique. Subsequently, the present inventors identified the structural epitopes that determine the binding to A56, and found that CAR-T cells that bind to such structural epitopes exhibited excellent effects in reducing the burden of cancer cells, thereby completing the present invention. [Means for solving the problem]

[0012] To solve the above-mentioned problems, in one aspect of the present invention, there is provided a genetically engineered immune cell that expresses a chimeric antigen receptor (CAR), comprising (i) an extracellular antigen-binding domain, (ii) a transmembrane domain, and (iii) an intracellular signaling domain, wherein the chimeric antigen receptor specifically binds to an antigen that is present on the surface of cancer cells and not present on the surface of normal cells, and the antigen is a protein that is not naturally expressed by cancer cells.

[0013] In another aspect of the present invention, there is provided a chimeric antigen receptor (CAR) comprising: (i) an extracellular antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, wherein the CAR specifically binds to protein A56 or a fragment thereof exposed on the surface of a cancer cell.

[0014] In yet another aspect of the present invention, a polynucleotide encoding a chimeric antigen receptor is provided.

[0015] In yet another aspect of the present invention, there is provided a vector comprising the polynucleotide.

[0016] In yet another aspect of the present invention, there is provided a genetically engineered immune cell into which a vector has been introduced.

[0017] In yet another aspect of the present invention, there is provided a method of producing a genetically engineered immune cell, comprising the step of introducing a vector into an immune cell.

[0018] In yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, comprising genetically engineered immune cells.

[0019] In yet another aspect of the present invention, a method of reducing cancer cell burden is provided, comprising administering genetically engineered immune cells.

[0020] In yet another aspect of the present invention, a kit for preventing or treating cancer is provided, comprising genetically engineered immune cells and an oncolytic virus.

[0021] In yet another aspect of the present invention, there is provided a method of treating cancer in an individual comprising administering to the individual i) an oncolytic virus and ii) genetically engineered immune cells.

[0022] In yet another aspect of the present invention, there is provided a use of genetically engineered immune cells for the prevention or treatment of cancer.

[0023] In yet another aspect of the present invention, there is provided a use of a genetically engineered immune cell for the manufacture of a medicament for preventing or treating cancer. [Effects of the Invention]

[0024] The chimeric antigen receptor-expressing immune cells of the present invention can effectively target the A56 protein, which is specifically expressed on the surface of cancer cells, enabling targeted treatment of cancer cells that survive infection with an oncolytic virus, thereby providing effective anticancer therapy. The chimeric antigen receptor-expressing immune cells of the present invention exhibit increased activation and proliferation ability specifically against the A56 protein and can exhibit excellent cytotoxic effects, thereby providing effective anticancer therapy against A56-expressing cancer cells. The immune cells are 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 depletion (e.g., cyclophosphamide and fludarabine), or immunotherapeutic agents). [Brief explanation of the drawings]

[0025] [Figure 1] FIG. 1 shows the results obtained by infecting a human lung cancer cell line (A549), a human colorectal cancer cell line (HCT-116), or a human melanoma cell line (SK-MEL-5) with an oncolytic virus containing a nucleic acid encoding the A56 protein, and then identifying the presence or absence of expression of the A56 protein on the cell surface of each cell line by immunofluorescence staining. [Figure 2] FIG. 1 shows the results obtained by infecting a human colorectal cancer cell line (HCT-116) with an oncolytic virus containing a nucleic acid encoding protein A56, and then identifying by immunofluorescence staining the presence or absence of expression of protein A56 on the cell surface of the infected HCT-116 cell line. [Figure 3] FIG. 1 shows the results obtained by intraperitoneally administering an oncolytic virus containing a nucleic acid encoding protein A56 to mice implanted with a human colorectal cancer cell line (HCT-116), followed by identifying the expression of protein A56 on the surface of each tissue. [Figure 4]FIG. 1 shows the results obtained by subjecting normal rabbits to intravascular administration of an oncolytic virus containing a nucleic acid encoding protein A56, followed by identification of the expression of protein A56 on the surface of each tissue. [Figure 5] This figure shows the results obtained by subjecting mice implanted with a mouse renal carcinoma cell line (Renca) to intratumoral administration of an oncolytic virus containing a nucleic acid encoding protein A56, and then identifying the expression of protein A56 on the surface of tumor tissue on days 7, 10, and 14. [Figure 6] FIG. 1 shows the results obtained by subjecting mice bearing a mouse renal carcinoma cell line (Renca) to intratumoral administration of hydroxyurea and an oncolytic virus containing a nucleic acid encoding protein A56, and then identifying the expression of protein A56 on the surface of tumor tissue on days 7, 10, and 14. [Figure 7] This figure shows the results obtained by subjecting mice implanted with a mouse renal carcinoma cell line (Renca) to a second administration of an oncolytic virus containing a nucleic acid encoding protein A56, and then identifying the expression of protein A56 on the surface of tumor tissue on days 21, 24, and 28. [Figure 8] FIG. 1 shows the results obtained by subjecting mice implanted with a mouse renal carcinoma cell line (Renca) to a second administration of hydroxyurea and an oncolytic virus containing a nucleic acid encoding protein A56, and then identifying the expression of protein A56 on the surface of tumor tissue on days 21, 24, and 28. [Figure 9] FIG. 1 shows an embodiment of protein A56 and fragments thereof. [Figure 10] FIG. 1 shows the results obtained by identifying whether protein A56 or a fragment thereof is expressed intracellularly and on the cell surface. [Figure 11] FIG. 1 shows the results obtained by identifying the expression level of the Fc-fused A56 fusion protein. [Figure 12]FIG. 1 shows the results obtained by measuring the affinity between anti-A56 antibodies produced in embodiments of the present invention and A56. [Figure 13] FIG. 1 shows the results obtained by measuring the affinity between anti-A56 antibodies produced in embodiments of the present invention and A56. [Figure 14] FIG. 1 shows the results obtained by measuring the affinity between anti-A56 antibodies produced in embodiments of the present invention and A56. [Figure 15] FIG. 1 shows the results obtained by measuring the affinity between anti-A56 antibodies produced in embodiments of the present invention and A56. [Figure 16] FIG. 1 shows the results obtained by measuring the affinity between anti-A56 antibodies produced in embodiments of the present invention and A56. [Figure 17] FIG. 1 shows the results obtained by measuring the affinity between anti-A56 antibodies produced in embodiments of the present invention and A56. [Figure 18A] FIG. 1 shows the results obtained by identifying the productivity of antibodies A56-01A02 to A56-02B06. [Figure 18B] 1 is a graph showing the productivity of antibodies A56-01A02 to A56-02B06. [Figure 18C] FIG. 1 shows the results obtained by identifying the productivity of antibodies A56-02D04 to A56-59E12. [Figure 19] FIG. 1 shows the results obtained by purifying anti-A56 antibodies produced in an embodiment of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 20] FIG. 1 shows the results obtained by purifying anti-A56 antibodies produced in an embodiment of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 21] FIG. 1 shows the results obtained by purifying anti-A56 antibodies produced in an embodiment of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 22] FIG. 1 shows the results obtained by purifying anti-A56 antibodies produced in an embodiment of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 23] FIG. 1 shows the results obtained by purifying anti-A56 antibodies produced in an embodiment of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 24] FIG. 1 shows the results obtained by purifying anti-A56 antibodies produced in an embodiment of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 25] FIG. 1 shows the results obtained by purifying anti-A56 antibodies produced in an embodiment of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 26] FIG. 1 shows the results obtained by purifying anti-A56 antibodies produced in an embodiment of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 27] FIG. 1 shows the results obtained by purifying anti-A56 antibodies produced in an embodiment of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 28] FIG. 1 shows the results obtained by purifying anti-A56 antibodies produced in an embodiment of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 29] FIG. 1 shows the results obtained by purifying anti-A56 antibodies produced in an embodiment of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 30] FIG. 1 shows the results obtained by purifying anti-A56 antibodies produced in an embodiment of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 31] FIG. 1 shows the results obtained by purifying anti-A56 antibodies produced in an embodiment of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 32] FIG. 1 shows the results obtained by purifying anti-A56 antibodies produced in an embodiment of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 33] FIG. 1 shows the results obtained by purifying anti-A56 antibodies produced in an embodiment of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 34] FIG. 1 shows the results obtained by purifying anti-A56 antibodies produced in an embodiment of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 35] FIG. 1 shows the results obtained by purifying anti-A56 antibodies produced in an embodiment of the present invention and then identifying these antibodies through SDS-PAGE. [Figure 36] FIG. 1 shows the results obtained by comparing the amino acid sequence homology of protein A56 depending on the vaccinia virus strain. [Figure 37] FIG. 1 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 the protein A56. [Figure 38] FIG. 1 shows a photograph taken after reacting protein A56 with a commercially available anti-A56 antibody followed by Western blotting, which was used as a control to determine whether the anti-A56 antibody has a structural epitope. [Figure 39] 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, and Ab16) and then performing Western blotting. [Figure 40] FIG. 1 shows the results obtained by analyzing the antigen-antibody binding complex (A56-C-His / Ab13) by high-mass MALDI. [Figure 41]FIG. 1 shows the results obtained by analyzing the antigen-antibody binding complex (A56-C-His / Ab16) by high-mass MALDI. [Figure 42] FIG. 1 shows the results obtained by analyzing the antigen-antibody binding complex (A56-C-His / Ab18) by high-mass MALDI. [Figure 43] FIG. 1 shows the results obtained by analyzing the antigen-antibody binding complex (A56-C-His / Ab01) by high-mass MALDI. [Figure 44] FIG. 1 shows the results obtained by analyzing the antigen-antibody binding complex (A56-C-His / Ab19) by high-mass MALDI. [Figure 45] FIG. 1 shows the results obtained by treating protein A56 with five proteolytic enzymes (trypsin, chymotrypsin, ASP-N, elastase, and thermolysin) followed by LTQ-Orbitrap MS (mass spectrometry) analysis. [Figure 46]

[0023] Figure 1 shows the location of the epitope on A56-C-His bound by anti-A56 antibody (Ab13), and a modeled structure of the protein A56-C-His. The amino acid numbering in A56 as shown here is based on the sequence obtained by excluding the N-terminal amino acids 1 to 16 from the amino acid sequence represented by SEQ ID NO: 1038 (e.g., the serine at position 30 corresponds to the serine at position 46 in the amino acid sequence represented by SEQ ID NO: 1038). [Figure 47]

[0023] Figure 1 shows the location of the epitope on A56-C-His to which anti-A56 antibody (Ab16) binds, and a modeled structure of the protein A56-C-His. The amino acid numbering in A56 as shown here is based on the sequence obtained by excluding the N-terminal amino acids 1 to 16 from the amino acid sequence represented by SEQ ID NO: 1038 (e.g., the serine at position 30 corresponds to the serine at position 46 in the amino acid sequence represented by SEQ ID NO: 1038). [Figure 48]

[0023] Figure 1 shows the location of the epitope on A56-C-His bound by anti-A56 antibody (Ab18), and a modeled structure of the protein A56-C-His. The amino acid numbering in A56 as shown here is based on the sequence obtained by excluding the N-terminal amino acids 1 to 16 from the amino acid sequence represented by SEQ ID NO: 1038 (e.g., the serine at position 30 corresponds to the serine at position 46 in the amino acid sequence represented by SEQ ID NO: 1038). [Figure 49]

[0023] Figure 1 shows the location of the epitope on A56-C-His bound by anti-A56 antibody (Ab01), and a modeled structure of the protein A56-C-His. The amino acid numbering in A56 as shown here is based on the sequence obtained by excluding the N-terminal amino acids 1 to 16 from the amino acid sequence represented by SEQ ID NO: 1038 (e.g., the serine at position 30 corresponds to the serine at position 46 in the amino acid sequence represented by SEQ ID NO: 1038). [Figure 50]

[0023] Figure 1 shows the location of the epitope on A56-C-His bound by anti-A56 antibody (Ab19), and a modeled structure of the protein A56-C-His. The amino acid numbering in A56 as shown here is based on the sequence obtained by excluding the N-terminal amino acids 1 to 16 from the amino acid sequence represented by SEQ ID NO: 1038 (e.g., the serine at position 40 corresponds to the serine at position 56 in the amino acid sequence represented by SEQ ID NO: 1038). [Figure 51] This figure summarizes the results of epitope mapping of five antibodies (Ab13, Ab16, Ab18, Ab01, and Ab19) to protein A56. The amino acid numbering for A56 shown here is based on the sequence obtained by excluding N-terminal amino acids 1 to 16 from the amino acid sequence represented by SEQ ID NO: 1038 (e.g., the serine at position 30 corresponds to the serine at position 46 in the amino acid sequence represented by SEQ ID NO: 1038). [Figure 52]This figure was obtained by modeling protein A56 into a 3D protein structure and then analyzing the structure with iCn3D. [Figure 53] FIG. 1 shows the location of the paratope on the anti-A56 antibody (Ab13) and the modeled structure formed upon binding of the antibody to the protein A56-C-His. [Figure 54] FIG. 1 shows the location of the paratope on the anti-A56 antibody (Ab16) and the modeled structure formed by binding of the antibody to the protein A56-C-His. [Figure 55] FIG. 1 shows the location of the paratope on the anti-A56 antibody (Ab18) and the modeled structure formed upon binding of the antibody to the protein A56-C-His. [Figure 56] FIG. 1 shows the location of the paratope on the anti-A56 antibody (Ab01) and the modeled structure formed upon binding of the antibody to the protein A56-C-His. [Figure 57] FIG. 1 shows the location of the paratope on the anti-A56 antibody (Ab19) and the modeled structure formed by binding of the antibody to the protein A56-C-His. [Figure 58A] FIG. 1 shows the formation of a hydrogen bond between K91 in protein A56 and T52 of the heavy chain in Ab13, and the measured bond distance. [Figure 58B] FIG. 1 shows the formation of a hydrogen bond between S62 in protein A56 and K57 of the heavy chain in Ab13, and the measured bond distance. [Figure 59A] This figure shows the results obtained by binding primary antibodies (SA2038, Ab13, A56-02A02) to an A56-C-His antigen-immobilized biosensor (NTA) to saturation, treating with nine antibodies, and then analyzing by surface plasmon resonance (SPR) whether additional binding of such antibodies to the biosensor occurs. [Figure 59B] FIG. 1 shows the results obtained by measuring the binding affinity (KD) between proteins A56 and Ab13 by OCTET (SPR) and ELISA. [Figure 60]FIG. 1 shows the binding of proteins A56 and Ab16 and the measured distance. [Figure 61A] This figure shows the results obtained by binding primary antibodies (SA2041, Ab16, A56-02B08) to an A56-C-His antigen-immobilized biosensor (NTA) to saturation, treating with nine antibodies, and then analyzing by SPR whether additional binding of such antibodies to the biosensor occurs. [Figure 61B] FIG. 1 shows the results obtained by measuring the binding affinity (KD) between proteins A56 and Ab16 by OCTET (SPR) and ELISA. [Figure 62A] FIG. 1 shows the formation of a hydrogen bond between S62 in protein A56 and R98 of the heavy chain in Ab18, and the measured bond distance. [Figure 62B] FIG. 1 shows the formation of a hydrogen bond between K91 in protein A56 and Y32 of the light chain in Ab18, and the measured bond distance. [Figure 63A] This figure shows the results obtained by binding primary antibodies (SA2043, Ab18, A56-02C06) to an A56-C-His antigen-immobilized biosensor (NTA) to saturation, treating with nine antibodies, and then analyzing by SPR whether additional binding of such antibodies to the biosensor occurs. [Figure 63B] FIG. 1 shows the results obtained by measuring the binding affinity (KD) between proteins A56 and Ab18 by OCTET (SPR) and ELISA. [Figure 64] FIG. 1 shows the binding of protein A56 to Ab01 and the measured distance. [Figure 65A] This figure shows the results obtained by binding primary antibodies (SA2026, Ab01, A56-01A02) to an A56-C-His antigen-immobilized biosensor (NTA) to saturation, treating with nine antibodies, and then analyzing using SPR methods to determine whether additional binding of such antibodies to the biosensor occurs. [Figure 65B]FIG. 1 shows the results obtained by measuring the binding affinity (KD) between protein A56 and Ab01 by OCTET (SPR) and ELISA. [Figure 66] FIG. 1 shows the binding of proteins A56 and Ab19 and the measured distance. [Figure 67A] This figure shows the results obtained by binding primary antibodies (SA2044, Ab19, A56-02C07) to an A56-C-His antigen-immobilized biosensor (NTA) to saturation, treating with nine antibodies, and then analyzing by SPR whether additional binding of such antibodies to the biosensor occurs. [Figure 67B] FIG. 1 shows the results obtained by measuring the binding affinity (KD) between proteins A56 and Ab19 by OCTET (SPR) and ELISA. [Figure 68] FIG. 1 is a schematic diagram of the structure of a chimeric antigen receptor constructed in an embodiment of the present invention. [Figure 69] FIG. 1 shows results depicting the cell viability observed when HeLa or HCT-116 cell lines were subjected to the administration of five types of CAR-T cells alone or in combination with oncolytic virus (OTS-412). [Figure 70] FIG. 1 shows results obtained by comparing the cytotoxicity observed when a HeLa cell line was subjected to the administration of five types of CAR-T cells alone or in combination with an oncolytic virus (OTS-412). [Figure 71] FIG. 1 shows results obtained by comparing the cytotoxicity observed when the NCI-H522 cell line was subjected to the administration of five types of CAR-T cells alone or in combination with an oncolytic virus (OTS-412). [Figure 72] FIG. 1 shows results obtained by comparing the cytotoxicity observed when the HCT-116 cell line was subjected to the administration of five types of CAR-T cells alone or in combination with an oncolytic virus (OTS-412). [Figure 73]FIG. 1 shows the results obtained by measuring the transduction efficiency of five types of CAR-T cells by flow cytometry (FACS). [Figure 74] FIG. 1 shows results depicting the cell viability observed when HCT-116 cell lines infected with various concentrations of oncolytic virus (OTS-412) were subjected to administration of UTD, Ab16 CAR-T cells, or Ab18 CAR-T cells. [Figure 75] Figure 1 shows photographs taken after A549 or HCT-116 cell lines infected with oncolytic virus (OTS-412) were administered with UTD, Ab16 CAR-T cells, or Ab18 CAR-T cells, followed by dead cell staining. [Figure 76] FIG. 1 shows results demonstrating the cytotoxicity observed when A549 or HCT-116 cell lines were subjected to administration of UTD, Ab16 CAR-T cells, or Ab18 CAR-T cells alone or in combination with oncolytic virus (OTS-412). [Figure 77] FIG. 1 shows results demonstrating the cytotoxicity observed when HeLa, MCF7, A549, or PC-3 cell lines were subjected to administration of UTD, Ab16 CAR-T cells, or Ab18 CAR-T cells alone or in combination with oncolytic virus (OTS-412). [Figure 78] This figure shows the results obtained by measuring the percentage of CD3 / CD25-expressing T cells by flow cytometry (FACS) in Ab16 CAR-T cells, Ab16 CAR-T cells cocultured with cancer cell lines, or Ab16 CAR-T cells cocultured with cancer cell lines infected with oncolytic virus (OTS-412) to check the activation of Ab16 CAR-T cells. [Figure 79]To check the proliferation ability of Ab18 CAR-T cells, the percentage of CD8-expressing T cells was measured by flow cytometry (FACS) in UTD (mock-T) cells, Ab18 CAR-T cells, UTD (mock-T) cells cocultured with a cancer cell line infected with oncolytic virus (OTS-412), and Ab18 CAR-T cells cocultured with a cancer cell line infected with oncolytic virus (OTS-412). [Figure 80] This figure shows the results obtained by administering HCT-116 cell line-implanted mice with oncolytic virus (OTS-412), Ab16 CAR-T cells or Ab18 CAR-T cells, and hydroxyurea (HU), followed by measuring the body weight of the mice on days 0, 3, 8, 10, 14, 17, and 21. "mOV" means that the oncolytic virus and HU are co-administered. Unless otherwise indicated, when used in the accompanying drawings herein, "mOV" or "mOTS-412" means co-administration with HU. [Figure 81] This figure shows the results obtained by administering an oncolytic virus (OTS-412), Ab16 CAR-T cells or Ab18 CAR-T cells, and HU to mice implanted with the HCT-116 cell line, and then measuring the tumor volume of the mice on days 0, 3, 8, 10, 14, 17, and 21. [Figure 82] This figure shows the results obtained by administering HCT-116 cell line-implanted mice with oncolytic virus (OTS-412), UTD or Ab16 CAR-T cells, and HU, and then measuring the body weight of the mice on days 0, 3, 8, 10, 14, 17, and 21. [Figure 83] This figure shows the results obtained by administering HCT-116 cell line-implanted mice with oncolytic virus (OTS-412), UTD or Ab16 CAR-T cells, and HU, and then measuring the tumor volume of the mice on days 0, 3, 8, 10, 14, 17, and 21. [Figure 84]FIG. 1 shows results demonstrating the cytotoxicity observed when MCF7 or A546 cell lines were subjected to administration of UTD, Ab16 CAR-T cells, or Ab18 CAR-T cells alone or in combination with oncolytic virus (WOTS-418). [Figure 85] This figure shows photographs taken after administering an oncolytic virus (OTS-412)-infected HCT-116 cell line with UTD and five types of CAR-T cells (Ab13 CAR-T, Ab16 CAR-T, Ab18 CAR-T, Ab01 CAR-T, and Ab19 CAR-T), followed by staining for dead cells. [Figure 86] This figure shows photographs taken after administering an oncolytic virus (WOTS-418)-infected HCT-116 cell line with UTD and five types of CAR-T cells (Ab13 CAR-T, Ab16 CAR-T, Ab18 CAR-T, Ab01 CAR-T, and Ab19 CAR-T), followed by staining for dead cells. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention will now be described in detail.

[0027] In an aspect of the present invention, there is provided a genetically engineered immune cell that expresses a chimeric antigen receptor (CAR), comprising (i) an extracellular antigen-binding domain, (ii) a transmembrane domain, and (iii) an intracellular signaling domain, wherein the chimeric antigen receptor specifically binds to an antigen that is present on the surface of a cancer cell and not present on the surface of a normal cell, and the antigen is a protein not naturally expressed by the cancer cell.

[0028] The antigen may be protein A56 or a fragment thereof.

[0029] The extracellular antigen-binding domain can specifically bind to a structural epitope of A56 or a fragment thereof, and the structural epitope may comprise a basic or nucleophilic amino acid present in the region from amino acid position 60 to amino acid position 63 in the amino acid sequence of protein A56 represented by SEQ ID NO: 1038, and may further comprise (i) a nucleophilic amino acid present in the region from amino acid position 44 to amino acid position 50 in the amino acid sequence of protein A56, (ii) a nucleophilic amino acid present in the region from amino acid position 53 to amino acid position 59 in the amino acid sequence of protein A56, (iii) a nucleophilic amino acid present in the region from amino acid position 85 to amino acid position 90 in the amino acid sequence of protein A56, or (iv) a basic or nucleophilic amino acid present in the region from amino acid position 91 to amino acid position 94 in the amino acid sequence of protein A56.

[0030] As used herein, the term "A56" refers to a protein translated from a gene designated A56, A56R, or HA (e.g., gene ID: 3707652) encoded in a poxviridae virus after the virus infects a host cell. Protein A56 or a fragment thereof may comprise the amino acid sequence set forth in SEQ ID NOs: 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1073, 1075, 1077, or 1079. A nucleic acid encoding protein A56 or a fragment thereof may comprise the base sequence represented by SEQ ID NO: 1039, 1041, 1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1072, 1074, 1076, or 1078. In the present invention, protein A56 may be used interchangeably with "UTTA".

[0031] In particular, the A56 protein may be a wild-type A56 protein or a variant thereof. The wild-type A56 protein may have the amino acid sequence represented by SEQ ID NO: 1038 or 1073. In addition, the nucleic acid encoding the wild-type A56 protein may have the base sequence represented by SEQ ID NO: 1039 or 1072.

[0032] In addition, a protein A56 variant may have one or more amino acid substitutions, deletions, or additions, so long as it can be localized on the surface of cancer cells like protein A56. A 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 identity with the amino acid sequence represented by SEQ ID NO: 1038 or 1073, and most preferably, a nucleotide sequence encoding an amino acid sequence having at least 95% sequence identity with the amino acid sequence represented by SEQ ID NO: 1038 or 1073. In particular, a protein A56 variant may comprise the amino acid sequence represented by SEQ ID NO: 1075 or 1079. A nucleic acid encoding a protein A56 variant may comprise the nucleotide sequence represented by SEQ ID NO: 1074 or 1078.

[0033] The protein A56 fragment may be a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, or 1077. In addition, the nucleic acid encoding the fragment may be a nucleotide sequence encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, or 1077. Specifically, a nucleotide sequence encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, or 1077 can be represented by SEQ ID NO: 1041, 1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, or 1076, respectively, in the order mentioned.

[0034] In addition, the nucleotide sequence encoding the protein A56 fragment 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 identity with the amino acid sequence represented by SEQ ID NO: 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, or 1077, and most preferably a nucleotide sequence encoding an amino acid sequence having at least 95% sequence identity thereto.

[0035] The binding molecule of the present invention refers to a biological molecule capable of specifically binding to A56, and representative examples thereof include antibodies and chimeric antigen receptors. More specifically, the binding molecule of 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 survive infection with oncolytic viruses as a secondary anticancer treatment. In addition, when used in cancer immunotherapy such as CAR-T cell therapy, the binding molecule itself exerts excellent cytotoxic effects, thereby enabling effective cancer treatment.

[0036] A structural epitope can include contiguous amino acids or amino acids that are not contiguous due to tertiary folding of a protein. Specifically, a structural epitope can include at least 2, 5, 8, 10, or 11 amino acids in a separate spatial three-dimensional structure.

[0037] The amino acid residues of an antigen involved in antigen / antibody binding are called an epitope, and the specific portion of an antibody that recognizes the epitope is called a paratope. Depending on how the paratope binds, epitopes can be broadly divided into linear epitopes consisting of a sequence of about 4 to about 12 consecutive amino acids, and structural epitopes consisting of a sequence of non-contiguous amino acids. In the case of structural epitopes, antigen / antibody binding occurs three-dimensionally in a folded structure. Therefore, in structural epitopes, the range of amino acid residues involved in binding is relatively broad and complex. Therefore, even if the structure of an antigen or its partial sequence is known, it is difficult to predict or mimic a monoclonal antibody that binds to the antigen three-dimensionally. Whether an antigen epitope is linear or structural can be identified through Western blot analysis by denaturing the antigen and then binding it with an antibody. In the case of a linear epitope, an antibody can bind to the epitope even if the sample (antigen) is denatured. However, in the case of a conformational epitope, if the antigen is denatured, it is difficult or impossible for the antibody to bind to the epitope three-dimensionally, and therefore a band corresponding to the expected molecular weight is not detected.

[0038] Protein A56 represented by SEQ ID NO: 1038 may preferably have the following secondary structural arrangement, consisting of a total of 8 sheets (green), 4 helices (red), and 7 loops (blue) (however, the structural arrangement does not have to be this): Region from amino acid 41 to amino acid 43: helices 1 and 2 Region from amino acid 44 to amino acid 50: Sheet 3 The region from amino acid 51 to amino acid 52: loop Region from amino acid 53 to amino acid 59: Sheet 4 The region from amino acid 60 to amino acid 63: loop Region from amino acid 64 to amino acid 66: Sheet 5 The region from amino acid 67 to amino acid 74: loop Region from amino acid 75 to amino acid 78: Sheet 6 The region from amino acid 79 to amino acid 80: loop Region from amino acid 81 to amino acid 83: helix 3 Amino acid at position 84: Loop Region from amino acid 85 to amino acid 90: Sheet 7 The region from amino acid 91 to amino acid 94: loop Region from amino acid 95 to amino acid 97: Helix 4 Amino acid at position 98: Loop Region from amino acid 99 to amino acid 105: Sheet 8 (Sheets 1 and 2 not shown)

[0039] A nucleophilic amino acid refers to an amino acid having a nucleophilic side chain that is susceptible to covalent reactions with electrophilic side chains, and may be, for example, cysteine (C), lysine (K), serine (S), threonine (T), or tyrosine (Y). A basic amino acid refers to an amino acid whose side chain is basic and has a (+) charge when dissociated in the neutral pH range, and may be, for example, histidine (H), arginine (R), or lysine (K).

[0040] The structural epitope may include a basic or nucleophilic amino acid present in the region from amino acid position 60 to amino acid position 63 in the amino acid sequence of protein A56 represented by SEQ ID NO: 1038, and may also include (i) a nucleophilic amino acid present in the region from amino acid position 44 to amino acid position 50 in the amino acid sequence of protein A56, and (ii) a nucleophilic amino acid present in the region from amino acid position 53 to amino acid position 59 in the amino acid sequence of protein A56.

[0041] Specifically, the structural epitope may include the nucleophilic amino acid serine at position 46 (S46), the amino acid serine at position 54 (S54), and the amino acid lysine at position 61 (K61) in the amino acid sequence of protein A56 represented by SEQ ID NO: 1038. 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: 1038. 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: 1038.

[0042] The structural epitope may include a basic or nucleophilic amino acid present in the region from amino acid 60 to amino acid 63 in the amino acid sequence of protein A56 represented by SEQ ID NO: 1038, and may further include (iii) a nucleophilic amino acid present in the region from amino acid 85 to amino acid 90 in the amino acid sequence of protein A56, and (iv) a basic or nucleophilic amino acid present in the region from amino acid 91 to amino acid 94 in the amino acid sequence of protein A56.

[0043] 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 SEQ ID NO: 1038. 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 SEQ ID NO: 1038. In addition, the structural epitope may include 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 SEQ ID NO: 1038.

[0044] In addition, the structural epitope may include the amino acids at positions 61 (K61) and 62 (S62) in the amino acid sequence of protein A56 represented by SEQ ID NO:1038.

[0045] We analyzed the binding characteristics of several different antibodies that bind to A56 with high affinity. As a result, we found that the basic amino acid at position 61 or 91 of A56 (K61, K91) commonly plays an important role in binding. Specifically, K61 or K91 forms strong hydrogen bonds with nucleophilic amino acids present in the paratope of each A56 antibody. As this binding progresses, the heavy and light chains of the antibody fold, causing a contraction, which then forms the remaining antigen-antibody binding. Separately, K61 or K91 may also contribute to antigen-antibody binding by affecting the nucleophilic serine amino acid (S62, S92) located immediately adjacent to it. Specifically, K61 or K91 excites the hydroxyl group of the immediately adjacent S62 or S92, generating an electrostatic force. Therefore, S62 or S92 is also thought to actively contribute to strong binding with the antibody.

[0046] The extracellular antigen-binding domain 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 extracellular antigen-binding domain may compete for binding to A56 with one or more of the following antigen-binding molecules: an A56 binding molecule (e.g., Ab13) comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 222, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 223, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 224, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 225, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 226, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 227; an A56 binding molecule (e.g., Ab16) comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 290, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 291, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 292, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 293, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 294, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 295; an A56 binding molecule (e.g., Ab18) comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 324, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 325, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 326, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 327, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 328, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 329; an A56 binding molecule (e.g., Ab01) comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6; an A56 binding molecule (e.g., Ab19) comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 341, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 342, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 343, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 344, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 345, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 346; an A56 binding molecule (e.g., Ab03) comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 35, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 36, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 37, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 38, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 39, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 40; an A56 binding molecule (e.g., Ab08) comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 120, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 121, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 123, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 124, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 125; an A56 binding molecule (e.g., Ab14) comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 239, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 240, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 241, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 242, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 243, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 244; an A56 binding molecule (e.g., Ab51) comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 851, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 852, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 853, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 854, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 855, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 856; and An A56 binding molecule (e.g., Ab55) comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 919, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 920, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 921, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 922, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 923, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 924.

[0047] "Compete 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 extent 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 it can be said to cross-compete according to the present invention, can be determined using standard competitive binding assays. One suitable assay uses Biacore technology, where 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 WO 2003 / 48731.

[0048] The A56 antibodies were identified to cross-inhibit binding to A56, even though their epitope sequences are not completely identical. This suggests that the specific higher-order structure formed between the A56-binding molecule and A56, i.e., the structural epitope of A56, is important for binding to A56. In particular, antibody Ab19 inhibited binding to A56 to a relatively lower extent 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 amino acid sequences such as 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 the stronger binding to A56.

[0049] The extracellular antigen binding domains are set forth in SEQ ID NOs: 1038, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307, 324, 341, 358, 375, 392, 409, 426, 443, 460, 477, 494, 511, 528, 545 , 562, 579, 596, 613, 630, 647, 664, 681, 698, 715, 732, 749, 766, 783, 800, 817, 834, 851, 868, 885, 902, 919, 936, 953, 970, 987, 1004, 1021, 1062, and 1063; SEQ ID NOs: 2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461, 478, 495, 512, 529, 546, 563, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 6 a heavy chain CDR2 selected from the group consisting of 80, 597, 614, 631, 648, 665, 682, 699, 716, 733, 750, 767, 784, 801, 818, 835, 852, 869, 886, 903, 920, 937, 954, 971, 988, 1005, 1022, and 1064; SEQ ID NOs: 3, 20, 37, 54, 71, 88, 105, 122, 139, 156, 173, 190, 207, 224, 241, 258, 275, 292, 309, 326, 343, 360, 377, 394, 411, 428, 445, 462, 479, 496, 513, 530, 547, 564, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1210, a heavy chain CDR3 selected from the group consisting of 81, 598, 615, 632, 649, 666, 683, 700, 717, 734, 751, 768, 785, 802, 819, 836, 853, 870, 887, 904, 921, 938, 955, 972, 989, 1006, 1023, and 1065; SEQ ID NOs: 4, 21, 38, 55, 72, 89, 106, 123, 140, 157, 174, 191, 208, 225, 242, 259, 276, 293, 310, 327, 344, 361, 378, 395, 412, 429, 446, 463, 480, 497, 514, 531, 548, 565, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, a light chain CDR1 selected from the group consisting of 82, 599, 616, 633, 650, 667, 684, 701, 718, 735, 752, 769, 786, 803, 820, 837, 854, 871, 888, 905, 922, 939, 956, 973, 990, 1007, 1024, and 1066; SEQ ID NOs: 5, 22, 39, 56, 73, 90, 107, 124, 141, 158, 175, 192, 209, 226, 243, 260, 277, 294, 311, 328, 345, 362, 379, 396, 413, 430, 447, 464, 481, 498, 515, 532, 549, 566, 58 a light chain CDR2 selected from the group consisting of: 3, 600, 617, 634, 651, 668, 685, 702, 719, 736, 753, 770, 787, 804, 821, 838, 855, 872, 889, 906, 923, 940, 957, 974, 991, 1008, 1025, and 1067; and SEQ ID NOs: 6, 23, 40, 57, 74, 91, 108, 125, 142, 159, 176, 193, 210, 227, 244, 261, 278, 295, 312, 329, 346, 363, 380, 397, 414, 431, 448, 465, 482, 499, 516, 533, 550, 567, a light chain CDR3 selected from the group consisting of 584, 601, 618, 635, 652, 669, 686, 703, 720, 737, 754, 771, 788, 805, 822, 839, 856, 873, 890, 907, 924, 941, 958, 975, 992, 1009, 1026, and 1068 may include:

[0050] In particular, the extracellular antigen-binding domain comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, 35, 120, 222, 239, 290, 324, 341, 851, 919, 1062, or 1063; a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, 36, 121, 223, 240, 291, 325, 342, 852, 920, or 1064; a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, 37, 122, 224, 241, 292, 326, 343, 853, 921, or 1065; a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, 38, 123, 225, 242, 293, 327, 344, 854, 922, or 1066; a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, 39, 124, 226, 243, 294, 328, 345, 855, 923, or 1067; and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6, 40, 125, 227, 244, 295, 329, 346, 856, 924, or 1068 may include:

[0051] As used herein, the term "antibody" refers to an immune protein 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 of which contains a heavy chain produced from a heavy chain constant region gene μ, δ, γ, α, or ε. IgG is primarily used in antibody technology. IgG includes four isotypes: IgG1, IgG2, IgG3, and IgG4, and each isotype may have different structural and functional properties.

[0052] IgG forms a highly stable Y-shaped structure (molecular weight: approximately 150 kDa) composed of two heavy chains (approximately 50 kDa) and two light chains (approximately 25 kDa). Antibodies have light and heavy chains, and each chain is divided into a variable region, whose amino acid sequence varies among antibodies, and a constant region, whose amino acid sequence is consistent among antibodies. The heavy chain constant region contains CH1, H (hinge), CH2, and CH3 domains. Each of these domains consists of two β-sheets, which are connected by intramolecular disulfide bonds. The two variable regions of the heavy and light chains associate together to form an antigen-binding site, which is located on each of the two arms of the Y-shape. The portion of the Y-shape that can bind to antigen is called the antibody-binding fragment (Fab), and the portion that does not bind to antigen is called the crystallizable fragment (Fc). Fab and Fc are connected by a flexible hinge region.

[0053] As used herein, the term "CDR" refers to an antigen-binding site, which is a hypervariable region present in the heavy and light chain variable regions of an antibody, and the amino acid sequence of this site varies depending on the antibody. Looking at the three-dimensional structure of an antibody, the CDRs are in the form of loops on the antibody surface. Beneath these loops are framework regions (FRs) that structurally support the CDRs. Each of the heavy and light chains has three loop structures, and these six loop region structures associate with each other and directly contact 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.

[0054] Additionally, the antibody fragment may be any 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 crystallizable region (Fc region) that has effector functions that transmit antigen-binding stimuli to cells, complement, etc., and may include third-generation antibody fragments such as single-domain antibodies or minibodies.

[0055] In addition, antibody fragments have the following advantages: they are smaller in size than fully-structured IgG, resulting in improved tissue or tumor penetration; and they can be produced in bacteria, which reduces production costs. Furthermore, antibody fragments lack Fc and are therefore used when the function of transmitting antigen-binding stimuli to cells, complement, etc. is undesirable. Antibody fragments have a short half-life in the human body and are therefore suitable for in vivo diagnostics. However, the replacement of some basic, acidic, or neutral amino acids among the amino acids constituting an antibody can alter the inherent isoelectric point (pI) of the antibody. Such a change in the isoelectric point of an antibody can lead to changes such as reducing the antibody's in vivo toxic side effects or increasing its water solubility. Therefore, for therapeutic antibodies, fully-structured IgG can be used, taking into account affinity or structural form.

[0056] The antibody can be easily produced by known monoclonal antibody production techniques. Methods for producing monoclonal antibodies can be carried out by preparing hybridomas using B lymphocytes obtained from immunized animals, or by using phage display technology. However, the present invention is not limited thereto.

[0057] The antibody or a fragment thereof can be used as a 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 the antibody or a fragment thereof can be used as the antigen-binding domain.

[0058] The extracellular antigen-binding domain refers to the region of an antibody that binds to an antigen. The extracellular antigen-binding domain may be an antibody or an antigen-binding fragment thereof. Preferably, the extracellular antigen-binding domain may be an antigen-binding fragment. Additionally, 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 via 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 extracellular antigen-binding domain may be scFv.

[0059] The extracellular antigen-binding domain may comprise a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 229, 297, 331, 8, and 348, and a light chain variable region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 230, 298, 332, 9, and 349.

[0060] The extracellular antigen-binding domain (i) can consist of any one amino acid sequence selected from SEQ ID NOs: 1081 to 1085.

[0061] The transmembrane domain refers to the region of a protein located in the cell membrane that connects the antigen-binding domain and the intracellular signal transduction domain and spans the cell membrane. The transmembrane domain can anchor the protein located in the cell membrane to the cell membrane. The transmembrane domain can be derived from any one selected from the group consisting of T cell receptor, 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 transmembrane domain can be derived from CD8α.

[0062] The transmembrane domain may consist of the amino acid sequence represented by SEQ ID NO: 1086. In addition, the nucleotide sequence encoding the transmembrane domain may be the nucleotide sequence represented by SEQ ID NO: 1096. In an embodiment of the present invention, a transmembrane domain derived from CD8α and consisting of the amino acid sequence represented by SEQ ID NO: 1086 was used.

[0063] The intracellular signaling domain refers to a region that transmits a signal to a cell when an antigen receptor (antigen-binding domain) present on the cell surface recognizes an extracellular antigen, thereby inducing responses such as cell activation, cytotoxic factor release, cytokine production, and proliferation. In addition, signals transmitted through only one antigen receptor (antigen-binding domain) are generally insufficient for cell activation, and therefore, a secondary or costimulatory signal is required. Therefore, the intracellular signaling domain may include a primary signaling domain as well as a secondary signaling domain and / or a costimulatory domain. In particular, the intracellular signaling domain may include a costimulatory domain and a primary signaling domain.

[0064] The costimulatory domain can be derived 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 costimulatory domain can be derived from CD137 (4-1BB).

[0065] The costimulatory domain may consist of the amino acid sequence of SEQ ID NO: 1087. In addition, the nucleotide sequence encoding the costimulatory domain may be the nucleotide sequence represented by SEQ ID NO: 1096. In an embodiment of the present invention, a costimulatory domain derived from CD137 (4-1BB) and consisting of the amino acid sequence represented by SEQ ID NO: 1087 was used.

[0066] The primary signaling domain can be derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, or CD66d. In particular, for T cells, signals are transmitted to cells through CD3 chains γ, δ, ε, or ζ. When generating chimeric antigen receptor T cells (CAR-T cells), CD3 chains γ, δ, ε, or ζ can be used as the primary signaling domain. In particular, the primary signaling domain can be derived from CD3ζ.

[0067] The primary signaling domain may consist of the amino acid sequence represented by SEQ ID NO: 1088. Additionally, the nucleotide sequence encoding the primary signaling domain may be the nucleotide sequence represented by SEQ ID NO: 1097. In an embodiment of the present invention, a primary signaling domain derived from CD3ζ and consisting of the amino acid sequence represented by SEQ ID NO: 1088 was used.

[0068] The immune cells may be selected from T cells, natural killer cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, macrophages, human embryonic stem cells, lymphocyte progenitor cells, T cell progenitor cells, and pluripotent stem cells capable of differentiating into lymphocytes. Specifically, the immune cells may be T cells, natural killer cells, or macrophages. In an embodiment of the present invention, T cells were used as the immune cells.

[0069] Genetically engineered immune cells can be administered together with oncolytic viruses to reduce cancer cell burden. Cancer cell burden can refer to the weight, volume, or number of cancer cells. Cancer cells that survive infection with oncolytic viruses express the protein A56 on their cell surface. Therefore, genetically engineered immune cells that specifically bind to A56 enable targeting for secondary anticancer therapy or secondary anticancer therapy. Genetically engineered immune cells can be administered before, simultaneously with, or after administration of oncolytic viruses.

[0070] The oncolytic virus may be a vaccinia virus, which 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).

[0071] The oncolytic virus can be a virus in which the thymidine kinase (TK) gene has been deleted. In particular, the oncolytic virus can be a recombinant vaccinia virus in which the thymidine kinase gene has been deleted.

[0072] As used herein, the term "thymidine kinase (TK)" refers to an enzyme called thymidine kinase and involved in nucleotide biosynthesis. TK is an enzyme used in nucleotide biosynthesis in both cells and viruses. Regarding cells, normal cells no longer divide, and therefore do not have TK. Even in rapidly dividing cells such as hair follicle cells, TK does not exist in sufficient amounts for viruses to utilize. From these perspectives, by deleting the TK gene, viruses can grow only in the presence of cancer cells in which TK is present, and as a result, cancer cells can be selectively killed.

[0073] The oncolytic virus may contain a nucleic acid encoding the A56 protein or a fragment thereof. The A56 protein or a fragment thereof is as described above. The A56 protein may be the wild-type A56 protein or a variant of the A56 protein. The nucleic acid encoding the A56 protein or a fragment thereof may be a nucleic acid encoding the wild-type A56 protein or a variant of the A56 protein. The oncolytic virus may be co-administered with hydroxyurea simultaneously, sequentially, or in the reverse order.

[0074] As used herein, unless otherwise indicated, the expression "in combination" includes both the administration of the therapeutic agents / agents in question in any order spaced apart in time, as well as the administration of the therapeutic agents / agents simultaneously.

[0075] In another aspect of the present invention, there is provided a chimeric antigen receptor (CAR) comprising: (i) an extracellular antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, wherein the CAR specifically binds to protein A56 or a fragment thereof exposed on the surface of a cancer cell.

[0076] The above (i) to (iii) refer to the descriptions made about genetically engineered immune cells. The chimeric antigen receptor may consist of any one of the amino acid sequences represented by SEQ ID NOs: 1081 to 1085.

[0077] In an embodiment of the present invention, CAR-T cells are designed to contain an antigen-binding domain (anti-UTTA scFv), a CD8 transmembrane domain (H+TM), and an intracellular signaling domain (4-1BB and CD3Z); and the CAR-T cells are named "Ab13 CAR-T," "Ab16 CAR-T," "Ab18 CAR-T," "Ab01 CAR-T," or "Ab19 CAR-T" depending on the antigen-binding domain. The sequence encoding the antigen-binding domain may include any one of the nucleotide sequences represented by SEQ ID NOs: 1090 to 1094.

[0078] In yet another aspect of the present invention, there is provided a polynucleotide encoding a chimeric antigen receptor, wherein the chimeric antigen receptor is as described above.

[0079] Polynucleotide refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a DNA / RNA hybrid. Polynucleotides can be single-stranded or double-stranded, recombinant, synthetic, or isolated. Non-limiting examples of polynucleotides include pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive-strand RNA (RNA(+)), negative-strand RNA (RNA(-)), synthetic RNA, synthetic mRNA, genomic DNA (gDNA), PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.

[0080] The polynucleotide may consist of a nucleotide sequence represented by any one of SEQ ID NOs: 1090 to 1094. In addition, the polynucleotide may have at least about 80%, 90%, 95%, or 99% homology to any one of the nucleotide sequences selected from SEQ ID NOs: 1090 to 1094, so long as the polynucleotide is capable of encoding a chimeric antigen receptor comprising an amino acid sequence represented by any one of SEQ ID NOs: 1090 to 1094.

[0081] A polynucleotide may be codon-optimized. As used herein, the term "codon optimization" refers to substituting codons in a polynucleotide encoding a polypeptide to increase the expression, stability, and / or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to: (i) variation in codon bias between two or more organisms or genes or synthetically constructed bias tables; (ii) variation in the degree of codon bias within an organism, gene, or gene set; (iii) synthetic variation of codons including context; (iv) variation of codons by decoding tRNA; (v) variation of codons by GC % either overall or at one position in a triplet; (vi) variation in the degree of similarity to a reference sequence, e.g., a naturally occurring sequence; (vii) variation in codon frequency cutoff; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence on which the design of the codon substitution set is to be based; (x) systematic variation of the codon set for each amino acid; and / or (xi) isolated removal of illogical translation start sites.

[0082] In yet another aspect of the present invention, there is provided a vector comprising a polynucleotide, wherein the polynucleotide is as described above.

[0083] Polynucleotides can be prepared, manipulated, expressed, and delivered using any of a variety of established techniques known and available in the art. To express the desired chimeric antigen receptor, a polynucleotide encoding the chimeric antigen receptor can be inserted into an appropriate vector.

[0084] Various vectors known in the art can be used. Depending on the type of host cell intended to produce the antigen receptor, expression control sequences such as promoters, terminators, and enhancers, sequences for membrane targeting or secretion, etc. can be appropriately selected and combined in various ways according to the purpose. Vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, virus vectors, etc. Suitable vectors contain expression control elements such as promoters, operators, initiation 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.

[0085] Preferably, the vector may be a viral vector, which may be derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, pox viruses, baculoviruses, papilloma viruses, and parvoviruses. In an embodiment of the present invention, a lentiviral vector was used.

[0086] The vector may further comprise a sequence encoding a signal peptide for exposing the antigen-binding domain on the outside of the cell membrane, and the sequence encoding the signal peptide is inserted before the sequence encoding the antigen-binding domain. The signal peptide may consist of the amino acid sequence represented by SEQ ID NO: 1080, and the nucleotide sequence encoding the signal peptide may be the nucleotide sequence represented by SEQ ID NO: 1089.

[0087] In yet another aspect of the present invention, there is provided a genetically engineered immune cell into which a vector has been introduced. In yet another aspect of the present invention, there is provided a method for producing a genetically engineered immune cell, the method comprising the step of introducing a vector into an immune cell. The vector and the immune cell are as described above.

[0088] Methods known in the art can be used to introduce vectors into immune cells. For example, vectors can be introduced into cells using transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, or a gene gun, or 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.

[0089] After vector introduction, the transduced or transfected immune cells are expanded ex vivo. In an embodiment, the transfected immune cells may be expanded by incubation for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days, preferably 12 to 14 days.

[0090] Methods for determining whether a vector has been successfully introduced into immune cells include, for example, molecular biological assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; biochemical assays, such as detecting the presence or absence of specific peptides by immunological methods (e.g., ELISA and Western blotting).

[0091] In yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, comprising genetically engineered immune cells.

[0092] The dosage of the pharmaceutical composition may vary depending on various factors, including the type of disease, the severity of the disease, the type and amount of the active ingredient and other ingredients contained in the composition, the type of formulation, as well as the patient's age, weight, general health condition, sex, diet, frequency of administration, route of administration, excretion rate of the composition, duration of treatment, and drugs used concomitantly.

[0093] In addition, pharmaceutical compositions can be administered to individuals by various methods known in the art. The administration route can be appropriately selected by those skilled in the art, taking into consideration the administration method, volume and viscosity of body fluids, etc.

[0094] The cancer 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, thymus 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.

[0095] In yet another aspect of the present invention, a method of reducing cancer cell burden is provided, comprising administering genetically engineered immune cells.

[0096] The individual may be a mammal, including a human, or a non-human animal. The term "non-human animal" refers to any vertebrate, and may include mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. Additionally, the individual may refer to an individual suffering from a cancer disease or a disease whose condition can be alleviated, inhibited, or treated by administering an oncolytic virus.

[0097] The dosage of the genetically engineered immune cells varies depending on the condition and weight of the individual, the severity of the disease, the type of drug, the route and duration of administration, and can be appropriately selected by one skilled in the art.

[0098] The genetically engineered immune cells can be administered parenterally, and such administration can be performed by any suitable method, such as intratumoral, intraperitoneal, subcutaneous, intradermal, intranodal, or intravenous administration, of which intratumoral, intraperitoneal, or intravenous administration may be preferred.

[0099] Regarding the administration route, dosage, and frequency of administration, the genetically engineered immune cells can be administered to a subject in various ways and amounts depending on the subject's condition and the presence or absence of side effects; the optimal administration route, dosage, and frequency of administration can be selected within an appropriate range by a person skilled in the art. In addition, the genetically engineered immune cells 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 combined preparation with other drugs. In particular, the genetically engineered immune cells can be provided in the form of an injectable solution.

[0100] In yet another aspect of the present invention, there is provided a kit for preventing or treating cancer, comprising genetically engineered immune cells and an oncolytic virus. The kit may further comprise hydroxyurea. In yet another aspect of the present invention, there is provided a method for treating cancer in an individual, comprising administering to the individual i) an oncolytic virus; and ii) genetically engineered immune cells, wherein the genetically engineered immune cells and the oncolytic virus are as described above.

[0101] The dosage of the oncolytic virus varies depending on the condition and weight of the subject, the severity of the disease, the type of drug, the route and duration of administration, and can be appropriately selected by those skilled in the art. 5 ~1×10 18 of virus particles, infectious viral units (TCID 50 ), or plaque-forming units (pfu) of oncolytic virus. Specifically, the dosage may be such that the patient receives 1×10 5 , 2 × 10 5 , 5×10 5 , 1×10 6 , 2 × 106 , 5×10 6 , 1×10 7 , 2 × 10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 , 5×10 8 , 1×10 9 , 2 × 10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 The amount of oncolytic virus to be administered may be such that the patient receives 1 x 10 or more viral particles, infectious viral units, or plaque forming units of oncolytic virus, and various values and ranges therebetween are also included. Preferably, the oncolytic virus is administered in an amount of 1 x 10 or more viral particles, infectious viral units, or plaque forming units. 5 ~1×10 10 More preferably, the oncolytic virus is administered at a dose of 1 x 10 pfu. 5 pfu or more and 1 x 10 9 Sub-pfu doses may be administered.

[0102] The oncolytic virus 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 which intratumoral, intraperitoneal, or intravenous administration may be preferred.

[0103] Regarding the administration route, dosage, and frequency of administration, the oncolytic virus can be administered to a subject in various ways and amounts depending on the subject's condition and the presence or absence of side effects; and the optimal administration route, dosage, and frequency of administration can be selected within an appropriate range by a person skilled in the art. In addition, the oncolytic virus 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 combined preparation with other drugs. In particular, the oncolytic virus can be provided in the form of an injectable solution.

[0104] In yet another aspect of the present invention, there is provided a use of genetically engineered immune cells for the prevention or treatment of cancer.

[0105] In yet another aspect of the present invention, there is provided a use of a genetically engineered immune cell for the manufacture of a medicament for preventing or treating cancer. [Example]

[0106] Preferred examples are presented below to help understand the present invention. However, the following examples are provided only for easier understanding of the present invention, and the scope of the present invention is not limited to the following examples.

[0107] I. Construction of a vector containing a nucleic acid encoding protein A56 or a fragment thereof, and identification of the expression of protein A56 on the surface of tumor cells Preparation 1.1. Construction of a vector containing a nucleic acid encoding protein A56 or a fragment thereof To construct plasmids encoding wild-type protein A56 and its fragments, overlapping PCR was performed by preparing primers to remove specific regions (signal, IgV-like, tandem repeats, stalk, transmembrane domain, and cytoplasmic tail) and form regions overlapping with GFP.

[0108] Preparation 1.2. Production of oncolytic vaccinia viruses containing nucleic acids encoding protein A56 or fragments thereof To generate oncolytic vaccinia virus containing protein A56, HeLaS3 (ATCC) cell line was cultured at 4 × 10 cells per well in a 6-well plate. 5 The cells were seeded with 1000 cells and then prepared in EMEM medium containing 10% fetal bovine serum. Treatment with wild-type vaccinia viruses (Wyeth and Western Reserve strains) was performed at an 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 a reporter gene and a gene for insertion using Xfect reagent buffer. The cells were cultured for 4 hours. Subsequently, the medium was replaced with EMEM medium containing 2% fetal bovine serum, and the cells were further cultured for 72 hours. Finally, the infected cells were harvested and then frozen and thawed three times. The cells were lysed by sonication, and free oncolytic vaccinia viruses (OTS-412, WOTS-418) containing nucleic acids encoding protein A56 or its fragments were obtained using the sucrose cushion method.

[0109] Reference Example 1. Comparison of the sequences of protein A56 for each vaccinia virus strain Macrogen was commissioned to sequence the A56 protein gene from the Wyeth strain OTS-412 and the Western Reserve strain WOTS-418, which were derived from wild-type vaccinia virus (NYC Department of Health strain VR-1536) obtained from the American Type Culture Collection (ATCC) by deleting the TK region. The A56 protein sequences from OTS-412 and WOTS-418 were aligned using NCBI Blast and Uniprot. No 100% identical sequences were found; specifically, a deletion of amino acids at positions 245-250 was identified. Comparison with four other strains with high sequence homology was also performed.

[0110] Experimental Example 1. Expression of Protein A56 on the Surface of Cancer Cells An oncolytic vaccinia virus containing the nucleic acid encoding protein A56 or a fragment thereof prepared in Preparation Example 1.2 was used to infect a human lung cancer cell line (A549), a human colorectal cancer cell line (HCT-116), or a human melanoma cell line (SK-MEL-5) to determine whether protein A56 is expressed on the surface of cancer cells.

[0111] 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 plated on cover glasses in 12-well plates at 3.5 × 10 cells per well. 4 Cells were seeded with 1000 cells. Subsequently, the cells were infected with oncolytic vaccinia virus at an MOI of 0.1 and then incubated at 37°C and 5% CO for 30 hours. Each incubated cell line was harvested and treated with 4% (v / v) paraformaldehyde (PFA), 1% (v / v) BSA, anti-A56 primary antibody (catalog no. ABIN1606294, Antibodies-Online) diluted 1:500, and secondary antibody (Alexa 594, catalog no. A21205, Invitrogen) diluted 1:200. DAPI staining was then performed. Samples of each cell line were then placed on slides and observed using a confocal microscope (Olympus, FV1000).

[0112] 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 (Figs. 1 and 2).

[0113] Experimental Example 2. Identification of the expression of protein A56 on the surface of mouse tumor tissue (I) A cancer-induced mouse model was subjected to intraperitoneal administration of an oncolytic vaccinia virus containing a nucleic acid encoding protein A56 or a fragment thereof, as prepared in Preparation Example 1.2, to determine whether protein A56 is expressed on the tissue surface.

[0114] Specifically, BALB / c nude mice were inoculated with 6.3 × 10 HCT-116 colorectal cancer cell lines (Korea Cell Line Bank, KCLB). 6 The cells were subcutaneously transplanted to induce tumor growth. The average tumor volume was 150 mm 3 ~200mm 3 When the mice reached 100 mg / kg, they were inoculated with 2 x 10 mL of the oncolytic vaccinia virus prepared in Preparation Example 1.2. 7 pfu. Then, on day 4, the mice were sacrificed, and tumor tissues, as well as brain, heart, lung, muscle, kidney, liver, and spleen tissues were collected from the mice.

[0115] Immunofluorescence staining for protein A56 was performed in the same manner as in Experimental Example 1. DAPI staining was performed. Then, each tissue sample was placed on a slide glass and observed using a fluorescence microscope.

[0116] As a result, we identified that protein A56 was expressed on the surface of tumor tissue in mice administered with oncolytic vaccinia virus. However, protein A56 was not expressed in the brain, heart, lung, muscle, kidney, liver, and spleen (Figure 3). These results indicate that if a binding molecule targeting protein A56 expressed on the surface of tumor tissue can be produced after administration of oncolytic vaccinia virus, this antibody could be used as an anti-cancer immunotherapy.

[0117] Experimental Example 3. Identification of the expression of protein A56 on the surface of rabbit tissues Normal rabbits were intravenously administered with the oncolytic vaccinia virus containing the nucleic acid encoding protein A56 or a fragment thereof prepared in Preparation Example 1.2 to determine whether protein A56 is expressed on the tissue surface, and thus analyze the toxicity risk.

[0118] Specifically, New Zealand rabbits were inoculated with 1 x 10 of the oncolytic vaccinia virus produced in Preparation Example 1.2. 8 pfu or 1 × 10 9 pfu. After 3 or 8 weeks, the rabbits were sacrificed and the brain, heart, lung, muscle, kidney, liver, and spleen tissues were collected from the rabbits.

[0119] Immunofluorescence staining for protein A56 was performed in the same manner as in Experimental Example 1. DAPI staining was performed. Then, each tissue sample was placed on a slide glass and observed using a fluorescence microscope.

[0120] As a result, it was determined that protein A56 was not expressed in the heart, lung, muscle, kidney, liver, and spleen tissues of rabbits administered oncolytic vaccinia virus.

[0121] On the other hand, a fluorescent reaction was detected in the brain tissue of rabbits administered with oncolytic vaccinia virus. To determine whether the detected fluorescent reaction was a non-specific reaction of anti-A56 antibody, the brain tissue of normal rabbits that were not administered with oncolytic vaccinia virus was subjected to immunofluorescence staining in the same manner as above, and then the fluorescent reaction in the brain tissue was examined using a fluorescence microscope.

[0122] As a result, the fluorescent reaction was detected even in the brain tissue of rabbits that had not been administered the oncolytic vaccinia virus, identifying such a fluorescent reaction as a nonspecific reaction (Figure 4).

[0123] In addition, when normal human brain tissue (Pusan National University Yangsan Hospital, Korea) that had not been administered oncolytic vaccinia virus was subjected to immunofluorescence staining using the same method as above, a weak fluorescent reaction was detected using a fluorescence microscope. However, considering 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 administered directly into the cerebral ventricles.

[0124] Experimental Example 4. Identification of the expression of protein A56 on the surface of mouse tumor tissue (II) A cancer-induced mouse model was subjected to simultaneous administration of an oncolytic vaccinia virus containing a nucleic acid encoding protein A56 or a fragment thereof, as prepared in Preparative Example 1.2, and hydroxyurea to determine whether protein A56 is expressed on the tissue surface.

[0125] Specifically, BALB / c nude mice were inoculated with 6.3 × 10 6 Renca cancer cell lines (Korea Cell Line Bank) were subcutaneously implanted to induce tumors. The average tumor volume was 150 mm. 3 ~200mm 3 When the mice reached 100 mg / kg, they were inoculated with 2 x 10 mL of the oncolytic vaccinia virus prepared in Preparation Example 1.2. 7 pfu and hydroxyurea at a dose of 30 mg / kg.

[0126] The renal cancer cell-implanted mice were divided into three groups (n = 4). The control group received intratumoral administration of saline, and the oncolytic vaccinia virus (1 × 10 7 pfu) alone, and a group receiving oncolytic vaccinia virus (1 × 10 7The experimental group was administered with 100 mg / kg of hydroxyurea (30 mg / kg pfu) and hydroxyurea (30 mg / kg). Oncolytic vaccinia virus was administered intratumorally twice, on days 0 and 14, and hydroxyurea was administered intraperitoneally six times a week, except for the days on which oncolytic vaccinia virus was administered, from 1 day before administration of oncolytic vaccinia virus to 21 days after administration of oncolytic vaccinia virus.

[0127] The mice were sacrificed on days 7, 10, and 14 after the first administration of oncolytic vaccinia virus, and tumor tissues were collected from the mice. The mice were also sacrificed on days 21, 24, and 28 after the second administration of oncolytic vaccinia virus, and tumor tissues were collected from the mice. Immunofluorescence staining for protein A56 was performed in the same manner as in Experimental Example 1. DAPI staining was performed. Then, each tissue sample was placed on a slide glass and observed under a fluorescent microscope.

[0128] As a result, it was identified that protein A56 was clearly expressed on the tumor surface of mice in the group administered oncolytic vaccinia virus alone and the group administered oncolytic vaccinia virus and hydroxyurea simultaneously by days 7, 10, and 14 after the first administration of oncolytic vaccinia virus (Figures 5 and 6).

[0129] In addition, when a second administration of oncolytic vaccinia virus was administered 14 days after the first administration, protein A56 was identified to be expressed on the tumor surface 7, 10, and 14 days after the second administration (Figures 7 and 8). Notably, protein A56 was identified to be expressed in both dead and live cells on day 24 (D10).

[0130] Experimental Example 5. Expression of Protein A56 or Fragments Thereof on the Cell Surface A HeLa cell line was treated with a plasmid encoding protein A56 or a fragment thereof to identify whether protein A56 or a fragment thereof is expressed intracellularly or on the cell surface.

[0131] Specifically, HeLa (cervical cancer cells, ATCC, USA) cell line was plated on a cover glass in a 12-well plate at 3.5 × 10 cells per well. 4 The cells were seeded with 1000 cells. Subsequently, the A56 fragment plasmid, diluted by mixing with Xfect reaction buffer and Xfect polymer, was incubated with HeLa cell lines at 37°C and 5% CO2 for 30 hours. Each incubated cell line was then harvested and treated with 4% (v / v) paraformaldehyde (PFA), 1% (v / v) BSA, anti-A56 antibody (catalog no. ABIN1606294, Antibodies-Online) diluted 1:500, and secondary antibody (Alexa 594, catalog no. A21205, Invitrogen) diluted 1:200. Nuclei were stained with DAPI, and the Golgi apparatus was stained with a fluorescent dye. Cell line samples were then placed on slides and observed using a confocal fluorescence microscope (Olympus, FV1000). The results are shown in Figures 9 and 10 and Table 1.

[0132] [Table 1]

[0133] Wild-type A56 (A56-G) and A56 fragments obtained by partial truncation of six regions of A56 were expressed on the cell surface. As a result, only wild-type A56 (A56-G) and A56-121, in which the IgV-like domain was truncated, were identified to reach the cell surface and be expressed there. Among A56 variants in which the IgV-like domain was omitted, variants in which the signal peptide, transmembrane domain, stalk region, and tandem repeat region were truncated individually or in combination were identified to be absent from the cell membrane. Furthermore, when the IgV-like domain was included, even variants containing only the transmembrane domain were identified to be absent from the cell membrane. That is, variants in which the IgV-like domain was omitted and five regions were included were identified to be expressed on the cell membrane.

[0134] II. Generation of antibodies that bind to protein A56 or fragments thereof Preparative Example 2. Purification and production of protein A56 The vector DNA (N293F-A56-C-HIS) was amplified and transfected into HEK293F cells for overexpression. Subsequently, primary purification was performed by affinity chromatography (Ni-NTA), followed by secondary purification by cation exchange chromatography (CEX). Thus, the A56-C-HIS protein was finally produced (Figure 11).

[0135] Preparation Example 3. Preparation of antibodies that bind to protein A56 or its fragments (I) We commissioned Ybiologics to generate anti-A56 antibodies that specifically bind to the A56 protein or its variants or fragments. Using phage library technology, 61 antibodies were generated and their CDRs were analyzed. The phage library was added to tubes coated with the A56 antigen, and biopanning was performed to find binding hits. Phages with specific binding were selected by washing three times on average. Three panning steps were performed. Subsequently, affinity tests were performed, and a small number of colonies showing high affinity were picked to identify whether the colonies exhibited affinity for the actual antigen. A set with a relatively high number of hits was selected, and an automated system was used for picking and hit selection.

[0136] The affinity between each of the anti-A56 antibodies thus produced and the protein A56 was measured. The results are shown in Figures 12 to 17. In addition, the productivity of each anti-A56 antibody is shown in Figures 18A to 18C. In addition, Figures 19 to 35 show the results obtained by identifying each of the produced anti-A56 antibodies through SDS-PAGE.

[0137] Experimental Example 6. Affinity Measurements for Anti-A56 Antibodies 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, diluted three-fold starting from 100 nM, at room temperature for a predetermined time. Then, the wells were washed three times with PBS, and then treated with a secondary antibody at room temperature for a predetermined time. Subsequently, the affinity of each anti-A56 antibody for protein A56 was measured at each concentration.

[0138] Experimental Example 7. Comparison of Amino Acid Sequence Homology Between Protein A56 for Each Vaccinia Virus Strain Comparison of amino acid sequence homology among protein A56 from vaccinia virus strains, i.e., Copenhagen, Ankara, Western Reserve (WR), International Health Department-J (IHD-J), Tiantan, and Wyeth (New York City Department of Health; NYCBOH). As a result, as shown in Figure 36, it was identified that the amino acids at positions 30 to 90 in the amino acid sequences of protein A56 from all strains are identical.

[0139] III. Identification of the structural epitope of protein A56 Example 1. Distinguishing between linear and structural epitopes on protein A56 To distinguish linear and conformational epitopes, Western blot analysis was performed on 10 anti-A56 antibodies (Ab18, Ab19, Ab01, Ab13, Ab14, Ab08, Ab03, Ab51, Ab55, and 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 and specifically binding to the A56 protein (Figure 37).

[0140] Specifically, HeLa cells were infected with OTS-412; after 24 hours, the HeLa cells were harvested, lysed, and total proteins were extracted. The proteins were subjected to a denaturing step and then loaded onto an SDS-PAGE gel for electrophoresis. After electrophoresis, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane and reacted with 10 anti-A56 antibodies, respectively. After washing with PBST (PBS: Donginbiotech Co., Ltd.; Tween 20: Sigma), the proteins were subsequently reacted with a secondary antibody (goat anti-human IgG Fc cross-adsorbed, HRP (Abcam, catalog no. ab98624)). After washing with PBST again, the membrane was treated with a luminescence reagent (ECL, Amersham Biosciences) and identified using an imaging system (chemiluminescence imaging system). 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 controls.

[0141] As a result, first, as shown in Figure 38, for the positive control group, protein A56 had a molecular weight corresponding to a band of 85 kD to 100 kD, while most of the 10 anti-A56 antibodies were found to have band sizes or intensities weaker than those of the positive control group (Figure 39). In particular, many weak bands with small sizes or no bands were observed for Ab03, Ab08, Ab13, Ab19, Ab51, Ab55, and Ab16. The absence of bands or bands with small sizes and intensities indicates that the epitope is formed three-dimensionally.

[0142] Furthermore, for epitope mapping of seven anti-A56 antibodies, Ab18, Ab13, and Ab16 were preferentially mapped, and then epitopes for two high-binding antibodies (Ab01 and Ab19) were identified.

[0143] Example 2. Analysis of intact protein A56 and anti-A56 antibodies and characterization of binding complexes Prior to epitope mapping, each sample (A56-C-His, three antibodies (Ab13, Ab16, and Ab18), and antigen-antibody binding complexes (A56-C-His / Ab13, A56-C-His / Ab16, and A56-C-His / Ab18) were characterized to analyze sample integrity and the degree of aggregation of the binding complexes.

[0144] Specifically, for intact mass analysis of the control group, protein A56 and anti-A56 antibodies (Ab13, Ab16, and Ab18) were mixed in 5 μl volumes. Then, 1 μl of the 10 μl mixture was mixed with a 1:1 ratio of acetonitrile / water and 0.1% TFA (K200 MALDI Kit) in a recrystallized sinapinic acid matrix (10 mg / ml) placed on a MALDI plate (SCOUT 384) and crystallized at room temperature. Molecular weight measurements were then performed in triplicate by MALDI-MS (mass spectrometry).

[0145] Here, mass analysis of the complex (A56-C-His / anti-A56 antibody) was performed by crosslinking 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 complex remaining after intact mass analysis was mixed with 1 μl of crosslinking reagent (K200 stabilizer, 2 mg / ml) and incubated at room temperature for 180 min. MALDI ToF MS was then performed using a standard nitrogen laser (MS: linear and positive mode, ion source 1: 20 kV, ion source 2: 17 kV, lens: 12 kV, pulsed ion extraction: 400 ns; HM4: gain voltage: 3.14 kV, acceleration voltage: 20 kV).

[0146] As a result, a molecular weight almost similar to that of the control group was detected in the sample identified by cross-linking (Table 2), and no non-covalent complexes were detected, thus confirming that the sample used for analysis was not clumped or damaged.

[0147] [Table 2]

[0148] Furthermore, the antigen-antibody binding complexes (A56-C-His / Ab13, A56-C-His / Ab16, A56-C-His / Ab18, A56-C-His / Ab01, and A56-C-His / Ab19) were stabilized by treatment with a dedicated reagent for noncovalent crosslinking of protein complexes, followed by high-mass MALDI analysis. As a result, two types of peaks were observed. These data were evaluated using Complex Tracker software to identify the presence of the A56·Ab complex and the 2A56·Ab complex (Figures 40–44). Additionally, the molecular weights of each sample and complex are shown in Table 3.

[0149] [Table 3]

[0150] Example 3. Epitope mapping of protein A56 to five anti-A56 antibodies The numbering of the amino acid sequence in A56 as shown in Examples 3.2 to 3.7 and the figures referred to therein is based on the sequence obtained by excluding the N-terminal amino acids 1 to 16 from the amino acid sequence represented by SEQ ID NO: 1038 (e.g., the serine at position 30 corresponds to the serine at position 46 in the amino acid sequence represented by SEQ ID NO: 1038).

[0151] Example 3.1. Sequencing of Protein A56 For epitope mapping of protein A56, the sequence of protein A56 was first identified. Protein A56 was fragmented into peptides by treatment with five proteolytic enzymes (trypsin, chymotrypsin, ASP-N, elastase, and thermolysin). Mass fingerprint information for the digested peptides was then obtained by LTQ-Orbitrap MS (mass spectrometry), and a comparative analysis was performed to determine whether the information matched existing sequence information for protein A56.

[0152] As a result, as shown in Figure 45, the amino acid fragments obtained by digestion of protein A56 with trypsin had a sequence coverage of 38.76%, the amino acid fragments obtained by digestion of protein A56 with chymotrypsin had a sequence coverage of 66.28%, the amino acid fragments obtained by digestion of protein A56 with ASP-N had a sequence coverage of 43.80%, the amino acid fragments obtained by digestion of protein A56 with elastase had a sequence coverage of 94.57%, and the amino acid fragments obtained by digestion of protein A56 with thermolysin had a sequence coverage of 83.33%. Considering all the sequences identified by the five proteases, the sequences were identified to be 99.61% identical to the existing protein A56 in terms of sequence information.

[0153] Example 3.2. Epitope mapping of protein A56 to anti-A56 antibody (Ab13) Samples of the antigen-antibody complex (A56-C-His / Ab13) were subjected to combined deuterium cross-linking and treatment with various proteases, resulting in alkylation and reduction. Subsequently, molecular weight data were obtained from them using high-mass MALDI MS and nLC-LTQ-Orbitrap MS, and then analyzed using software (XQuest, Stavrox).

[0154] As a result, the sequences of the direct contact sites between protein A56 and each anti-A56 antibody (Ab13) were identified using cross-linking. Based on this, the epitopes and paratopes shown in Table 4 were identified as follows:

[0155] [Table 4]

[0156] As shown in Table 4, the epitopes on the binding site of A56-C-His and the anti-A56 antibody (Ab13) were identified to be located at positions 38, 46, 50, 70, 71, 75, 76, 78, 80, 84, and 85 in the amino acid sequence of A56-C-His.

[0157] Specifically, as shown in Figure 46, the epitope positions from amino acid residues 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 (positions 2 to 148 in the amino acid sequence) was represented in silico using Swiss-Model software, the binding sites between Ab13 and A56-C-His are shown in blue. The binding sites in the amino acid sequence of A56-C-His were identified to correspond to positions 38 to 50 (SIILLAAKSDVLY) and positions 70 to 85 (TTITIKSLTARDAGTY).

[0158] Example 3.3. Epitope mapping of protein A56 to anti-A56 antibody (Ab16) Samples of the antigen-antibody complex (A56-C-His / Ab16) were subjected to combined deuterium cross-linking and treatment with various proteases, resulting in alkylation and reduction of the samples. Subsequently, molecular weight data were obtained from them using high-mass MALDI MS and nLC-LTQ-Orbitrap MS, and then analyzed using software (XQuest, Stavrox).

[0159] The sequences of the direct contact sites between protein A56 and each anti-A56 antibody (Ab16) were identified using cross-linking, and the epitopes and paratopes shown in Table 5 were identified as follows.

[0160] [Table 5]

[0161] As shown in Table 5, the epitopes on the binding site of A56-C-His and the anti-A56 antibody (Ab16) were identified to be located at positions 30, 33, 38, 45, 46, 55, 56, 58, and 60 in the amino acid sequence of A56-C-His.

[0162] Specifically, as shown in Figure 47, the epitope positions from amino acid residue 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 (positions 2 to 148 in the amino acid sequence) was represented in silico using Swiss-Model software, the binding sites between Ab16 and A56-C-His are shown in blue. The binding sites were identified to correspond to positions 30 to 46 (SAWYKEPNSIILLAAKS) and 55 to 60 (TKDKIS) in the amino acid sequence of A56-C-His.

[0163] Example 3.4. Epitope mapping of protein A56 to anti-A56 antibody (Ab18) Samples of the antigen-antibody complex (A56-C-His / Ab18) were subjected to combined deuterium cross-linking and treatment with various proteases, resulting in alkylation and reduction. Subsequently, molecular weight data were obtained from them using high-mass MALDI MS and nLC-LTQ-Orbitrap MS, and then analyzed using software (XQuest, Stavrox).

[0164] The sequences of the direct contact sites between protein A56 and each anti-A56 antibody (Ab18) were identified using cross-linking, and the epitopes and paratopes shown in Table 6 were identified as follows.

[0165] [Table 6]

[0166] As shown in Table 6, the epitopes on the site where A56-C-His and the anti-A56 antibody (Ab18) bind were identified to be located at positions 46, 50, 55, 56, 60, 71, 75, and 76 in the amino acid sequence of A56-C-His.

[0167] Specifically, as shown in Figure 48, the epitope positions from amino acid residues 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 (positions 2 to 148 in the amino acid sequence) was represented in silico using Swiss-Model software, the binding sites between Ab18 and A56-C-His are shown in blue. The binding sites in the amino acid sequence of A56-C-His were identified to correspond to positions 46 to 60 (SDVLYTKDKIS) and positions 71 to 76 (TITIKS).

[0168] Example 3.5. Epitope mapping of protein A56 to anti-A56 antibody (Ab01) A sample of the antigen-antibody complex (A56-C-His / Ab01) was subjected to combined deuterium cross-linking and treatment with various proteases, resulting in alkylation and reduction of the sample. Subsequently, molecular weight data were obtained from them using high-mass MALDI MS and nLC-LTQ-Orbitrap MS, and then analyzed using software (XQuest, Stavrox).

[0169] The sequences of the direct contact sites between protein A56 and each anti-A56 antibody (Ab01) were identified using cross-linking, and the epitopes and paratopes shown in Table 7 were identified as follows.

[0170] [Table 7]

[0171] As shown in Table 7, the epitopes on the binding site between A56-C-His and the anti-A56 antibody (Ab01) were identified to be located at positions 30, 38, 45, 75, and 84 in the amino acid sequence of A56-C-His.

[0172] Specifically, as shown in Figure 49, the epitope positions from amino acid residues 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 (positions 2 to 148 in the amino acid sequence) was represented in silico using Swiss-Model software, the binding sites between AbO1 and A56-C-His are shown in blue. The binding sites in the amino acid sequence of A56-C-His were identified to correspond to positions 30 to 45 (SAWYKEPNSIILLAAK) and positions 75 to 84 (KSLTARDAGT).

[0173] Example 3.6. Epitope mapping of protein A56 to anti-A56 antibody (Ab19) Samples of the antigen-antibody complex (A56-C-His / Ab19) were subjected to combined deuterium cross-linking and treatment with various proteases, resulting in alkylation and reduction of the samples. Subsequently, molecular weight data were obtained from them using high-mass MALDI MS and nLC-LTQ-Orbitrap MS, and then analyzed using software (XQuest, Stavrox).

[0174] The sequences of the direct contact sites between protein A56 and each anti-A56 antibody (Ab19) were identified using cross-linking, and the epitopes and paratopes shown in Table 8 were identified as follows.

[0175] [Table 8]

[0176] As shown in Table 8, the epitope on the site where A56-C-His and the anti-A56 antibody (Ab19) bind was identified to be located at positions 45 and 46 in the amino acid sequence of A56-C-His.

[0177] Specifically, as shown in Figure 50, the epitope position from amino acid residues 30 to 90 in the amino acid sequence of A56-C-His is shown in red (positions 45 and 46); and when the protein structure of UTTA-C-His (positions 2 to 148 in the amino acid sequence) was represented in silico using Swiss-Model software, the binding site between Ab19 and A56-C-His is shown in blue. The binding site in the amino acid sequence of A56-C-His was identified to correspond to positions 45 and 46 (KS).

[0178] Example 3.7. Comparison of epitope mapping of protein A56 to anti-A56 antibodies (Abl3, Ab16, Ab18, Ab01, Ab19) The results obtained by epitope mapping in Examples 3.2 to 3.6 were compared and shown in Figures 51 and 52. As a result, it was identified that the epitope in the amino acid sequence of protein A56 was mapped to positions 30 to 85, which corresponds to the IgG-like domain region.

[0179] In addition, the IgG-like domain of protein A56 was modeled for its primary 3D protein structure using SWISS-MODEL and structurally analyzed using iCn3D. As a result, the domain was identified to consist of a total of eight sheets (green), four helices (red), and seven loops (blue). In particular, helices 1 and 2 were identified to be structurally folded immediately adjacent to helix 3.

[0180] Structural analysis of the A56 epitope involved in binding with anti-A56 antibodies (Ab13, Ab16, Ab18, Ab01, and Ab19) has shown that the epitope consists of the positively charged basic amino acid residues lysine (K / Lys) and arginine (R / Arg), the nucleophilic amino acid residues serine (S / Ser) and threonine (T / Thr), and the aromatic tyrosine (Y / Tyr). Basic amino acids readily form hydrogen bonds with their positively charged side chains, while nucleophilic and aromatic amino acids can form hydrogen bonds due to partial negative charges imparted by adjacent amino acids. The main physicochemical properties of the cross-linking amino acid residues in protein A56 and anti-A56 antibodies are summarized below.

[0181] Example 4. Analysis of the paratope of protein A56 for five anti-A56 antibodies The numbering of the amino acid sequence in A56 as shown in Examples 4.1 to 4.5 and the figures referred to therein is based on the sequence obtained by excluding the N-terminal amino acids 1 to 16 from the amino acid sequence represented by SEQ ID NO: 1038 (e.g., the serine at position 30 corresponds to the serine at position 46 in the amino acid sequence represented by SEQ ID NO: 1038).

[0182] Example 4.1. Paratope analysis of protein A56 for anti-A56 antibody (Ab13) The primary 3D protein structure of Ab13 was modeled using SWISS-MODEL software, and structural analysis of the amino acids involved in binding was performed using iCn3D. The results showed that the nucleophilic amino acids serine (S / Ser) and threonine (T / Thr) and aromatic tyrosine (Y / Tyr) are distributed in the exposed terminal portion of the heavy chain and therefore have strong binding affinity with the strongly positively charged lysine (K91) and arginine (R96) in protein A56; the strongly positively charged lysine (K26) is located in CDR1 of the light chain, and lysine (K57) is located in the loop between sheet 5 (S5) and sheet 6 (S6). The nucleophilic amino acids serine (S) and tyrosine (Y / Tyr) in protein A56 have binding affinity with the lysines (K26, K57) (Figure 53).

[0183] Example 4.2. Paratope analysis of protein A56 for anti-A56 antibody (Ab16) The primary 3D protein structure of Ab16 was modeled using SWISS-MODEL software, and structural analysis of the amino acids involved in binding was performed using iCn3D. As a result, it was predicted that arginine (R56) and lysine (K58) in CDR2, located in the loop between sheet 5 (S5) and sheet 6 (S6), the terminal exposed portion of the heavy chain, have a stronger positive charge (blue mash in Figure 58) due to the nucleophilic amino acid serine (S57) located between them, which is advantageous for preferential binding to the nucleophilic amino acid serine (S46, S54) in protein A56; and that this antibody has a high level of binding affinity to the nucleophilic amino acid 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 54).

[0184] Example 4.3. Paratope analysis of protein A56 for anti-A56 antibody (Ab18) Ab18 was modeled into a primary 3D protein structure using SWISS-MODEL software, and structural analysis of the amino acids involved in binding was performed using iCn3D. As a result, the nucleophilic amino acids serine (S / Ser) and threonine (S / Ser) and aromatic tyrosine (Y / Tyr) are distributed in the exposed helix of the heavy chain CDR1, which is relatively negatively charged. Therefore, it was predicted that this region would bind to the strongly positively charged lysine (K72) in protein A56; and the strongly positively charged arginine (R98) was identified to be located in CDR3. Additionally, it was analyzed that the nucleophilic amino acid serine (S30) in 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 the tyrosine (Y / Tyr, T71) in protein A56 (Figure 55).

[0185] Example 4.4. Paratope analysis of protein A56 for anti-A56 antibody (Ab01) Ab01 was modeled into a primary 3D protein structure using SWISS-MODEL software, and 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 aromatic tyrosine (Y106) are distributed in the terminal exposed portion of the heavy chain and therefore have binding affinity with 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 binding affinity with the strongly positively charged lysine (K61) in protein A56 (Figure 56).

[0186] Example 4.5. Paratope analysis of protein A56 for anti-A56 antibody (Ab19) The primary 3D protein structure of Ab19 was modeled using SWISS-MODEL software, and structural analysis of the amino acids involved in binding was performed using iCn3D. As a result, it was found that this antibody has a relatively low binding affinity due to simple hydrogen bonds caused by serines (S93, S94), and that this antibody may not overlap with other anti-A56 antibodies in terms of epitope due to the binding site (K61, S62) located in the loop between sheet 4 (S4) and sheet 5 (S5) in protein A56 (Figure 57).

[0187] Example 5. Analysis of binding of anti-A56 antibodies to protein A56 The amino acid sequence numbering in A56 as shown in Examples 5.1.1 to 5.5.3 and the figures referred to therein is based on the sequence obtained by excluding the N-terminal amino acids 1 to 16 from the amino acid sequence set forth in SEQ ID NO: 1038 (e.g., the serine at position 30 corresponds to the serine at position 46 in the amino acid sequence set forth in SEQ ID NO: 1038).

[0188] Example 5.1.1. Electrostatic Force Analysis of the Binding of Anti-A56 Antibody (Ab13) to Protein A56 To analyze the electrostatic forces of binding between the anti-A56 antibody (Ab13) and the protein A56, we used DelPhi potential, a scientific application that calculates the electrostatic potential and corresponding electrostatic energy in and around a macromolecule. DelPhi potential is a commonly used analytical method for visualizing electrostatic changes along the surface of a protein or other macromolecule and for calculating the electrostatic components of various energies. DelPhi potential incorporates the effect of ionic strength-mediated screening by evaluating the Poisson-Boltzmann equation at a finite number of points in a three-dimensional lattice box. Here, blue and red indicate positive and negative potentials, respectively, and field lines indicate the direction and strength of the electrostatic forces surrounding the protein.

[0189] Specifically, the positively charged basic amino acid lysine (K91) in protein A56, which has a strong DelPhi surface electrostatic potential, excited the hydroxyl group of the serine (S92) residue immediately adjacent to the lysine, resulting in the generation of an electrostatic force that created a relatively low level of DelPhi surface electrostatic potential, which then became strongly positive. Meanwhile, the negatively charged D25 and D55 around K26 and K57 of the light chain in Ab13 created a relatively strong positive DelPhi surface electrostatic potential.

[0190] In addition, T52 and T58 of the heavy chain in Ab13 were highly exposed to the outside due to the adjacent hydrophobic residues I51 (isoleucine) and F54 (phenylalanine), which is aromatic and does not contain a hydrophilic reactive group, and the high level of DelPhi surface electrostatic potential of T52 and T58 was strongly negatively charged due to the negatively charged D56.

[0191] As a result, the electrostatic properties shown in Table 9 were identified.

[0192] [Table 9]

[0193] In this table, the letter (H or L) following the number of the amino acid position shown in the rightmost column indicates heavy chain (H) or light chain (L).

[0194] Example 5.1.2. Analysis of the binding distance between anti-A56 antibody (Ab13) and protein A56 To analyze the binding distance between anti-A56 antibody (Ab13) and protein A56, a structural alignment of protein A56 with Ab13 was predicted using Swiss-PdbViewer (4.1.0), and the H-bonds, angles, and distances between atoms were predicted by comparing the active site or other related parts.

[0195] As a result, the NH residue, i.e., the terminal functional group of K91, which has a strong positive charge in protein A56, forms a hydrogen bond with the O- of T52, which has an appropriate level of negative charge in the heavy chain of Ab13, and the distance between them was measured to be 4.88 Å (Figure 58A). In addition, the NH residue, i.e., the terminal functional group of K57, which has a strong positive charge in the heavy chain of Ab13, forms a hydrogen bond with the O- of S62, which has an appropriate level of negative charge in protein A56, and the distance between them was measured to be 3.73 Å (Figure 58B).

[0196] As two hydrogen bonds are formed, the negatively charged O- of Y66 in protein A56 is predicted to bond with the strongly positively charged K26 in the light chain of Ab 13. In addition, as two hydrogen bonds are formed, the heavy and light chains are predicted to undergo a contraction folding, resulting in the formation of the remaining bonds (S54 (A56) ⇔ heavy chain in antibody) and R96 (A56) ⇔ heavy chain in antibody) electrostatically.

[0197] Example 5.1.3. Competitive Assay of Binding of Anti-A56 Antibody (Ab13) to Protein A56: Binning Study To determine whether the 10 anti-A56 antibodies (Ab18, Ab19, Ab01, Ab13, Ab14, Ab08, Ab03, Ab51, Ab55, and Ab16) have the same or different epitopes on the protein A56, analysis was performed by surface plasmon resonance (SPR) using an Octet instrument.

[0198] Specifically, the A56-C-His antigen was immobilized on a biosensor (NTA), and primary antibodies (SA2038, Ab13, and A56-02A02) were allowed to bind to the antigen until saturation. The remaining nine antibodies were then used as secondary antibodies to further bind to the antigen. If the antibodies had the same epitope, competition would occur, making further binding difficult; if the antibodies had different epitopes, further binding would likely occur. In addition, the results were rechecked by performing an additional experiment in which the binding order of the secondary and primary antibodies to the A56-C-His antigen was reversed.

[0199] As a result, as shown in Figure 59A, no additional binding was identified, indicating that the antibodies have the same epitope.

[0200] In addition, Western blotting was performed to check the binding affinity in the two-dimensional structure using polyacrylamide gel electrophoresis (PAGE), and it was confirmed that no binding occurred for the linear protein A56. From these results, it was confirmed that the anti-A56 antibody (Ab13) recognizes and binds only to the three-dimensional structure of protein A56. In addition, the binding affinity (K ) calculated by OCTET (SPR) and ELISA was D ) values are shown in Figure 59B.

[0201] Example 5.2.1. Electrostatic Force Analysis of the Binding of Anti-A56 Antibody (Ab16) to Protein A56 We used DelPhi potential to analyze the electrostatic forces involved in the binding of anti-A56 antibody (Ab16) and protein A56. Specifically, the basic amino acid lysine (K61), which has a strong positive DelPhi surface electrostatic potential, excited the hydroxyl group of the serine (S62) residue immediately adjacent to the lysine, resulting in the generation of an electrostatic force, resulting in a relatively low level of DelPhi surface electrostatic potential. Here, the positively charged DelPhi surface electrostatic potential for S59 and K60 of the light chain of Ab16 was formed due to the physicochemical properties of protein A56.

[0202] In addition, arginine (R56) and lysine (K58) in CDR2, located in the loop between sheet 5 (S5) and sheet 6 (S6), the terminal exposed portion of the heavy chain in Ab16, have a stronger positive charge due to the nucleophilic amino acid serine (S57) located between these two amino acids, and as a result, it was predicted that binding to the nucleophilic amino acid serine (S46, S54) in protein A56 would proceed preferentially; the electrostatic properties were identified as shown in Table 10.

[0203] [Table 10]

[0204] In this table, the letter (H or L) following the number of the amino acid position shown in the rightmost column indicates heavy chain (H) or light chain (L).

[0205] 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, a structural alignment of protein A56 with Ab16 was predicted using Swiss-PdbViewer (4.1.0), and the H-bonds, angles, and distances between atoms were predicted by comparing the active site or other related parts.

[0206] As a result, as shown in Figure 60, the NH residue, i.e., the terminal functional group of K61, which is strongly positively charged in protein A56, forms a hydrogen bond with the O- of S59, which is negatively charged at an appropriate level, in the light chain of Ab16, and the distance between them is measured to be 4.78 Å. At the same time, the NH residue, i.e., the terminal functional group of K60, which is strongly positively charged in the light chain of Ab16, forms a hydrogen bond with the O- of S62, which is negatively charged at an appropriate level, in protein A56, and the distance between them is measured to be 5.07 Å.

[0207] As the two hydrogen bonds proceed, the negatively charged O- of S46 / S54 in protein A56 is predicted to bind to the strongly positively charged R56 / K58 in the heavy chain of Ab 16. In addition, as the binding proceeds, the heavy and light chains are predicted to undergo a contraction folding, resulting in a strong electrostatic bond between the remaining bonds.

[0208] Example 5.2.3. Competitive Assay of Binding of Anti-A56 Antibody (Ab16) to Protein A56: Binning Study To determine whether the 10 anti-A56 antibodies (Ab18, Ab19, Ab01, Ab13, Ab14, Ab08, Ab03, Ab51, Ab55, and Ab16) have the same or different epitopes on the protein A56, analysis was performed by surface plasmon resonance (SPR) using an Octet instrument.

[0209] Specifically, the A56-C-His antigen was immobilized on a biosensor (NTA), and primary antibodies (SA2041, Ab16, and A56-02B08) were allowed to bind to the antigen until saturation. The remaining nine antibodies were then used as secondary antibodies to further bind to the antigen. If the antibodies had the same epitope, competition would occur, making further binding difficult; if the antibodies had different epitopes, further binding would likely occur. In addition, the results were rechecked by performing an additional experiment in which the binding order of the secondary and primary antibodies to the A56-C-His antigen was reversed.

[0210] As a result, as shown in Figure 61A, no additional binding was identified, indicating that the antibodies have the same epitope.

[0211] In addition, Western blotting was performed to check the binding affinity in the two-dimensional structure using PAGE, and multiple linear bands were identified. This indicates that there is 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. In addition, the binding affinity (K) calculated by OCTET (SPR) and ELISA was D ) values are shown in Figure 61B.

[0212] Example 5.3.1. Electrostatic Force Analysis of the Binding of Anti-A56 Antibody (Ab18) to Protein A56 We used DelPhi potential to analyze the electrostatic forces of binding between the anti-A56 antibody (Ab18) and the A56 protein. Specifically, the basic amino acid lysine (K61), which has a strong positive DelPhi surface electrostatic potential, excites the hydroxyl group of the serine (S62) residue immediately adjacent to the lysine, resulting in a relatively low level of DelPhi surface electrostatic potential. Here, R98 of the heavy chain of Ab18 is highly exposed to the outside due to the methyl groups (CH3) of the adjacent A97 and the short tryptophan residue W99, which is aromatic and does not contain a hydrophilic reactive group, resulting in a high level of DelPhi surface electrostatic potential of R98. In addition, the negatively charged Y49 and S52 residues surrounding K50 of the light chain of Ab18 formed a relatively strong positive DelPhi surface electrostatic potential.

[0213] As a result, the electrostatic properties shown in Table 11 were identified.

[0214] [Table 11]

[0215] In this table, the letter (H, L, or U) following the number of the amino acid position shown in the rightmost column indicates heavy chain (H), light chain (L), or UTTA (or A56) (U).

[0216] Example 5.3.2. Analysis of the Binding Distance between Anti-A56 Antibody (Ab18) and Protein A56 To analyze the binding distance between anti-A56 antibody (Ab18) and protein A56, a structural alignment of protein A56 with Ab18 was predicted using Swiss-PdbViewer (4.1.0), and the H-bonds, angles, and distances between atoms were predicted by comparing the active site or other related parts.

[0217] As a result, the NH residue, i.e., the terminal functional group of K91, which has a strong positive charge in protein A56, forms a hydrogen bond with Y32L, which has a suitable negative charge in the light chain of Ab18, and the distance between them was measured to be 3.46 Å (Figure 62B). In addition, it was predicted that the structure would fold by electrostatically attracting S30, which has a suitable negative charge in the light chain of Ab18. In addition, the NH residue, i.e., the terminal functional group of R98, which has a strong positive charge in the heavy chain of Ab18, forms a hydrogen bond with the O- of S62, which has a suitable negative charge in protein A56, and the distance between them was measured to be 3.77 Å (Figure 62A).

[0218] As the two hydrogen bonds proceed, it is predicted that the strongly positively charged K50 of the light chain of Ab18 will fold and bond with the negatively charged O- of S30 in protein A56.

[0219] Example 5.3.3. Competitive Assay of Binding of Anti-A56 Antibody (Ab18) to Protein A56: Binning Study To determine whether the 10 anti-A56 antibodies (Ab18, Ab19, Ab01, Ab13, Ab14, Ab08, Ab03, Ab51, Ab55, and Ab16) have the same or different epitopes on the protein A56, analysis was performed by the SPR method using an Octet instrument.

[0220] Specifically, the A56-C-His antigen was immobilized on a biosensor (NTA), and primary antibodies (SA2043, Ab18, and A56-02C06) were allowed to bind to the antigen until saturation. The remaining nine antibodies were then used as secondary antibodies to further bind to the antigen. If the antibodies had the same epitope, competition would occur, making further binding difficult; if the antibodies had different epitopes, further binding would likely occur. In addition, the results were rechecked by performing an additional experiment in which the binding order of the secondary and primary antibodies to the A56-C-His antigen was reversed.

[0221] As a result, as shown in Figure 63A, no additional binding was identified, indicating that the antibodies have the same epitope.

[0222] In addition, Western blotting was performed to check the binding affinity in the two-dimensional structure using PAGE, and multiple linear bands were identified. These results confirmed that this antibody recognizes and binds not only to the amino acid sequence of the two-dimensional structure of protein A56, but also to the three-dimensional motif in protein A56. In addition, the binding affinity (K D ) values are shown in Figure 63B.

[0223] Example 5.4.1. Electrostatic Force Analysis of the Binding of Anti-A56 Antibody (Ab01) to Protein A56 We used DelPhi potential to analyze the electrostatic forces involved in the binding of anti-A56 antibody (Ab01) to protein A56. Specifically, the basic amino acid lysine (K61), which has a strong positive charge and a strong DelPhi surface electrostatic potential, excites the hydroxyl group of the serine (S62), the residue immediately adjacent to the lysine, resulting in a relatively low level of DelPhi surface electrostatic potential and a strong positive charge. Meanwhile, the DelPhi surface electrostatic potentials of S32 and T102 in the light chain of Ab01 were relatively negatively charged at appropriate levels.

[0224] In addition, S103 and Y106 of the heavy chain in Ab01 were highly exposed to the outside due to the adjacent hydrophobic residues L107 and F101, which is aromatic and does not contain a hydrophilic reactive group, and the high level of DelPhi surface electrostatic potential was strongly negatively charged due to the nucleophilic amino acid S103.

[0225] As a result, the electrostatic properties shown in Table 12 were identified.

[0226] [Table 12]

[0227] In this table, the letter (H or L) following the number of the amino acid position shown in the rightmost column indicates heavy chain (H) or light chain (L).

[0228] Example 5.4.2. Analysis of the Binding Distance between Anti-A56 Antibody (Ab01) and Protein A56 To analyze the binding distance between anti-A56 antibody (Ab01) and protein A56, a structural alignment of protein A56 with Ab01 was predicted using Swiss-PdbViewer (4.1.0), and the H-bonds, angles, and distances between atoms were predicted by comparing the active site or other related parts.

[0229] As a result, as shown in Figure 64, the NH residue, i.e., the terminal functional group of K61, which is strongly positively charged in protein A56, forms a hydrogen bond with the O- of T102, which is negatively charged at an appropriate level, in the light chain of Ab01, and the distance between them is measured to be 3.69 Å. In addition, the NH residue, i.e., the terminal functional group of K91, which is strongly positively charged in protein A56, forms a hydrogen bond with the O- of Y106, which is negatively charged at an appropriate level, in the heavy chain of Ab01, and the distance between them is measured to be 3.12 Å.

[0230] As two hydrogen bonds are formed, S54 in protein A56 is predicted to bind to T57, T100, and S103 of the heavy chain and S32 of the light chain in Ab01. Additionally, as two hydrogen bonds are formed, the heavy and light chains are predicted to undergo a contraction folding, resulting in the remaining bonds being formed electrostatically.

[0231] Example 5.4.3. Competitive Assay of Binding of Anti-A56 Antibody (Ab01) to Protein A56: Binning Study To determine whether the 10 anti-A56 antibodies (Ab18, Ab19, Ab01, Ab13, Ab14, Ab08, Ab03, Ab51, Ab55, and Ab16) have the same or different epitopes on the protein A56, analysis was performed by the SPR method using an Octet instrument.

[0232] Specifically, the A56-C-His antigen was immobilized on a biosensor (NTA), and the primary antibodies (SA2026, Ab01, and A56-01A02) were allowed to bind to the antigen until saturation. The remaining nine antibodies were then used as secondary antibodies to further bind to the antigen. If the antibodies had the same epitope, competition would occur, making further binding difficult; if the antibodies had different epitopes, further binding would likely occur. In addition, the results were rechecked by performing an additional experiment in which the binding order of the secondary and primary antibodies to the A56-C-His antigen was reversed.

[0233] As a result, as shown in Figure 65A, no additional binding was identified, indicating that the antibodies have the same epitope.

[0234] Additionally, Western blotting was performed to check the binding affinity in the two-dimensional structure using PAGE, and a linear single band was identified. In addition, the binding affinity (K) calculated by OCTET (SPR) and ELISA was also obtained. D ) values are shown in Figure 65B.

[0235] Example 5.5.1. Electrostatic Force Analysis of the Binding of Anti-A56 Antibody (Ab19) to Protein A56 We used DelPhi potential to analyze the electrostatic forces involved in the binding of anti-A56 antibody (Ab19) to protein A56. Specifically, the positively charged basic amino acid lysine (K61) in protein A56, which has a strong DelPhi surface electrostatic potential, excites the hydroxyl group of the serine (S62) residue immediately adjacent to the lysine, resulting in a relatively low level of DelPhi surface electrostatic potential, which becomes strongly positively charged. Among the amino acids 90 to 95 of the light chain of Ab19 (DSSSD), the DelPhi surface electrostatic potential of serines 93 and 94 (S93 and S94) is relatively negatively charged due to the strong negative charges of the acidic aspartic acid residues (D90 and D95).

[0236] In addition, heavy chain Y32 in Ab19 was highly exposed to the outside due to its immediate neighboring hydrophobic amino acids V (valine) and L (leucine), and F (phenylalanine), which is aromatic and does not contain a hydrophilic reactive group, and its low DelPhi surface electrostatic potential was negatively charged due to the nucleophilic amino acid S (serine).

[0237] As a result, the electrostatic properties shown in Table 13 were identified.

[0238] [Table 13]

[0239] Example 5.5.2. Analysis of the Binding Distance between Anti-A56 Antibody (Ab19) and Protein A56 To analyze the binding distance between anti-A56 antibody (Ab19) and protein A56, a structural alignment of protein A56 with Ab19 was predicted using Swiss-PdbViewer (4.1.0), and the H-bonds, angles, and distances between atoms were predicted by comparing the active site or other related parts.

[0240] As a result, as shown in Figure 66, the terminal functional groups of K61 and S62 in protein A56 are ionized to form NH+ (K61) and O- (S62), and S93 and S94 of the light chain in Ab19 have a strongly negatively charged electrostatic surface DelPhi potential of O- (S93, S94) due to the adjacent aspartic acid residues (D91 and D95), which is predicted to result in bond formation. NH2+ (K61) and O- (S62), spaced 9.9 Å apart in protein A56, are located between O- (S93) of the light chain and O- of Y32 of the heavy chain, spaced 19.5 Å apart in Ab19. As a result, K61 in protein A56 binds to S93 in Ab19, and the bond distance is measured to be 4.59 Å (indicated by the pink circle). The bond distance of S62 in protein A56 bound to Y32 in Ab19 was measured to be 6.08 Å (indicated by the white circle).

[0241] In addition, the binding of protein A56 to Ab19 indicates that the two molecules bind horizontally. Therefore, it was predicted that multiple binding would be possible due to the presence of different epitopes on protein A56 if another antibody with a specific paratope other than the binding site had a complementary electrostatic surface DelPhi potential.

[0242] Example 5.5.3. Competitive Assay of Binding of Anti-A56 Antibody (Ab19) to Protein A56: Binning Study To determine whether the 10 anti-A56 antibodies (Ab18, Ab19, Ab01, Ab13, Ab14, Ab08, Ab03, Ab51, Ab55, and Ab16) have the same or different epitopes on the protein A56, analysis was performed by the SPR method using an Octet instrument.

[0243] Specifically, the A56-C-His antigen was immobilized on a biosensor (NTA), and primary antibodies (SA2044, Ab19, and A56-02C07) were allowed to bind to the antigen until saturation. The remaining nine antibodies were then used as secondary antibodies to further bind to the antigen. If the antibodies had the same epitope, competition would occur, making further binding difficult; if the antibodies had different epitopes, further binding would likely occur. In addition, the results were rechecked by performing an additional experiment in which the binding order of the secondary and primary antibodies to the A56-C-His antigen was reversed.

[0244] As a result, as shown in Figure 67A, analysis revealed that slight additional binding of two antibodies (SA2041 (Ab16) and SA2043 (Ab18)) occurred during the secondary antibody binding step. These results identified that the two antibodies (SA2041 (Ab16) and SA2043 (Ab18)) have epitopes other than the epitope of Ab19.

[0245] Additionally, Western blotting was performed to check the binding affinity in the two-dimensional structure using PAGE, and a linear single band was identified. In addition, the binding affinity (K) calculated by OCTET (SPR) and ELISA was also obtained. D ) values are shown in Figure 67B.

[0246] III. Generation of CAR-T cells using the antigen-binding region of the anti-A56 antibody Example 6.1. Structural Design of Chimeric Antigen Receptors The chimeric antigen receptor structure was designed to contain a signal peptide, an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. As an example, as shown in Figure 68, a chimeric antigen receptor was designed to contain an antigen-binding domain (anti-UTTA scFv), a CD8 transmembrane domain (H+TM), and an intracellular signaling domain (4-1BB and CD3Z).

[0247] Example 6.2. Construction of a vector encoding a chimeric antigen receptor The pLVX-EF1α-IRES-mCherry vector (Spel / Notl) was used as the vector, and a gene encoding a chimeric antigen receptor (CAR) containing a signal peptide (sp), a single-chain variable fragment (anti-UTTA scFv) that specifically binds to A56, the transmembrane domain of human CD8 (H+TM), and intracellular signaling domains (4-1BB and CD3ζ) was inserted into the vector.

[0248] The amino acid and nucleotide sequences of the chimeric antigen receptors used in this experiment are shown in Table 14.

[0249] [Table 14] JPEG0007721661000015.jpg201149 JPEG0007721661000016.jpg197149 JPEG0007721661000017.jpg197149 JPEG0007721661000018.jpg96149

[0250] Example 6.3. Generation of CAR-T cells With IRB approval for human-derived material research, CAR-T cells were generated from blood donated by Pusan National University Yangsan Hospital (Korea) using the MACS cell separation system by negative selection, in which all blood cells except T cells were labeled with a marker. CD3+ T cells (≥97%) were then isolated using MACS Pan T cell Ab. Subsequently, 1 × 10 6 CD3+ T cells were cultured in medium containing 20 IU / ml rhIL-2 and TransAct (CD3 / CD28 agonist) for 24 hours to induce T cell activation.

[0251] Activated T cells were treated with each of the five types of lentivirus cloned into the vector constructed in Experimental Example 6.2 at an MOI of 50 and further cultured in the same medium for 48 hours. T cells were then cultured at 1 x 10 in medium containing 20 IU / ml rhIL-2. 6 The cells were resuspended at 1000 cells / ml. The medium was replaced with fresh medium containing rhIL-2 for cell growth, and the cells were counted every 2 or 3 days. CAR-T cells generated using a lentivirus directed against the antigen-binding domain of Ab13 were named "Ab13 CAR-T," while CAR-T cells generated using a lentivirus directed against the antigen-binding domain of Ab16 were named "Ab16 CAR-T." CAR-T cells generated using a lentivirus directed against the antigen-binding domain of Ab18 were named "Ab18 CAR-T," and CAR-T cells generated using a lentivirus directed against the antigen-binding domain of Ab01 were named "Ab01 CAR-T." CAR-T cells generated using a lentivirus directed against the antigen-binding domain of Ab19 were named "Ab19 CAR-T." Additionally, T cells not transduced with any CAR (UTD, untransduced) were used as a control group.

[0252] Experimental Example 8. Identification of the anti-cancer effects of oncolytic vaccinia virus (OTS-412) and CAR-T cells: in vitro (I) HCT-116 cell line (5 × 10 3 cells / well) and HeLa cell line (5 × 10 3Cells (cells / well) were seeded into a 96-well plate. Then, while incubating at 37°C, the cells were either untreated or infected with OTS-412 (prepared in Example 1.1) at an MOI of 1 to ensure that the A56 protein was expressed on the cancer cell surface. After 2 hours, the medium was replaced with 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, and Ab19 CAR-T) were administered at a T cell:cancer cell ratio of 1:1. After 48 hours, cytotoxicity was measured using a CCK-8 kit.

[0253] As a result, for the HCT-116 cell line and HeLa cell line infected with OTS-412, the groups treated with Ab13 CAR-T, Ab16 CAR-T, Ab18 CAR-T, and Ab19 CAR-T cells (excluding Ab01 CAR-T cells) among the five types of CAR-T cells showed statistically significant specific cytotoxic effects compared to the groups treated with UTD cells (Figure 69).

[0254] Experimental Example 9. Identification of the anti-cancer effects of oncolytic vaccinia virus (OTS-412) and CAR-T cells: in vitro (II) The anti-cancer effects of the 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).

[0255] In detail, three cancer cell lines, HeLa (3 × 10 3 cells / well), NCI-H522 (6 × 10 3 cells / well), and HCT-116 (9 × 10 3 The cells were then seeded in a 96-well plate with OTS-412 (1.55 × 10 cells) of Preparation Example 1.1 at an MOI of 0.05 while incubating at 37°C. 8The cells were infected with 1000 pfu / ml of CAR-T cells, resulting in the expression of the A56 protein on the cancer cell surface. After two hours, the medium was replaced with medium containing 2% FBS. Four hours later, UTD cells and five types of CAR-T cells (Ab13 CAR-T, Ab16 CAR-T, Ab18 CAR-T, Ab01 CAR-T, and Ab19 CAR-T) were administered at a T cell:cancer cell ratio of 3:1. The T cells were administered with medium (10% FBS) containing a red fluorescent dye. Cell death image data were then acquired at 30-minute intervals for five days using an Incucyte system and analyzed using software.

[0256] As a result, for the OTS-412-infected HeLa cell line, when compared with the group treated with UTD cells, the groups treated with each of the five types of CAR-T cells showed statistically significant specific cytotoxic effects (Figure 70; * indicates p<0.033, ** indicates p<0.002, and *** indicates p<0.001 versus the UTD group, and ## indicates p<0.002 versus 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 statistically significant differences in protein A56-specific cytotoxicity compared with the cytotoxicity of the group not infected with OTS-412.

[0257] For the NCI-H522 cell line infected with OTS-412, when compared with the group treated with UTD cells, among the five types of CAR-T cells, the groups treated with Ab01 CAR-T, Ab13 CAR-T, Ab16 CAR-T, and Ab19 CAR-T showed statistically significant specific cytotoxic effects (Figure 71; * indicates p<0.033, ** indicates p<0.002, and *** indicates p<0.001 compared with the UTD group, and ## indicates p<0.002 compared with the 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 with the cytotoxicity of the group not infected with OTS-412.

[0258] For the OTS-412-infected HCT-116 cell line, when compared with the group treated with UTD cells, the groups treated with each of the five types of CAR-T cells showed statistically significant CAR-T cell-specific cytotoxicity effects (Figure 72; * indicates p<0.033, ** indicates p<0.002, and *** indicates p<0.001 vs. the UTD group, and ## indicates p<0.002 vs. 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 with the cytotoxicity of the group not infected with OTS-412.

[0259] Furthermore, when the transduction efficiencies of the five types of CAR-T cells were measured by flow cytometry (FACS), distinct 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, no distinct peaks were clearly observed, likely due to the low intensity of CAR expressed on the T cell surface, and their transduction efficiencies were also low (Figure 73). The differences in transduction efficiencies among the five CAR-Ts were similar to those observed for blood derived from subjects of different ages and genders. The transduction efficiencies of Ab13 CAR-T, Ab16 CAR-T, and Ab19 CAR-T were consistently higher than those of Ab01 CAR-T and Ab18 CAR-T; among these, the transduction efficiency of Ab16 CAR-T was consistently the highest.

[0260] Experimental Example 10. Identification of the anti-cancer effects of oncolytic vaccinia virus (OTS-412) and CAR-T cells: in vitro (III) Among the five types of CAR-T cells, Ab16 CAR-T cells and Ab18 CAR-T cells exhibit antigen-specific cytotoxic effects in various types of cancer cells and relatively low cytotoxicity against cancer cells that do not express the A56 protein. The cytotoxic effects of these CAR-T cells, which depend on the expression level of the A56 protein, were analyzed by changing the dose of OTS-412.

[0261] Specifically, the HCT-116 cell line (1 × 10 4 Cells (cells / well) were seeded into a 96-well plate. Then, while incubating at 37°C, the cells were either untreated or infected with OTS-412 (prepared in Example 1.1) at MOIs of 0, 0.0625, 0.0125, 0.25, 0.5, and 1, respectively, to express the A56 protein on the cancer cell surface. After 2 hours, the medium was replaced with medium containing 2% FBS. After 4 hours, UTD cells, Ab16 CAR-T, and Ab18 CAR-T were administered at a T cell:cancer cell ratio of 1:1. After 48 hours, cytotoxicity was measured using a CCK-8 kit. A cancer cell line that was not treated with OTS-412 or CAR-T cells served as a control.

[0262] As a result, for the OTS-412-infected HCT-116 cell line, the groups treated with Ab16 CAR-T cells and AB18 CAR-T cells showed statistically significant CAR-T cell-specific cytotoxicity effects compared to the group treated with UTD cells at all MOIs of 0.0625, 0.0125, 0.25, 0.5, and 1 (Figure 74).

[0263] Experimental Example 11. Identification of the anti-cancer effects of oncolytic vaccinia virus (OTS-412) and CAR-T cells: in vitro (IV) The anti-cancer effect of CAR-T cells was analyzed by measuring cell death in real time using a fluorescence-based cell imaging system (Incucyte® Live Cell Analysis System, Sartorius).

[0264] Specifically, the cancer cell line A546 (1 × 10 4 cells) and HCT-116 (3 × 10 4 The cells were then seeded in a 96-well plate with OTS-412 (1.55 × 10 cells) of Preparation Example 1.1 at an MOI of 0.05 while incubating at 37°C. 8 The cells were infected with 1000 CAR-T cells (pfu / ml), resulting in the expression of the A56 protein on the cancer cell surface. After two hours, the medium was replaced with medium containing 2% FBS. Four hours later, UTD cells, Ab16 CAR-T cells, and Ab18 CAR-T cells were administered at a T cell:cancer cell ratio of 3:1. The T cells were administered with medium (10% FBS) containing a red fluorescent dye. Cell death image data were then acquired at 30-minute intervals for five days using an Incucyte system and analyzed using software.

[0265] As a result, for the OTS-412-infected A549 and HCT-116 cell lines, more dead cells were stained in the groups treated with Ab16 CAR-T or Ab18 CAR-T cells than in the group treated with UTD cells, as shown in Figure 75. In addition, for the OTS-412-infected A549 and HCT-116 cell lines, both the groups treated with Ab16 CAR-T and Ab18 CAR-T cells, respectively, showed statistically significant CAR-T cell-specific cytotoxicity effects compared to the group treated with UTD cells (Figure 76).

[0266] Experimental Example 12. Identification of the anti-cancer effects of oncolytic vaccinia virus (OTS-412) and CAR-T cells: in vitro (V) HeLa cell line (5 × 10 3 cells / well), MCF7 cell line (5 × 10 3 cells / well), A549 cell line (5 × 10 3 cells / well), or PC-3 cell line (5 × 10 3Cells (cells / well) were seeded into a 96-well plate. Then, while incubating at 37°C, the cells were infected with OTS-412 (prepared in Example 1.1) at an MOI of 0.05 to express the A56 protein on the cancer cell surface. After 2 hours, the medium was replaced with medium containing 2% FBS. After 4 hours, UTD cells, Ab16 CAR-T, and Ab18 CAR-T were administered at a T cell:cancer cell ratio of 1:1. After 44 hours for the HeLa cell line and 72 hours for the other cell lines, cytotoxicity was measured using a CCK-8 kit. Here, cancer cell lines treated with T cells alone without treatment with oncolytic vaccinia virus were used as controls.

[0267] As a result, it was identified that the combined treatment of OTS-412 and Ab16 CAR-T cells or Ab18 CAR-T cells significantly increased cytotoxicity in all cancer cell lines compared to treatment with T cells alone (Figure 77).

[0268] Experimental Example 13. Identification of CAR-T cell activity To determine whether the CAR-T cells were activated, measurements were performed by flow cytometry (FACS) using the indicator CD25 expressed on the surface of T cells.

[0269] Specifically, the HCT-116 cell line (4 × 10 5 Cells (cells / well) were infected with OTS-412 at an MOI of 1. After 4 hours, Ab16 CAR-T cells were administered at a T cell:cancer cell ratio of 1:1. After 48 hours, measurements were performed using flow cytometry (FACS).

[0270] As a result, as shown in Figure 78, for Ab16 CAR-T cells that were not co-cultured with HCT-116 cell line, the percentage of T cells expressing CD3+CD25+ was 16.03%, while for Ab16 CAR-T cells that were co-cultured with HCT-116 cell line, the percentage of T cells expressing CD3+CD25+ increased to 24.83%. In particular, for Ab16 CAR-T cells that were co-cultured with HCT-116 cell line infected with OTS-412, the percentage of T cells expressing CD3+CD25+ increased to 39.39%.

[0271] Experimental Example 14. Identification of the proliferation capacity of CAR-T cells To identify the proliferation capacity of CAR-T cells, measurements were performed by flow cytometry (FACS) using the indicator CD28 expressed on the surface of T cells.

[0272] Specifically, the HCT-116 cell line (4 × 10 5 Cells (cells / well) were infected with OTS-412 at an MOI of 1. Four hours later, UTD (mock-T) cells and Ab18 CAR-T cells were administered at a T cell:cancer cell ratio of 1:1. Measurements were performed by flow cytometry (FACS) on days 1 and 5.

[0273] As a result, as shown in Figure 79, it was identified that on day 1, the percentage of T cells expressing CD4+ was higher in UTD cells (32.15%) than in Ab18 CAR-T cells (28.03%), and on day 5, the percentage of T cells expressing CD4+ was higher in Ab18 CAR-T cells (49.70%) than in UTD cells (37.21%). These results identified that Ab18 CAR-T cells have an increased proliferative capacity specifically against cancer cells expressing protein A56.

[0274] Experimental Example 15. Identification of the anti-cancer effects of oncolytic vaccinia virus (OTS-412) and CAR-T cells in HCT-116 cell line-implanted mice (I) Female NOD SCID mice (NOD.CB17-Prkdcs scid / NCrKoat, 7 weeks old) were subjected to a 1-week acclimation period, after which 2.5 × 10 6 HCT-116 cell line (Korea Cell Line Bank) was allogeneically transplanted at 100 cells / mL. The tumor volume was 150 mm. 3 After that, add OTS-412 to 1 × 10 5 Three days after OTS-412 administration, Ab16 CAR-T cells and Ab18 CAR-T cells were administered at a dose of 5 × 10 pfu each. 6 The cells were intratumorally administered at a dose of 100 μl. The control group received intratumoral administration of saline. The control group received intraperitoneal administration of 30 mg / kg / day six times a week until the sixth day after administration of OTS-412, except for the day on which OTS-412 was administered.

[0275] The body weight of the mice in each group was measured on days 3, 8, 10, 14, 17, and 21. As a result, no trend toward weight loss was observed in any group (Figure 80). In addition, on days 3, 8, 10, 14, 17, and 21, the mice in each group were sacrificed and the tumor volume was measured. As a result, it was identified that tumor growth was significantly inhibited in the mice in the groups that received co-administration of OTS-412 and Ab16 CAR-T cells or Ab18 CAR-T cells (Figure 81).

[0276] Experimental Example 16. Identification of the anti-cancer effects of oncolytic vaccinia virus (OTS-412) and CAR-T cells in HCT-116 cell line-implanted mice (II) Female NOD SCID mice (NOD.CB17-Prkdcs scid / NCrKoat, 7 weeks old) were subjected to a 1-week acclimation period, after which 2.5 × 10 6 HCT-116 cell line (Korea Cell Line Bank) was allogeneically transplanted at 100 cells per 1000 mcg. 3 After that, add OTS-412 to 1 × 10 6On day 3 after OTS-412 administration, UTD cells and Ab16 CAR-T cells were administered at a dose of 5 × 10 6 The cells were intratumorally administered at a dose of 100 μl. The control group received intratumoral administration of saline. The control group received intraperitoneal administration of 30 mg / kg / day six times a week until the sixth day after administration of OTS-412, except for the day on which OTS-412 was administered.

[0277] The body weights of the mice in each group were measured on days 3, 8, 10, 14, 17, and 21. As a result, no trend toward weight loss was observed in any group (Figure 82). In addition, on days 3, 8, 10, 14, 17, and 21, the mice in each group were sacrificed and tumor volumes were measured. As a result, it was identified that tumor growth was significantly inhibited in mice receiving co-administration of OTS-412 and UTD cells or Ab16 CAR-T cells; and a statistically significant difference was observed when comparing tumors in mice receiving co-administration of OTS-412 and Ab16 CAR-T cells with tumors in mice receiving co-administration of OTS-412 and UTD cells (Figure 83).

[0278] Experimental Example 17. Identification of the anti-cancer effects of oncolytic vaccinia virus (WOTS-418) and CAR-T cells MCF7 cell line (5 × 10 3 cells / well) or A549 cell line (5 × 10 3Cells (cells / well) were seeded into a 96-well plate. Then, while incubating at 37°C, the cells were infected with WOTS-418 (prepared in Example 1.1) at an MOI of 0.05 to express the A56 protein on the cancer cell surface. After 2 hours, the medium was replaced with medium containing 2% FBS. After 4 hours, UTD cells, Ab16 CAR-T, and Ab18 CAR-T were administered at a T cell:cancer cell ratio of 1:1. After 72 hours, cytotoxicity was measured using a CCK-8 kit. Here, a cancer cell line treated with T cells alone without treatment with oncolytic vaccinia virus was used as a control group.

[0279] As a result, it was identified that combined treatment with WOTS-418 and Ab16 CAR-T cells or Ab18 CAR-T cells significantly increased cytotoxicity in all cancer cell lines compared to treatment with T cells alone (Figure 84).

[0280] Experimental Example 18. Identification of the anti-cancer effects of oncolytic vaccinia viruses (OTS-412, WOTS-418) and CAR-T cells The anti-cancer effect of CAR-T cells was analyzed by measuring cell death in real time using a fluorescence-based cell imaging system (Incucyte® Live Cell Analysis System, Sartorius).

[0281] In detail, the cancer cell line HCT-116 (1 × 10 4Cells (number of cells) were seeded into a 96-well plate. Then, while incubating at 37°C, the cells were infected with OTS-412 and WOTS-418 (Preparation Example 1.1) at an MOI of 0.05, resulting in the expression of protein A56 on the cancer cell surface. After 2 hours, the medium was replaced with 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, and Ab19 CAR-T) were administered at a T cell:cancer cell ratio of 3:1. The T cells were administered with medium (10% FBS) containing a red fluorescent staining reagent. Cell death image data were then acquired at 30-minute intervals for 5 days using an Incucyte system and analyzed using software.

[0282] As a result, for the HCT-116 cell line infected with OTS-412 or WOTS-418, it was identified that 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, and Ab19 CAR-T) than in the group treated with UTD cells (Figures 85 and 86).

Claims

1. A genetically engineered T cell expressing a chimeric antigen receptor (CAR) comprising (i) an extracellular antigen-binding domain, (ii) a transmembrane domain, and (iii) an intracellular signaling domain, wherein the chimeric antigen receptor specifically binds to an antigen that is present on the surface of a cancer cell and not present on the surface of a normal cell, wherein the antigen is a protein not naturally expressed by the cancer cell; The extracellular antigen-binding domain is (a) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 222, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 223, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 224, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 225, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 226, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 227; (b) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 290, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 291, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 292, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 293, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 294, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 295; (c) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 324, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 325, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 326, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 327, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 328, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 329; (d) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6; (e) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 341, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 342, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 343, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 344, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 345, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 346; (f) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 35, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 36, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 37, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 38, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 39, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 40; (g) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 120, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 121, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 123, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 124, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 125; (h) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 239, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 240, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 241, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 242, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 243, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 244; (j) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 851, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 852, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 853, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 854, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 855, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 856; or (k) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 919, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 920, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 921, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 922, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 923, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

924. , genetically engineered T cells.

2. The extracellular antigen-binding domain is (a) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 222, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 223, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 224, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 225, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 226, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 227; (b) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 290, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 291, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 292, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 293, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 294, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 295; (c) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 324, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 325, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 326, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 327, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 328, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 329; (d) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6; or (e) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 341, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 342, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 343, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 344, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 345, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

346.

2. The genetically engineered T cell of claim 1, wherein

3. The extracellular antigen-binding domain comprises: (f) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 35, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 36, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 37, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 38, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 39, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 40; (g) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 120, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 121, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 123, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 124, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 125; (h) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 239, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 240, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 241, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 242, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 243, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 244; (j) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 851, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 852, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 853, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 854, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 855, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 856; or (k) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 919, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 920, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 921, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 922, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 923, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

924.

2. The genetically engineered T cell of claim 1, wherein

4. 2. The genetically engineered T cell of claim 1, wherein (i) the extracellular antigen-binding domain consists of any one amino acid sequence selected from SEQ ID NOs: 1081-1085.

5. The genetically engineered T cell of claim 1, wherein the extracellular antigen-binding domain is an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:290, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:291, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:292, a light chain CDR1 having the amino acid sequence of SEQ ID NO:293, a light chain CDR2 having the amino acid sequence of SEQ ID NO:294, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

295.

6. The genetically engineered T cell of claim 1, wherein the extracellular antigen-binding domain is an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 222, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 223, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 224, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 225, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 226, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

227.

7. The genetically engineered T cell of claim 1, wherein the extracellular antigen-binding domain is an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 324, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 325, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 326, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 327, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 328, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

329.

8. The genetically engineered T cell of claim 1, wherein the extracellular antigen-binding domain is an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

6.

9. The genetically engineered T cell of claim 1, wherein the extracellular antigen-binding domain is an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 341, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 342, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 343, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 344, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 345, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

346.

10. 2. The genetically engineered T cell of claim 1, wherein the intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

11. 11. The genetically engineered T cell of claim 10, wherein the costimulatory domain is derived from at least one molecule selected from the group consisting of CD137 (4-1BB), CD28, and CD134 (OX40).

12. The genetically engineered T cell of claim 10, wherein the costimulatory domain consists of the amino acid sequence represented by SEQ ID NO: 1087.

13. 11. The genetically engineered T cell of claim 10, wherein the primary signaling domain is derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, or CD66d.

14. 11. The genetically engineered T cell of claim 10, wherein the primary signaling domain is derived from CD3ζ.

15. 11. The genetically engineered T cell of claim 10, wherein the primary signaling domain consists of the amino acid sequence set forth in SEQ ID NO: 1088.

16. The genetically engineered T cells of claim 1, wherein the T cells are administered together with an oncolytic virus to reduce cancer cell burden, and wherein administration of the T cells is performed before, simultaneously with, or after administration of the oncolytic virus.

17. 17. The genetically engineered T cell of claim 16, wherein the oncolytic virus is a vaccinia virus.

18. The vaccinia virus may be any of the following: 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 (IHD-W).

18. The genetically engineered T cell of claim 17, wherein the T cell is an Immunoglobulin D1 / D2 (IHD-D2) Division-White (IHD-W).

19. A genetically engineered T cell expressing a chimeric antigen receptor (CAR) comprising (i) an extracellular antigen-binding domain, (ii) a transmembrane domain, and (iii) an intracellular signaling domain, wherein the chimeric antigen receptor specifically binds to an antigen present on the surface of a cancer cell and not present on the surface of a normal cell, wherein the antigen is a protein not naturally expressed by the cancer cell; The extracellular antigen-binding domain is (a) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 222, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 223, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 224, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 225, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 226, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 227; (b) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 290, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 291, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 292, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 293, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 294, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 295; (c) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 324, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 325, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 326, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 327, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 328, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 329; (d) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6; (e) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 341, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 342, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 343, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 344, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 345, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 346; (f) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 35, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 36, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 37, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 38, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 39, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 40; (g) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 120, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 121, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 123, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 124, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 125; (h) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 239, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 240, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 241, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 242, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 243, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 244; (j) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 851, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 852, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 853, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 854, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 855, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 856; or (k) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 919, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 920, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 921, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 922, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 923, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

924. and The transmembrane domain consists of the amino acid sequence represented by SEQ ID NO: 1086; A genetically engineered T cell, wherein the intracellular signaling domain comprises a costimulatory domain consisting of the amino acid sequence represented by SEQ ID NO: 1087, and a primary signaling domain consisting of the amino acid sequence represented by SEQ ID NO: 1088.

20. (i) an extracellular antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain A chimeric antigen receptor (CAR) comprising: specifically binds to protein A56 or a fragment thereof exposed on the surface of cancer cells, The extracellular antigen-binding domain is (a) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 222, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 223, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 224, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 225, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 226, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 227; (b) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 290, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 291, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 292, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 293, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 294, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 295; (c) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 324, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 325, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 326, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 327, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 328, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 329; (d) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6; (e) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 341, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 342, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 343, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 344, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 345, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 346; (f) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 35, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 36, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 37, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 38, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 39, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 40; (g) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 120, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 121, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 122, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 123, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 124, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 125; (h) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 239, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 240, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 241, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 242, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 243, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 244; (j) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 851, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 852, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 853, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 854, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 855, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 856; or (k) an A56 binding molecule comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 919, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 920, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 921, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 922, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 923, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:

924. , a chimeric antigen receptor.

21. The chimeric antigen receptor of claim 20, wherein the extracellular antigen-binding domain consists of an amino acid sequence represented by any one of SEQ ID NOs: 1081 to 1085.

22. A polynucleotide encoding the chimeric antigen receptor of claim 20.

23. The polynucleotide according to claim 22, comprising a base sequence represented by any one selected from SEQ ID NOs: 1090 to 1094.

24. The polynucleotide of claim 22 A vector comprising:

25. Signal peptide coding sequence 25. The vector of claim 24, further comprising:

26. 26. The vector of claim 25, wherein a sequence encoding a signal peptide is inserted before a sequence encoding an extracellular antigen-binding domain.

27. A genetically engineered T cell into which the vector of claim 24 has been introduced.

28. 25. A step of introducing the vector of claim 24 into T cells.

1. A method for producing genetically engineered T cells, comprising:

29. A pharmaceutical composition for preventing or treating cancer, comprising the genetically engineered T cells of any one of claims 1 to 19.

30. 30. The pharmaceutical composition of claim 29, wherein the cancer is 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, thymus 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.

31. A genetically engineered T cell according to any one of claims 1 to 19; and A kit for preventing or treating cancer, comprising an oncolytic virus.

32. 20. Use of a genetically engineered T cell according to any one of claims 1 to 19 for the manufacture of a medicament for preventing or treating cancer.

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