T cell receptor, immune cell including t cell receptor and method for using the same

Abstract

To provide a T cell receptor which is specific to a specific antigen for promoting targeting of a NK cell to a solid cancer.SOLUTION: There is provided a method for promoting targeting of a NK cell to a solid cancer, the method is configured to: identify a T cell receptor which can recognize a specific molecule modification to the solid cancer; deform a gene so as to express the T cell receptor in a NK cell (NK-92) and cell strain, for improving targeting of the NK cell to the solid cancer. In addition to an antigen-specific T cell receptor, there are also provided: a nucleic acid; a vector; an immune cell including the vector; and a method using the immune cell.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to T cell receptors, NK cells containing the T cell receptors, and methods of using same. [Background technology]

[0002] As of 2018, cell therapy drugs used in cancer treatment, such as Axicabtagene ciloleucel, Tisagenlecleucel, and Immuncell-LC, which are blood cancer treatment drugs developed by Gilead, Novartis, and MOLMED, are already being used in the market for cancer treatment, and clinical trials of cell therapy drugs that have progressed to phase 2 / 3 are also underway. More than 50% of all cell therapy development is being conducted in North America (USA) (344 cases as of 2018), and a considerable portion of research is also underway in China (203 cases).

[0003] Various types of cell therapy drugs, such as CAR-T, which is suitable for recognizing and activating the surface of cancer cells, TCR-T, which has a modified TCR, autologous T cells that recognize tumor antigens, and TIL, have been developed and are undergoing preclinical and clinical research. In particular, cell therapy drugs targeting CD19 have been developed the most, followed by TAA / TSA, BCMA, GD2, and HER2. CD19 is almost the only cell therapy drug that has made it to the market, and the IND application process is currently underway in conjunction with most clinical and preclinical research.

[0004] When comparing cell therapy with currently used immune checkpoint inhibitors, there are limitations to immunotherapy, as the lack of intratumoral T cells makes immune checkpoint inhibitors less effective, and the number of intratumoral T cells that target tumor antigens is low, at around 5%. Therefore, cell therapy has the advantage of being able to attack tumors intensively by multiplying and utilizing only immune cells that target tumors.

[0005] Lung cancer has a high mortality rate worldwide, and among lung cancer patients in Asia in particular, EGFR mutation tumors account for a significant proportion (50%), of which 27% have the L858R mutation. A characteristic of EGFR mutations is that they are often effective against EGFR-targeted drugs in the early stages, but after 8-10 months, most patients develop resistance, and there are no treatments available for acquired resistance. In particular, it is known that combination therapy with immune checkpoint inhibitors is ineffective, and the development of new therapeutic elements is necessary.

[0006] Thus, there is a need for therapeutic agents that specifically target the critical genetic lesions that direct the growth of such tumors.

[0007] The Background of the Invention is provided to facilitate a better understanding of the present invention and should not be construed as an admission that any of the material described in the Background of the Invention is prior art. Summary of the Invention [Problem to be solved by the invention]

[0008] Meanwhile, natural killer cells (NK cells) are cytotoxic lymphocytes that constitute the main component of the innate immune system. Generally, NK cells, which account for about 10% to 15% of circulating lymphocytes, are non-specific to antigens and can target and bind to many malignant cells, including virus-infected cells, without prior immune sensitization, and can induce apoptosis of these cells. In this case, apoptosis of target cells can be generated by inducing cell lysis.

[0009] Therefore, NK cells are isolated from peripheral blood lymphocytes of a subject for therapeutic purposes, and the isolated NK cells are cultured again to obtain a large number of cells, and then reinjected into the subject. Such NK cell therapy, i.e., in vitro and in vivo infusion therapy, has attracted attention because it can induce very effective cell death in infected or tumor cells. However, NK cell infusion therapy has a limitation in that the targets are limited.

[0010] Therefore, the inventors of the present invention focused on NK cells through genetic modification to overcome this. More specifically, conventional NK cell-based cell therapy drugs have adopted strategies such as inducing antibody-dependent cellular cytotoxicity via expression of Fc receptors or targeting cancer via expression of chimeric antigen receptors. However, such strategies have not been very effective in treating solid cancers.

[0011] Therefore, in order to overcome the limitations of the above-mentioned solid tumor treatment, the inventors of the present invention focused on T cell receptors specific to specific antigens, and attempted to enhance the targeting of NK cells to solid tumors through this.

[0012] Ultimately, the inventors of the present invention were able to identify a T cell receptor capable of recognizing specific molecular mutations in solid tumors, genetically engineer this to be expressed in NK cells (NK-92) and cell lines, and improve the targeting of NK cells to solid tumors.

[0013] Furthermore, the inventors of the present invention recognized that further comprising an Fc receptor in the above-mentioned NK cells would further improve the immune response against the targeted solid cancer. Therefore, the inventors of the present invention developed NK cells that contain both a T cell receptor and an Fc receptor, and have further improved targeting and anti-cancer effect against solid cancers.

[0014] More specifically, the inventors of the present invention confirmed an analysis platform that showed an antigen prediction rate of 90% or more based on the results of WES and RNA seq of cancer patients. At this time, the algorithm applied to the analysis confirmed a group of antigen candidates through Neopepsee based on the Net-MHC algorithm. Next, the predicted antigens were synthesized, processed into DCs and CD8 T cells, and the antigens were verified.

[0015] Based on this, we discovered the major HLA-A alleles that can recognize the antigen for L858R. As a result, not only was the HVKITDFGR antigen binding affinity for A*33:03 high, but it was also predicted to function sufficiently as an antigen based on more than 10 parameter criteria provided by neopepsee.

[0016] Based on this, we constructed a validation platform that can predict tumor-specific antigens and confirmed the reaction to the antigen. In the validation platform, dendritic cells (DCs) were differentiated from peripheral blood mononuclear cells (PBMCs) of healthy subjects with HLA-A matching, and the DCs recognized the antigens. Based on this, the antigen reactivity to L858R was measured.

[0017] As expected, T cells that reacted specifically to the tumor were produced in patients with HLA-A that recognized L858R, and responses to the antigen (IFN-gamma ELISPOT) were confirmed.

[0018] It was also confirmed that the TCR of the T cells identified through the above process could be expressed in immune cells.

[0019] Therefore, the problem to be solved by the present invention is to provide T cells or NK-92 cells having a TCR that targets the MHC-1-L858R neoantigen complex that increases on the surface of cancer cells, and to provide an EGFR mutant-targeting cell therapy that can directly induce apoptosis in cancer cells.

[0020] Another problem to be solved by the present invention is to provide a cell therapy agent having a further improved apoptotic function against cancer cells by further comprising an Fc receptor in the T cells or NK-92 cells having the above-mentioned TCR, thereby further improving the immune response thereto.

[0021] Furthermore, the Asian ratio of the aforementioned HLA-A is 20% or more, and it is expected that many patients with EGFR mutations will benefit from the present invention.

[0022] The object of the present invention is not limited to the above-mentioned objects, and other objects not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0023] According to one embodiment of the present invention, the present invention provides a T cell receptor, (i) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAFIGHGGSQGNLIF (SEQ ID NO: 1) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSMQGAMSEQFF (SEQ ID NO: 13) or a variant thereof; or (ii) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAATGTYKYIF (SEQ ID NO: 2) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSPEFARALDNQPQHF (SEQ ID NO: 14) or a variant thereof; or (iii) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAYGGGSEKLVF (SEQ ID NO: 3) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSSATGTQGYTF (SEQ ID NO: 15) or a variant thereof; or (iv) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CALINARLMF (SEQ ID NO: 4) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSFTNTGELFF (SEQ ID NO: 16) or a variant thereof; or (v) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAVNGGSQGNLIF (SEQ ID NO: 5) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSMWQGNGEQYF (SEQ ID NO: 17) or a variant thereof; or (vi) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAMREGYGGATNKLIF (SEQ ID NO: 6) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSVGPGTTSYNEQFF (SEQ ID NO: 18) or a variant thereof; or (vii) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAYNNGDGGSQGNLIF (SEQ ID NO: 7) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CATSRDRSTDTQYF (SEQ ID NO: 19) or a variant thereof; or (viii) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CATDGGSARQLTF (SEQ ID NO: 8) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSLGLSGYTF (SEQ ID NO: 20) or a variant thereof; or (ix) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CATLYNTDKLIF (SEQ ID NO: 9) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSQSMNTEAFF (SEQ ID NO: 21) or a variant thereof; or (x) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAMRGPWRGSSGSARQLTF (SEQ ID NO: 10) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASRTGLSYEQYF (SEQ ID NO: 22) or a variant thereof; or (xi) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CALSVRGFKTSYDKVIF (SEQ ID NO: 11) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSFGSAYNEQFF (SEQ ID NO: 23) or a variant thereof; or (xii) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAVNMMDSSYKLIF (SEQ ID NO: 12) or a variant thereof; and A T cell receptor is provided, comprising a complementarity determining region (CDR) 3 of a T cell receptor (TCR) β chain variable region comprising the amino acid sequence CASSFPTARSNTEAFF (SEQ ID NO: 24) or a variant thereof.

[0024] According to a feature of the invention, the T cell receptor comprises: HVKITDFGR (SEQ ID NO: 49) or an epitope contained within its MHC-binding form, It can have binding affinity to at least one of HLA-A*33:03 and HLA-A*31:01.

[0025] According to another feature of the invention, the T cell receptor is capable of targeting the EGFR L858R mutation.

[0026] According to another feature of the invention, the TCR alpha chain variable region consists of the amino acid sequence of SEQ ID NO:55, which may comprise the amino acid sequence of SEQ ID NO:1.

[0027] According to another feature of the invention, the TCR alpha chain variable region consists of the amino acid sequence of SEQ ID NO:57, which may comprise the amino acid sequence of SEQ ID NO:2.

[0028] According to another feature of the invention, the TCR β chain variable region consists of the amino acid sequence of SEQ ID NO:56, which may comprise the amino acid sequence of SEQ ID NO:13.

[0029] According to another feature of the invention, the TCR β chain variable region consists of the amino acid sequence of SEQ ID NO:58, which may comprise the amino acid sequence of SEQ ID NO:14.

[0030] According to another feature of the present invention, the T cell receptor may be, but is not limited to, a single chain type.

[0031] According to another feature of the invention, the TCR α chain variable region and the TCR β chain variable region may be linked by a linker sequence.

[0032] According to one embodiment of the present invention, there is provided a nucleic acid encoding a TCR according to the above.

[0033] According to a feature of the present invention, the nucleic acid may comprise at least one nucleic acid sequence selected from SEQ ID NOs: 51 to 54; or a nucleic acid sequence having at least 80% or more identity to at least one nucleic acid sequence selected from SEQ ID NOs: 51 to 54.

[0034] In this case, SEQ ID NOs: 51 and 53 may represent nucleic acid sequences encoding the TCR α chain variable region, and SEQ ID NOs: 52 and 54 may represent nucleic acid sequences encoding the TCR β chain variable region.

[0035] According to another feature of the present invention, the nucleic acid can further comprise, but is not limited to, Furin, 2A and IRES sequences.

[0036] According to one embodiment of the present invention, there is provided a vector comprising a nucleic acid according to the above.

[0037] In this case, the vector is an expression vector, which may be a lentiviral vector, but is not limited thereto, and may include any expression vector available in the art.

[0038] According to a feature of the invention, the vector may comprise the nucleic acid sequence of SEQ ID NO:50; or a nucleic acid sequence having at least 80% or more identity to the nucleic acid sequence of SEQ ID NO:50.

[0039] According to one embodiment of the present invention, there is provided an immune cell comprising a T cell receptor according to the above.

[0040] In this case, the immune cells may include the TCR, nucleic acid, and vector according to the above, and may be NK-92 cells. That is, the immune cells may be NK-92 cells in which the TCR is expressed by including the TCR, nucleic acid, and vector according to the above.

[0041] Furthermore, the immune cells may be, but are not limited to, NK-92 cells, and may include any of a variety of immune cells that can be modified with an inserted TCR.

[0042] According to one embodiment of the present invention, a cellular therapeutic agent comprising immune cells containing the above-mentioned T cell receptor is provided.

[0043] According to a feature of the invention, the cellular therapy agent may further comprise effector T cells.

[0044] According to another aspect of the present invention, the cellular therapy agent may be, but is not limited to, a solid tumor therapy agent, and may be a therapy agent for any carcinoma comprising the HVKITDFGR antigen of SEQ ID NO: 49 according to one embodiment of the present invention.

[0045] Hereinafter, the present invention will be described in more detail with reference to the following embodiments. However, these embodiments are merely for illustrative purposes and should not be construed as limiting the scope of the present invention. Effect of the Invention

[0046] The present invention can be proposed as a novel therapeutic agent for tumors with acquired resistance. More specifically, conventional lung cancer therapeutic agents show therapeutic effects in the early stages, but after 8 to 10 months, most patients develop acquired resistance. However, there are no alternative therapeutic agents for this.

[0047] Therefore, the present invention can be proposed as a novel therapeutic agent that can overcome these limitations. In addition, the present invention has the effect of targeting and treating not only acquired resistance but also harboring EGFR where mutations have occurred.

[0048] Furthermore, unlike drugs with a half-life, the present invention has the effect of enhancing the tumor-killing effect by increasing other immune responses together with the initial activation of T cells.

[0049] More specifically, the present invention has the effect of inducing the differentiation and proliferation of memory T cells, evading the defense mechanisms of cancer cells, and treating cancer or neoplastic conditions and preventing recurrence, progression, and metastasis.

[0050] In addition, the present invention has the effect of enhancing the effects of conventional therapeutic drugs such as TKIs and PD-1 / PD-L1 drugs in parallel, thereby increasing the survival rate of patients.

[0051] The effects of the present invention are not limited to the above-mentioned examples, and a wider variety of effects are incorporated into the present specification. [Brief description of the drawings]

[0052] [Figure 1] The present invention provides an exemplary system for predicting antigens in cancer patients and a method for producing TCR-T or TCR-NK cells based on the system. [Diagram 2] 1 shows an exemplary process for validating predicted antigens in Neopepsee. [Diagram 3] This shows the results of validating antigen-specific T cells analyzed using the validation platform. [Figure 4a] The results of HLA-A and antigen sequences targeting L858R are shown. [Figure 4b] The results of HLA-A and antigen sequences targeting L858R are shown. [Figure 5a] Results for tumor-specific T cells that specifically bind to the EGFR-MT-L858R-HVKITDFGR-A*33:03 tetramer. [Figure 5b]Results for tumor-specific T cells that specifically bind to the EGFR-MT-L858R-HVKITDFGR-A*33:03 tetramer. [Figure 5c] Results for tumor-specific T cells that specifically bind to the EGFR-MT-L858R-HVKITDFGR-A*33:03 tetramer. [Figure 5d] Results for tumor-specific T cells that specifically bind to the EGFR-MT-L858R-HVKITDFGR-A*33:03 tetramer. [Figure 6] These are the results for the cell killer factor of T cells whose activity was specifically induced by the antigen (EGFR-MT-L858R-HVKITDFGR-A*33:03 tetramer). [Figure 7] This is a distribution map of the receptors of T cells whose activity was specifically induced by an antigen (EGFR-MT-L858R-HVKITDFGR-A*33:03 tetramer). [Figure 8] These are the results for the NK-92 cell line when cultured in a T flask. [Figure 9] This shows the results on the proliferation efficiency of NK-92 depending on the concentration of FBS and IL-2. [Figure 10] The results show the cell proliferation efficiency of the NK-92 cell line depending on the culture method. [Figure 11a] Results on activation and virulence factors of NK-92 cell lines and NK-92 cell lines expressing Fc receptors depending on culture conditions. [Figure 11b] Results on activation and virulence factors of NK-92 cell lines and NK-92 cell lines expressing Fc receptors depending on culture conditions. [Figure 12] This shows the results on the efficiency of GFP expression in NK-92 cells using a protein expression promoter. [Figure 13a] Results for chemokine receptor expression in NK-92 cells. [Figure 13b] Results for chemokine receptor expression in NK-92 cells. [Figure 14a]The results are for the expression of CD3 molecules and T cell receptor molecules in NK-92 cells. [Figure 14b] The results are for the expression of CD3 molecules and T cell receptor molecules in NK-92 cells. [Figure 15a] 1 is a lentiviral vector map for expressing TCR sequences according to one embodiment of the present invention. [Figure 15b] FIG. 1 is a schematic diagram of the nucleic acid sequences of CD3 molecules and T cell receptors for expressing TCR sequences according to one embodiment of the present invention. [Figure 16a] 1 shows the results of confirming the cellular activity of a TCR according to one embodiment of the present invention. [Figure 16b] 1 shows the results of confirming the cellular activity of a TCR according to one embodiment of the present invention. [Figure 17a] 1 shows the results of confirming the cell killer effect on NK cells containing a TCR according to one embodiment of the present invention. [Figure 17b] 1 shows the results of confirming the cell killer effect on NK cells containing a TCR according to one embodiment of the present invention. [Figure 18] 1 shows the results of confirming the tumor-suppressing effect of NK cells containing a TCR according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] The advantages and features of the present invention, as well as the methods for achieving them, will become clearer with reference to the following detailed embodiments in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, and may be embodied in various forms, and the present embodiments are provided so that the disclosure of the present invention is complete and the scope of the invention can be fully understood by those skilled in the art to which the present invention pertains, and the present invention is defined only by the claims. As used herein, the term "T-cell receptor (TCR)" includes not only natural TCRs, but also TCR variants, fragments, and constructs. Thus, TCRs optionally include additional domains and / or portions, and include heterodimers including TCR α and β chains, as well as multimers and single chain constructs.

[0054] The term "epitope" as used herein generally refers to the site on an antigen, typically a (poly-)peptide, that is recognized by a binding domain. In this context, a binding domain refers to an antigen-binding site, i.e., the domain of a molecule that binds to and interacts with a specific epitope of an antigen target.

[0055] As used herein, the term "natural killer cell (NK cell)" refers to a cell of the immune system that induces apoptosis of target cells without restriction according to major histocompatibility complex (MHC) class in the absence of specific antigenic stimulation. The target cell may be a cancer or tumor cell. NK cells are characterized by the presence of CD56 and the absence of CD3 surface markers. Therefore, we plan to artificially insert CD3-gamma, delta, zeta, etc. and conduct research.

[0056] The term "vector" as used herein refers to a non-chromosomal nucleic acid that contains an intact replicon such that the vector can replicate when placed in a permissive cell by a transformation process. Vectors can replicate from bacteria but have limited replication capacity in mammalian cells and may be viral or non-viral. Non-viral vectors for delivery of nucleic acids include naked DNA, DNA complexed with cationic lipids alone or in combination with cationic polymers, anionic and cationic liposomes, cationic polymers contained in liposomes, DNA protein complexes and particles containing heterologous polylysine, oligopeptides of defined length, DNA condensed with polyethyleneamine, ternary complexes containing viruses and polylysine DNA.

[0057] The term "immune cells" as used herein can include T cells, natural killer T cells (NKT), natural killer cells (NK), human embryonic stem cells, hematopoietic stem cells (HSC) and induced pluripotent stem cells (iPS). In this case, the T cells can be cytotoxic T cells (CTL), regulatory T lymphocytes, inflammatory T lymphocytes, helper T lymphocytes and gamma-delta T cells, innate lymphocytes (ILC1, ILC2), and the aforementioned T cells can be CD4+, CD8+ and mixed populations thereof.

[0058] As used herein, the term "human leukocyte antigen (HLA)" refers to human genes that encode major histocompatibility complex (MHC) proteins on the surface of cells that are responsible for regulating the immune system.

[0059] As used herein, the term "dendritic cells (DCs)" refers to members of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. These cells are characterized by their unique morphology and high expression levels of surface MHC class I and class II molecules, proteins that present antigenic peptides to T cells. APCs (such as macrophages) and T cells, including DCs, can be conveniently isolated or derived from peripheral blood and numerous tissues and differentiated as peripheral blood mononuclear cells (PBMCs) derived from peripheral blood.

[0060] As used herein, the term "treatment" includes, without limitation, any action that improves or provides benefit to the symptoms of cancer.

[0061] As used herein, the term "specifically binds" generally means that a TCR is more likely to bind to its intended antigen target through its antigen binding site than to a random, unrelated, non-target antigen. Additionally, "specifically binds" can mean that the binding specificity of the TCR for its antigen target is at least about 5-fold, preferably 10-fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more greater than its binding specificity for a non-target antigen.

[0062] Antigen prediction and validation process

[0063] Below, the process of antigen prediction and validation via Neopepsee will be described with reference to Figures 1 to 3(b).

[0064] FIG. 1 illustrates an exemplary system for predicting antigens in cancer patients and a method for producing TCR-T or TCR-NK cells based on the system. For example, after obtaining somatic mutations occurring in the patient's tumor through RNA sequencing, whole exome sequencing, and reference WES in normal blood, mononuclear cells derived from the patient are differentiated into dendritic cells, exposing epitopes identified from the previously identified mutant genes, proliferating T cells through antigen presentation, confirming the reactivity of the proliferated T cells, and obtaining the CDR3 sequence of the TCR through the TCR sequence of the T cells based on the confirmed reactivity, and then converting the T cells into tumor-specific cells through a gene carrier containing this sequence. Thus, a cell therapy drug can be produced and verified based on autologous T cells or NK-92 cells.

[0065] More specifically, antigen prediction is the first step to be taken prior to the development of tumor vaccines and cell therapy drugs. Antigen prediction is based on abnormal mutant proteins identified by mutation analysis (WES: whole exome sequencing) for tumors, and the expression of mutant antigens is demonstrated through RNA seq. Increased affinity between tumor antigens and MHC-1 can mean that they are potential antigens that can be recognized by immune cells. In this study, we used Neopepsee as an analysis pipeline to analyze antigens.

[0066] Next, a peptide against the predicted antigen is synthesized / purified with 9 amino acids at a high purity of over 95%, and then used as a solvent in DMSO, which is the most commonly used solvent for dissolving antigens. The peptide is then exposed to LPS and differentiated into mature DCs, after which it is used to educate T cells.

[0067] Next, verification will be carried out based on the level of IFN-gamma expression.

[0068] Conventionally, pVAC seq is the most commonly used validation platform for antigen analysis, which is an open analysis platform based on Linux and Python programs. The core algorithm of this program is NetMHC-pan, which can quantify the binding affinity of HLA-A, and pVAC seq additionally includes the expression level of tumor antigens through RNA seq. Neopepsee also includes a similar algorithm and 12 additional parameters, making it an improved analysis platform. It is an analysis platform that has secured excellent results in benchmarking analysis between previously reported antigen discovery programs. Neopepsee is an open source program and an analysis technology that has been recently academically published.

[0069] Therefore, we performed experiments based on patients with EGFR mutations, determining the presence or absence of L858R and E19Del mutations.

[0070] Referring to Figure 1, first, WES of the patient's tumor was performed, and a reference analysis was performed using matched normal blood to analyze germline mutations or SNVs in the WES. The tool used to determine whether there was a mutation in the WES was the GATK2 pipeline, and the analysis was performed based on the mapping data for hg38.

[0071] 50 ml of blood was collected from the patient, and the recognition rate of antigens by DCs present in the blood via T cells was verified based on the IFNg ELISPOT data.

[0072] Next, once validation of the candidate antigens presented by the computer is complete, the TCR sequencing of the T cells is analyzed to identify the dominant TCR that can best recognize the tumor antigen.

[0073] Next, we will generate CDR3 recombinant TCRs through TCR sequencing of the verified T cells to produce and verify cell therapy drugs that can specifically recognize antigens in autologous / allogeneic T cells and NK-92 cells.

[0074] Figure 2 shows an example of the antigen validation process predicted by Neopepsee. In this case, the antigen validation process takes approximately 21 days in total, which is a platform that can validate antigens that react specifically with tumors from patient PBMCs in a fairly short period of time. Antigens predicted by Neopepsee are validated based on the expression level of IFN-gamma after differentiation into mDCs together with 9-mer peptide antigens synthesized by iDCs differentiated from CD14, and the differentiated DCs are co-cultured with T cells and then proliferated into antigen-specific T cells. At each stage, the important stages in the development of cell therapy drugs are represented on a 10-point scale, and QC points are specified.

[0075] More specifically, referring to Figure 2, first, PBMCs are isolated from the blood of the patient on the first day. At this time, the protocol used to isolate PBMCs is a method that can secure lymphocytes and monocytes in the blood, and CD14-positive cells, i.e., monocytes, can be secured by a magnet and antibody-microbead method.

[0076] Thereafter, the CD14+ cells undergo a process of differentiation into DCs, and differentiation into imDCs is induced using GM-CSF and IL-4.

[0077] Next, CD8+ cells are also collected in the same manner as CD14- cells, stored at -80°C, and then used to co-culture with imDCs capable of presenting antigens.

[0078] Then, on days 7 to 8, the cells are treated with LPS together with 10 μg / ml of the already synthesized tumor antigen and cultured for 16 hours to allow sufficient recognition of the tumor antigen in DCs.

[0079] After a washing process, the cells are cultured together with CD8-positive cells.

[0080] Next, DC-CD8+ cells are co-cultured for approximately 11 days in a dedicated medium supplemented with IL-7 / IL-15.

[0081] Then, cell counting of the T cells collected after the 11th day is performed, and after cell counting is completed, the antigen is re-treated and the expression of IFN-gamma in the T cells is analyzed using ELISPOT.

[0082] Figure 3 shows the results of antigen-specific T cell verification using the verification platform. In order to verify the antigen verification capability of Neopepsee, dendritic cells were made to recognize representative antigens from 20 patients (patients with HLA-A type A*:24:02), and the T cells were educated. An ELISPOT verification experiment was then conducted to verify whether there was a reaction to tumor antigens. As a result, it was confirmed that the antigen verification capability was over 90%.

[0083] More specifically, the experiment was conducted with HLA-A type A*24.02, which is the most prevalent type in the world. The specific antigens of 20 patients were predicted using Neopepsee, and DCs were made to recognize a total of 41 antigens. After educating T cells, the degree of reaction to these antigens was checked. All but three of the 41 antigens showed a reaction to each tumor antigen. Furthermore, the activity can be expressed by the number of spots and surface area of ​​IFN-gamma against the antigen, and the more spots there are, the better the presentation of the antigen is, and the surface area can represent the amount of IFN-gamma against the antigen. Furthermore, the degree of IFN-gamma expression in T cells in the untreated group was used as a cut-off to determine positivity.

[0084] Referring to Figure 3, the prediction rate of IFN-gamma expression against antigens, as predicted by Neopepsee, is over 90% based on the expression of IFN-gamma. Specific antigens were predicted for each of 20 patients using Neopepsee, and reactivity against 41 types of antigens was predicted, confirming reactivity against over 90% of tumor antigens.

[0085] From the above results, it is possible to select tumor-specific antigens with high predictive power, which can be used as a basis for the development and verification of cell therapy drugs.

[0086] Obtaining specific antigens for EGFR-mutated tumors

[0087] In the following, specific antigens for EGFR mutated tumors are described with reference to Figures 4a and 4b.

[0088] Figures 4a and 4b show the results of HLA-A and antigen sequences targeting L858R.

[0089] Referring to FIG. 4a, antigens capable of recognizing the L858R mutation in EGFR were selected using Neopepsee, and L858R mutant peptides and wild-type (WT) peptides for EGFR were produced. The antigens capable of recognizing the L858R mutation in EGFR were applied to 12 major HLA-As, and the degree of antigen prediction for 9-11mers was verified through MHC binding affinity.

[0090] Through the above process, HLA-A including HLA-A*2402, HLA-A*0201, HLA-A*3303, HLA-A*1101, HLA-A*0206 and HLA-A*3101 were selected, all of which were shown to have a high frequency of 5% or more. Among these, HLA-A*2402, HLA-A*0201 and HLA-A*3303, which have a frequency of 10% or more, were selected to attempt to derive antigens from the samples of a total of 10 patients containing the L858R mutation against EGFR.

[0091] Therefore, referring to FIG. 4b, it was shown that the samples of a total of 10 patients containing the L858R mutation against EGFR all contained HLA-A*3303 in common, unlike HLA-A*2402 and HLA-A*0201, and thus HLA-A*3303 was selected and the antigen for it was identified.

[0092] Eventually, the target antigen for the L858R mutation in EGFR was selected as HVKITDFGR, as it was shown that the antigen with the HVKITDFGR sequence had a similar reaction (result) from patient samples based on HLA-A*3303.

[0093] From the above results, it is possible to select tumor-specific antigens with high predictive power, which can be used as a basis for the development and validation of cell therapy drugs.

[0094] T cell receptor (TCR)

[0095] Hereinafter, with reference to Figures 5a to 7, a TCR capable of targeting a specific antigen for EGFR mutant tumors will be described. In order to confirm a TCR capable of targeting a specific antigen for EGFR mutant tumors, EGFR-L858R-specific T cells were produced, and the produced T cells were sorted using tetramer. Furthermore, the separated cells were analyzed by single-cell RNA analysis.

[0096] More specifically, CD14+ positive cells were isolated from the blood of each individual and cultured with EGFR-MT antigen (9mer) to induce antigen presentation, then co-cultured with CD8+ positive cells from the same individual for 14 days, and the presence of EGFR-L858R-specific T cells was confirmed by ELISPOT. Furthermore, T cells that could bind to the EGFR-MT 9mer-MHC-1 complex were sorted using tetramers, and the sequence for the TCR was obtained by scRNA seq / VDJ.

[0097] First, the process of selecting T cells specific to the EGFR L858R mutant antigen will be described with reference to FIGS. 5a to 5d.

[0098] Figure 5a shows the results for tumor-specific T cells that specifically adhere to the EGFR-MT-L858R-HVKITDFGR-A*33:03 tetramer. For convenience of explanation, Figures 5b to 5d are referred to. Furthermore, to confirm the TCR, samples were taken from individuals containing A*33:03 / A*02.06.

[0099] Tumor-specific T cells that specifically adhere to the EGFR-MT-L858R-HVKITDFGR-A*33:03 tetramer (EGFR-L858R-A*33:03 tetramer-positive T cells) are memory T cells and express CD44 + CCR7 + Central memory cells (Tcm) and CD44 + CCR7 - Effector memory T cells (Tem) and their mixed cells (Tcm and Tem mixed, CD44 + CCR7 + / - ) was shown to contain

[0100] More specifically, referring to Figures 5b-5d, expression results for the cellular activation factors in T cells described in 1a are shown.

[0101] First, referring to FIG. 5b, tumor-specific T cells that specifically bind to the EGFR-MT-L858R-HVKITDFGR-A*33:03 tetramer (EGFR-L858R-A*33:03 tetramer-positive T cells) were shown to express CD8a, CD4, CD44 and CD62L, which are mature T cell factors, and therefore may mean that tumor-specific T cells that specifically adhere to the EGFR-MT-L858R-HVKITDFGR-A*33:03 tetramer (EGFR-L858R-A*33:03 tetramer-positive T cells) are mature T cells.

[0102] Next, referring to FIG. 5c, tumor-specific T cells (EGFR-L858R-A*33:03 tetramer-positive T cells) that specifically adhere to the EGFR-MT-L858R-HVKITDFGR-A*33:03 tetramer are shown to express CD25 and CD69, which are cell activation factors, and therefore, can mean that tumor-specific T cells (EGFR-L858R-A*33:03 tetramer-positive T cells) that specifically adhere to the EGFR-MT-L858R-HVKITDFGR-A*33:03 tetramer are activated effector T cells.

[0103] Next, referring to FIG. 5d, tumor-specific T cells that specifically adhere to the EGFR-MT-L858R-HVKITDFGR-A*33:03 tetramer (EGFR-L858R-A*33:03 tetramer-positive T cells) are shown to express CCR7, IFN-gamma and Granzyme-B.

[0104] In this case, since CCR7 is a memory cell factor, it can be said that tumor-specific T cells (EGFR-L858R-HVKITDFGR-A*33:03 tetramer-positive T cells) that specifically adhere to the EGFR-MT-L858R-HVKITDFGR-A*33:03 tetramer are memory T cells, which can survive in the body for a long time and induce a secondary immune response.

[0105] Furthermore, since IFN-gamma and Granzyme-B are cytotoxic factors, it can be said that tumor-specific T cells that specifically adhere to the EGFR-MT-L858R-HVKITDFGR-A*33:03 tetramer (EGFR-L858R-A*33:03 tetramer-positive T cells) are T cells that can secrete cytokines.

[0106] Thus, tumor-specific T cells (EGFR-L858R-A*33:03 tetramer-positive T cells) that specifically adhere to the EGFR-MT-L858R-HVKITDFGR-A*33:03 tetramer according to one embodiment of the present invention represent activated T cells in an in vivo immune response against tumors containing the L858R mutation, and can thereby have an anti-cancer effect against tumors containing the L858R mutation.

[0107] Hereinafter, the receptor (TCR) for T cells specific to the EGFR L858R mutant antigen will be described with reference to Figures 6 and 7. Hereinafter, the receptor sequence for T cells specific to the EGFR L858R mutant antigen was derived based on the expression of cell killer factors in T cells selected through the process of Figures 5a to 5d.

[0108] More specifically, FIG. 6 shows the results for cell killer factors of T cells whose activity was induced specifically to an antigen (EGFR-MT-L858R-HVKITDFGR-A*33:03 tetramer).

[0109] First, referring to (a) of FIG. 6, it is shown that among the tumor-specific T cells (EGFR-L858R-A*33:03 tetramer-positive T cells) that specifically adhere to the EGFR-MT-L858R-A*33:03 tetramer, the cells expressing the cell killer factor are about 10-15% of about 800 total cells, and are about 27% of the total activated cells. That is, it can be said that the tumor-specific T cells (EGFR-L858R-A*33:03 tetramer-positive T cells) that specifically adhere to the EGFR-MT-L858R-HVKITDFGR-A*33:03 tetramer can express a cytotoxic (killer) factor at a high rate when activated, and thus can have an effective cytotoxic (killer) effect on the tumor.

[0110] Therefore, referring to FIG. 6(b), among the T cells expressing the above-mentioned cell killer factors, only the cells expressing the highest levels of the cell killer factors were selected, and the correlation between them was analyzed.

[0111] Referring to FIG. 7, a distribution map of T cell receptors in which activity was specifically induced by the antigen (EGFR-MT-L858R-HVKITDFGR-A*33:03 tetramer) is shown. At this time, the analysis of the T cell receptor was performed based on the cells selected in FIG. 2, and the V, D, J and C constituent sequences of the T cell receptor chain were analyzed through single cell analysis, and sequence analysis of the CDR3 sequence was performed based on this. First, the analyzed T cell receptors have a total of 295 types of diversity, and the analysis results of the top 10 T cell receptor chains are shown in [Table 1] below, which shows that the combination of TRAV23 / DV6 and TRBV18 and the combination of TRAV24 and TRBV19 have the highest frequency.

[0112] [Table 1] [Table 1]

[0113] Furthermore, based on the analysis results of [Table 1] described above, the sequences for the generated CDR3 are shown in the following [Table 2], all of which were selected based on T cells expressing cell killer factors, and therefore, when they contain the CDR3 of the TCR α chain variable region composed of the amino acid sequences of SEQ ID NOs: 1 to 12 and the CDR3 of the TCR β chain variable region composed of the amino acid sequences of SEQ ID NOs: 13 to 24, they can have an anticancer effect against EGFR L858R mutation tumors. Here, the order of [Table 2] is listed according to the expression rate of cell killer factors (IFN-gamma, Granzyme-B) (rank 1 is the highest expression rate).

[0114] Furthermore, a TCR having a more effective anticancer effect against EGFR L858R mutant tumors may be a TCR comprising a CDR3 of a TCR α chain variable region composed of the amino acid sequences of SEQ ID NOs: 1 to 8 and a CDR3 of a TCR β chain variable region composed of the amino acid sequences of SEQ ID NOs: 13 to 20.

[0115] It has been disclosed that the expression level (secretion ability) of cell killer factors is highest for TCRs comprising a CDR3 of a TCR α chain variable region composed of the amino acid sequences of SEQ ID NOs: 1 and 2 and a CDR3 of a TCR β chain variable region composed of the amino acid sequences of SEQ ID NOs: 13 and 14, but it has been shown that the expression levels of cell killer factors for TCRs comprising a CDR3 of a TCR α chain variable region composed of the amino acid sequences of SEQ ID NOs: 3 to 12 and a CDR3 of a TCR β chain variable region composed of the amino acid sequences of SEQ ID NOs: 15 and 24 are similar.

[0116] Therefore, the cell killer effect of a TCR having a CDR3 of a TCR α chain variable region composed of the amino acid sequences of SEQ ID NOs: 1 to 12 may be the same, and the cell killer effect of a TCR having a CDR3 of a TCR β chain variable region composed of the amino acid sequences of SEQ ID NOs: 13 to 24 may also be the same.

[0117] The TCR having the most effective anti-cancer effect against EGFR L858R mutant tumors may be a TCR comprising a CDR3 of a TCR α chain variable region composed of the amino acid sequences of SEQ ID NOs: 1 and 2 and a CDR3 of a TCR β chain variable region composed of the amino acid sequences of SEQ ID NOs: 13 and 14.

[0118] [Table 2] [Table 2] [Table 3]

[0119] Based on the above results, a TCR according to one embodiment of the present invention, which includes a CDR3 of a TCR α chain variable region composed of the amino acid sequences of SEQ ID NOs: 1 to 12 and a CDR3 of a TCR β chain variable region composed of the amino acid sequences of SEQ ID NOs: 13 to 24, can more accurately target the EGFR-L858R mutant antigen (HVKITDFGR-A*33:03) by including a CDR3 that is expressed at a high frequency, and the TCR according to one embodiment of the present invention also has very high expression of cell killer factors, so can have a more effective anti-cancer response (tumor cell apoptosis effect) in tumors containing the EGFR-L858R mutant antigen.

[0120] NK cells

[0121] In the following, with reference to Figures 8 to 14b, NK cells containing TCR and Fc receptors that can target specific antigens for EGFR mutant tumors will be described.

[0122] First, referring to FIG. 8, the results for the NK-92 cell line when cultured in a T flask are shown. In this case, the NK cells are NK-92 cells, and are cultured using X-vivo10 medium or Xuri T cell medium. More specifically, the culture medium is X-vivo10 or Xuri T cell medium supplemented with 1-10% human AB serum, 100-2000 U / ml IL-2, 0.1-5 mM L-aspartic acid, 0.1-5 mM L-glutamine, and 0.1-5 mM L-serine, although the volumes are not limited thereto. Furthermore, the NK-92 cells are cultured at a concentration of 2.5×10 5 The cells were subcultured at a concentration of 10000000000 cells / ml, cultured in a T flask, and subcultured every 3 days. Furthermore, cell proliferation was measured by counting the number of cells using a hematocytometer, cell viability was measured by staining with trypan blue or the cell death marker PI and then using flow cytometry, and cell proliferation efficiency was measured by staining with the cell growth factor KI67 and then using flow cytometry. The above-mentioned process was performed in the same manner as in Figures 8 and 9.

[0123] The cell density of NK-92 cells was maintained at 1 × 10 5 ~1×10 6 It was shown that the number of NK-92 cells was maintained constant at 80-100% until the 22nd day of culture, and the number of NK-92 cells was increased by 126 times in terms of fold expansion after 22 days of culture. Furthermore, it was shown that the cell viability was maintained constant at 80-100% until the 22nd day of culture, and the cell proliferation was also maintained constant at 70-85% until the 22nd day of culture.

[0124] Next, referring to FIG. 9, the results on the proliferation efficiency of NK-92 depending on the concentration of FBS and IL-2 are shown.

[0125] First, referring to (a) and (b) of FIG. 9, it was shown that the proliferation efficiency of NK-92 depending on the FBS concentration was best when 5% FBS was added. Furthermore, referring to FIG. 13a and FIG. 13b, it was shown that the proliferation efficiency of NK-92 depending on the IL-2 concentration was best when 1000U / ml IL-2 was added. That is, it was shown that the higher the FBS and IL-2 in NK-92 cells, the higher the proliferation efficiency, and the concentrations of FBS and IL-2 that can most preferably increase the proliferation efficiency of NK-92 cells may be 5% and 1000U / ml, respectively, but are not limited thereto.

[0126] Referring to FIG. 10, the results of the cell proliferation efficiency of the NK-92 cell line according to the culture method are shown. At this time, the NK-92 cells were cultured using a cell proliferation device, the Xuri W-25 device, for effective proliferation. At this time, the NK-92 culture medium was the same as the culture medium used in the above-mentioned FIGS. 11a to 15b, and a 2L to 10L perfusion bag was attached to the Xuri W-25 device, after which the culture medium was inoculated and stabilized. Furthermore, after the temperature and pH of the culture medium were stabilized, 0.5 to 1.0×10 8 NK-92 cells / bag were inoculated, and the culture environment conditions were set to 2 to 2 RPM shaking. Cell viability, cell proliferation rate, and cell count were measured every 12 hours after cell inoculation. Half of the culture medium was removed through a filter every two days, and new culture medium was inoculated. Finally, cells were cultured in 1 L of culture medium in a 2 L perfusion bag and 5 L of culture medium in a 10 L perfusion bag. Furthermore, 0.5 to 1.0 × 10 10 When the cell number was reached, NK-92 cells were harvested using a cell harvester, Sefia S-2000, and then cell viability, cell proliferation rate, and cell number were measured.

[0127] Referring to FIG. 10(a), the cell density increased most rapidly to 1*10 when cultured at the optimization 10L scale. 2It was shown that the cell density reached 100 times the normal T-flask culture time, and it was shown that the time to reach 100 times was shortened by 11 days compared to general T-flask culture. Furthermore, referring to FIG. 10(b), the proliferation rate was the fastest at 1*10 when cultured at the optimization 10L scale. 10 It has been shown that the multiplication factor reaches 1*10 10 It was shown that the time to reach maturity was 11 days shorter than that of general T-flask culture. Therefore, NK-92 cells can be obtained in large numbers in a shorter time when cultured at an optimization 10L scale in the Xuri W-25 device.

[0128] 11a and 11b show the results of the activating and toxic factors of NK-92 cell lines and NK-92 cell lines expressing Fc receptors according to the culture conditions. In this case, the cytoactivating surface factors, cytotoxic factors and chemokine receptors of NK-92 cells were confirmed by staining with each antibody and then confirming using flow cytometry. More specifically, the cytoactivating surface factors were confirmed using NKG2D, NKp30, NKp44 and NKp46, and the cytotoxic factors were confirmed through CD107, Granzyme B and Perforin.

[0129] Referring to Figure 11a, the results of the activating and toxic factors of the NK-92 cell line according to the culture conditions are shown. When NK-92 is cultured in Xuri medium, it is shown that the cell activating surface factors NKG2D, NKp30, NKp44 and NKp46 and the cytotoxic factors CD107, granzyme B and perforin are all expressed. Similarly, when NK-92 is cultured in X-vivo medium, it is shown that the cell activating surface factors NKG2D, NKp30, NKp44 and NKp46 and the cytotoxic factors CD107, granzyme B and perforin are all expressed.

[0130] Further, referring to FIG. 11b, the results of the activation and toxicity factors of the NK-92 cell line expressing Fc receptors according to the culture conditions are shown. When NK-92 expressing Fc receptors is cultured in Xuri medium, it is shown that the cell activation surface factors NKG2D, NKp30, NKp44, and NKp46, and the cytotoxic factors CD107, granzyme B, and perforin are all expressed. Similarly, when NK-92 expressing Fc receptors is cultured in X-vivo medium, it is shown that the cell activation surface factors NKG2D, NKp30, NKp44, and NKp46, and the cytotoxic factors CD107, granzyme B, and perforin are all expressed.

[0131] The above results indicate that the NK-92 cell line and the NK-92 cell line expressing Fc receptors can fully express the functions of cell activity and cytotoxicity when cultured in both Xuri medium and X-vivo medium.

[0132] Referring to FIG. 12, the results of GFP expression efficiency of NK-92 cells by protein expression promoters are shown. At this time, in order to select promoters for the expression of T cell receptors in NK-92 cells, protein expression by each promoter was confirmed. More specifically, NK-92 cells were transformed using electroporation and pCAG-GFP, pEF-1α-GFP, and pCMV-GFP plasmids. At this time, the concentration of the plasmid was 1×10 6 The amount may be, but is not limited to, 2ug per cell. Next, 24 hours after transduction, GFP protein expression was confirmed using flow cytometry and CAG, EF-1α, CMV, etc.

[0133] Since the expression rate for GFP was shown to be highest with the pCMV promoter, pCMV is the most preferred promoter for expressing T cell receptors in NK-92 cells.

[0134] Figures 13a and 13b show the results of chemokine receptor expression in NK-92 cells. In this case, the expression of chemokine receptors was confirmed through flow cytometry and chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CX3CR1 in the same manner as in Figures 11a to 11b. First, referring to Figure 13a, it is shown that NK-92 cells cultured under the above-mentioned culture conditions and method express chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, and CCR9.

[0135] Furthermore, referring to FIG. 13b, NK-92 cells were shown to express the chemokine receptors CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6 and CX3CR1.

[0136] Thus, NK-92 cells cultured under the above-mentioned culture conditions and methods can activate acquired immune responses by expressing chemokine receptors.

[0137] Figures 14a and 14b show the results for the expression of CD3 molecules and T cell receptor molecules in NK-92 cells. At this time, NK-92 cells were transfected with pCMV-CD3γT2AδF2AεP2Aζ plasmid and pCMV-TCRαT2Aβ plasmid by electroporation. At this time, the concentration of the plasmid was 1×10 6 The amount of transfection may be, but is not limited to, 2 ug per cell. Then, 24 hours after transduction, the expression of CD3 molecules and T cell receptor molecules was confirmed using flow cytometry.

[0138] First, referring to Figure 14a, NK-92 cells expressing CD3 molecules account for 28.8% of all NK-92 cells. This means that TCR has been stably transduced into NK-92 cells by the above-mentioned process, since CD3, an accessory protein chain of the T cell antigen receptor, is expressed in NK-92 cells.

[0139] Next, referring to Figure 14b, it is shown that NK-92 cells expressing T cell receptors account for 8.3% of the total NK-92 cells. In other words, the expression of T cell antigen receptors in NK-92 cells means that TCRs have been stably transduced into NK-92 cells by the above-mentioned process.

[0140] Through the above process, the present invention can provide a TCR and immune cells containing the same, i.e., NK cells, that can more effectively target solid cancers, particularly lung cancers with EGFR mutations, thereby improving the anti-cancer effect.

[0141] Ultimately, the immune cells of the present invention, by containing the above-mentioned TCR, can more effectively target lung cancers containing EGFR mutations, thereby further improving the anti-cancer effect against targeted cancer cells.

[0142] TCR and NK cells comprising the same according to one embodiment of the present invention

[0143] Hereinafter, with reference to Figs. 15a and 15b, the configuration of a TCR according to one embodiment of the present invention (a TCR including CDR3 of a TCR α chain variable region composed of the amino acid sequences of SEQ ID NOs: 1 to 12 and CDR3 of a TCR β chain variable region composed of the amino acid sequences of SEQ ID NOs: 13 to 24 and an NK cell including the same will be specifically described.

[0144] FIG. 15a is a lentiviral vector map for expressing TCR sequences according to one embodiment of the present invention.

[0145] In this case, a lentiviral vector is used to transduce NK-92 cells (expressing a TCR sequence whose activity is specifically induced by the EGFR-L858R mutant antigen (HVKITDFGR-A*33:03)), and the vector that can be used for transduction is not limited to the lentiviral vector, and all of the various vectors that can be used for transduction in the art can be used.

[0146] Lentiviral vector sequence for expression of TCR sequences according to one embodiment of the present invention: JPEG2025038010000005.jpg168134JPEG2025038010000006.jpg167134JPEG2025038010000007.jpg168134JPEG2025038010000008.jpg167130JPEG202 5038010000009.jpg167130JPEG2025038010000010.jpg168133JPEG2025038 010000011.jpg168131JPEG2025038010000012.jpg167130JPEG20250380100 00013.jpg167132JPEG2025038010000014.jpg166131JPEG2025038010000015.jpg167130JPEG2025038010000016.jpg167131JPEG2025038010000017.j pg167129JPEG2025038010000018.jpg166130JPEG2025038010000019.jpg166130JPEG2025038010000020.jpg169130JPEG2025038010000021.jpg169130

[0147] Additionally, the vector may contain components of the CD3 molecule and components of TCR alpha and beta.

[0148] Referring now to FIG. 15b, there is shown a schematic diagram of the nucleic acid sequences of the CD3 molecule and the T cell receptor for expressing the TCR sequences according to one embodiment of the present invention.

[0149] In one embodiment of the present invention, components for the CD3 molecule and T cell receptor nucleic acid sequences for expressing the TCR sequence are linked by Furin+x2A and IRES sequences for independent expression of each element.

[0150] Sequence of a TCR according to one embodiment of the present invention TCR alpha nucleic acid sequence for expression (TRAV23 / DV6): 5'-ATGGAGACCCTCTTGGGCCTGCTTATCCTTTGGCTGCAGCTGCAATGGGTGAGCAGCAAACAGGAGGTGACGCAGATTCCTGCAGCTCTGAGTGTCCCAGAAGGAGAAAACTTGGTTCTCAACTGCAGTTTCACTGATAGCGCTATTTACAACCTCCAGTGGTTTAGGCAGGACCCTGGGGAAAGGTCTCACATCTCTGTTGCTTATTCAGTCAAGTCAGAGAGAGCAAACAAGTGGAAGACTTAATGCCTCGCTGGATAAATCATCAGGACGTAGTACTTTATACATTGCAGCTTCTCAGCCTGGTGACTCAGCCACCTACCTC[TGTGCAGCAACAGGAACCTACAAATACATCTTT]GGAAGAGGAACCAGCCTTATTGTTCATC CGTATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCA TGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC-3' (SEQ ID NO: 51)

[0151]

[0152] In this case, SEQ ID NOs: 51 and 52 contain nucleic acid sequences (SEQ ID NOs: 25 and 37) corresponding to the amino acid sequences of SEQ ID NOs: 1 and 13, respectively, which are TCRs according to one embodiment of the present invention, and the amino acid sequences for SEQ ID NOs: 51 and 52 are as follows: SEQ ID NOs: 55 and 56 (shown in [ ]).

[0153] Sequence of a TCR according to one embodiment of the present invention for expression TCR alpha amino acid sequence (TRAV23 / DV6): METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYL[CAATGTYKYIF]GRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 55)

[0154] Sequence of a TCR according to one embodiment of the present invention for expression TCR beta amino acid sequence (TRBV18): MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYF[CASSPEFARALDNQPQHF]GPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG-3' (SEQ ID NO:56)

[0155] Sequence of a TCR according to one embodiment of the present invention TCR alpha nucleic acid sequence for expression (TRAV24): 5'-ATGGAGACCCTCTTGGGCCTGCTTATCCTTTGGCTGCAGCTGCAATGGGTGAGCAGCAAACAGGAGGTGACGCAGATTCCTGCAGCTCTGAGTGTCCCAGAAGGAGAAAACTTGGTTCTCAACTGCAGTTTCACTGATAGCGCTATTTACAACCTCCAGTGGTTTAGGCAGGACCCTGGGGAAAGGTCTCACATCTCTGTTGCTTATTCAGTCAAGTCAGAGAGAGCAAACAAGTGGAAGACTTAATGCCTCGCTGGATAAATCATCAGGACGTAGTACTTTATACATTGCAGCTTCTCAGCCTGGTGACTCAGCCACCTACCTC[TGTGCCTTTATAGGCCATGGAGGAAGCCAAGGAAATCTCATCTTT]GGAAGAGGAACCAGCCTTATTGTT CATCCGTATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTG CATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC-3' (SEQ ID NO: 53)

[0156] TCRβ nucleic acid sequence (TRBV19) for TCR array expression according to an embodiment of the present invention: 5'-ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTC[TGTGCCAGTAGTATGCAGGGGGCTATGAGTGAGCAGTTCTTC]GGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTATGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAG-3' (SEQ ID NO: 54)

[0157] In this case, SEQ ID NOs: 53 and 54 contain nucleic acid sequences (SEQ ID NOs: 26 and 38) corresponding to the amino acid sequences of SEQ ID NOs: 2 and 14, respectively, which are TCRs according to one embodiment of the present invention, and the amino acid sequences for SEQ ID NOs: 53 and 54 are as follows: SEQ ID NOs: 57 and 58 (shown in [ ]).

[0158] TCR alpha amino acid sequence for sequence expression of a TCR according to one embodiment of the present invention (TRAV24): METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYL[CAFIGHGGSQGNLIF]GRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:57)

[0159] Sequence of a TCR according to one embodiment of the present invention for expression TCR beta amino acid sequence (TRBV19): MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYF[CASSMQGAMSEQFF]GPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO:58)

[0160] The above-mentioned TCR sequence according to one embodiment of the present invention can be expressed and used in various immune cells including not only NK cells but also T cells, natural killer T cells (NKT), human embryonic stem cells, hematopoietic stem cells (HSC), induced pluripotent stem cells (iPS), etc., and thus, immune cells including such a TCR sequence according to one embodiment of the present invention can have a more effective immune response and anti-cancer effect against various tumor cells including the EGFR-L858R mutant antigen (HVKITDFGR-A*33:03).

[0161] Verification of TCR and NK cells containing the same according to one embodiment of the present invention

[0162] Hereinafter, with reference to Figures 16a to 18, the anti-cancer effect of a TCR according to one embodiment of the present invention (including CDR3 of a TCR α chain variable region composed of the amino acid sequences of SEQ ID NOs: 1 to 12 and CDR3 of a TCR β chain variable region composed of the amino acid sequences of SEQ ID NOs: 13 to 24) and NK cells containing the same will be specifically described.

[0163] In this case, the TCR expressed in the NK cells was a TCR containing a CDR3 of a TCR α chain variable region composed of the amino acid sequences of SEQ ID NOs: 1 and 2 and a CDR3 of a TCR β chain variable region composed of the amino acid sequences of SEQ ID NOs: 13 and 14. However, this is merely an example, and there is no difference in the cell killer effect between TCRs containing a CDR3 of a TCR α chain variable region composed of the amino acid sequences of SEQ ID NOs: 1 to 12 and a CDR3 of a TCR β chain variable region composed of the amino acid sequences of SEQ ID NOs: 13 to 24, and therefore they can be used selectively.

[0164] 16a and 16b show the results of confirming the cellular activity of a TCR according to one embodiment of the present invention.

[0165] First, referring to Figure 16a, knockout Jurkat cells were used in Comparative Example 1 (control, TCRKO), and cells (NY-ESO-1 2:01 TCR) in which a TCR specific to the NY-ESO-1_HLA-A02:01 antigen was expressed in a lentivirus-knockout Jurkat cell were used in Comparative Example 2. Furthermore, in embodiments 1 (L858R1 33:03 TCR) and 2 (L858R2 33:03 TCR), cells in which a TCR according to one embodiment of the present invention was expressed in a lentivirus-knockout Jurkat cell were used. More specifically, embodiment 1 (L858R1 33:03 TCR) is a cell expressing a TCR based on the nucleic acid sequence of SEQ ID NOs: 51 and 52, which include the amino acid sequences of SEQ ID NOs: 1 and 13 (nucleic acid sequences of SEQ ID NOs: 25 and 37 for TRAV23 / DV6 and TRBV18), and embodiment 2 (L858R2 33:03 TCR) is a cell expressing a TCR based on the nucleic acid sequence of SEQ ID NOs: 53 and 54, which include the amino acid sequences of SEQ ID NOs: 2 and 14 (nucleic acid sequences of SEQ ID NOs: 26 and 38 for TRAV24 and TRBV19).

[0166] Furthermore, in the case of embodiments 1 and 2 (L858R1 33:03 TCR and L858R2 33:03 TCR), they specifically react to the EGFR-L858R mutant antigen (HVKITDFGR-A*33:03) tetramer and secrete cell killer factors such as IFN-gamma. Therefore, comparative examples 1 and 2 and embodiments 1 and 2 were exposed to the NY-ESO-1_HLA-A02:01 antigen tetramer and the EGFR-L858R mutant antigen (HVKITDFGR-A*33:03) tetramer, and the expression of IFN-gamma was measured.

[0167] Referring to Figure 16b, in the case of Comparative Example 1 (TCRKO Jurkat T cell), since it does not contain a TCR capable of recognizing the antigen, it is shown that there are no IFN-gamma expressing cells (IFN-γ positive cells), and in the case of Comparative Example 2 (NY-ESO-1-TCR Jurkat T cell), since it contains a TCR capable of recognizing only the NY-ESO-1_HLA-A02:01 antigen, it is shown that IFN-gamma expressing cells are present only in the NY-ESO-1_HLA-A02:01 antigen.

[0168] Furthermore, in the case of embodiments 1 and 2 (L858R1 33:03 TCR and L858R2 33:03 TCR), by including a TCR according to one embodiment of the present invention capable of recognizing the EGFR-L858R mutant antigen (HVKITDFGR-A*33:03), it is shown that the TCR specifically responds to the EGFR-L858R mutant antigen (HVKITDFGR-A*33:03) tetramer and IFN-gamma is expressed.

[0169] In other words, it can mean that the TCR according to one embodiment of the present invention is expressed in immune cells, specifically reacts to the EGFR-L858R mutant antigen (HVKITDFGR-A*33:03), and can secrete cell killer factors (cytokines) such as IFN-gamma.

[0170] Furthermore, the TCR according to one embodiment of the present invention does not induce expression of IFN-gamma in NY-ESO-1_HLA-A02:01 antigens other than the EGFR-L858R mutant antigen (HVKITDFGR-A*33:03), which may mean that high-level targeting of the EGFR-L858R mutant antigen (HVKITDFGR-A*33:03) that the present invention is intended to target is possible.

[0171] Thus, a TCR according to one embodiment of the present invention can more effectively target tumors containing the EGFR-L858R mutant antigen (HVKITDFGR-A*33:03), and thus immune cells containing a TCR according to one embodiment of the present invention can have improved anti-cancer effects.

[0172] 17a and 17b show the results of confirming the cell-killing effect of NK cells containing the TCR according to one embodiment of the present invention. In order to confirm the cell-killing effect, a real-time cell analyzer (RTCA) was used, and tumor cell samples were obtained from YU1154 (antigen-specific target cells) and YU1094 (antigen-nonspecific target cells, EGFR), which are tumor cell lines derived from a patient expressing the EGFR-L858R mutant antigen (HVKITDFGR-A*33:03). mut HLA11:01 More specifically, tumor cells (target cells, T), which are tumor cell lines derived from the above-mentioned patients, were cultured in a cell culture plate of the analyzer, and then co-cultured with NK-92 cells (effector cells, E) at a certain ratio (T:E ratio), and the degree of cell killing (cytolysis) according to the culture time was measured. In this case, the NK-92 cells were wild type (WT) NK-92 cells and TCR (EGFR) cells according to one embodiment of the present invention. mut HLA33:03 The NK-92 cells containing a TCR according to one embodiment of the present invention were used as an embodiment, and wild type (WT) NK-92 cells were used as a comparative example, and their killer activity was compared. For the sake of convenience, the NK-92 cells containing a TCR according to one embodiment of the present invention will be referred to as an embodiment, and wild type (WT) NK-92 cells will be referred to as a comparative example.

[0173] First, referring to FIG. 17a, the cell killer effect against YU1154 (antigen-specific target cells) is shown, and it is shown that NK cells containing a TCR according to one embodiment of the present invention (TCR-NK), i.e., in the case of the embodiment, have a higher killer effect than the comparative example (wild-type NK cells).

[0174] More specifically, referring to Fig. 17a(a), the embodiment of the present invention was shown to have a killer effect of about 60% or more for 6 hours when co-cultured with target cells (tumor cells) at ratios of 10 and 50 (TCR-NK1:10, 1:50) to target cells (tumor cells). In contrast, the comparative example shows a killer effect of 50% or less in all cases, regardless of the ratio to target cells (tumor cells).

[0175] Furthermore, referring to Fig. 17a(b), the embodiment of the present invention was shown to have a killer effect of about 70% or more for 12 hours when co-cultured with target cells (tumor cells) at ratios of 10 and 50 (TCR-NK1:10, 1:50) to target cells (tumor cells). In contrast, the comparative example shows a killer effect of 50% or less in all cases, regardless of the ratio to target cells (tumor cells).

[0176] Furthermore, referring to FIG. 17a(c), the embodiment of the present invention was shown to have a killer effect of about 85% or more for 72 hours when co-cultured with target cells (tumor cells) 1 at a ratio of 10 and 50 (TCR-NK1:10, 1:50). In contrast, the comparative example was shown to have a killer effect of 10% or less in all cases except when co-cultured with target cells (tumor cells) 1 at a ratio of 50 (WT-NK1:50), and even when co-cultured with target cells (tumor cells) 1 at a ratio of 50 (WT-NK1:50), it was shown to have a killer effect of only about 80%, which is lower than the embodiment of the present invention described above.

[0177] Ultimately, NK cells comprising a TCR according to one embodiment of the present invention can more effectively induce cell killers in EGFR-L858R mutant antigen (HVKITDFGR-A*33:03), thereby having improved anti-cancer effects in tumors containing EGFR-L858R mutant antigen (HVKITDFGR-A*33:03).

[0178] Next, referring to FIG. 17b, the cell killer effect against YU1094 (antigen non-specific target cells) is shown, and it is shown that NK cells containing a TCR according to one embodiment of the present invention (TCR-NK), i.e., in the case of the embodiment, have a higher killer effect than the comparative example (wild-type NK cells).

[0179] More specifically, referring to (a) of Figure 17b, the embodiment of the present invention was shown to have a killer effect of about 40% or more for 6 hours when co-cultured with target cells (tumor cells) at ratios of 10 and 50 (TCR-NK1:10, 1:50) to target cells (tumor cells). In contrast, the comparative example shows a killer effect of less than 40% in all cases, regardless of the ratio to target cells (tumor cells).

[0180] Furthermore, referring to FIG. 17b(b), the embodiment of the present invention was shown to have a killer effect of about 50% or more for 12 hours when co-cultured with target cells (tumor cells) at a ratio of 10 and 50 (TCR-NK1:10, 1:50). In contrast, the comparative example was shown to have a killer effect of less than 40% in all cases except when co-cultured with target cells (tumor cells) at a ratio of 1:50 (WT-NK1:50), and even when co-cultured with target cells (tumor cells) at a ratio of 1:50 (WT-NK1:50), it was shown to have a killer effect about 5% lower than the embodiment of the present invention described above.

[0181] Furthermore, referring to (c) of Figure 17b, it was shown that the embodiment of the present invention had a killer effect of about 70% or more for 72 hours when co-cultured at a ratio of 10 and 50 (TCR-NK 1:10, 1:50) to target cells (tumor cells) 1. In contrast, all of the comparative examples were shown to have a lower killer effect than the embodiment of the present invention described above (TCR-NK 1:10, 1:50).

[0182] Ultimately, NK cells comprising a TCR according to one embodiment of the present invention can more effectively induce cell killers in EGFR-L858R mutant antigen (HVKITDFGR-A*33:03), thereby having improved anti-cancer effects in tumors containing EGFR-L858R mutant antigen (HVKITDFGR-A*33:03).

[0183] 18 shows the results of confirming the tumor-suppressing effect of NK cells containing a TCR according to one embodiment of the present invention. The tumor-suppressing effect was confirmed in a tumor animal model (NOG mice implanted lung adenocarcinoma) derived from a patient who was induced to express the EGFR-L858R mutant antigen (HVKITDFGR-A*33:03). The tumor animal model was transplanted with tumor tissue derived from a patient containing the EGFR-L858R mutant antigen (HVKITDFGR-A*33:03), and the tumor size was 100 to 150 mm. 3 After rearing for 4 to 5 months to reach the target size, NK-92 cells containing the TCR according to one embodiment of the present invention were injected when the target size was reached. Furthermore, the injection of the NK cells containing the TCR according to one embodiment of the present invention was performed twice a week for a total of 4 weeks, and the injection amount was 1*10 7 ~5*10 7 The injection volume of the NK cells containing the TCR according to one embodiment of the present invention can be selected and used in various ways depending on the method of cell therapy. Also, as a comparative example, wild-type NK-92 cells and an anti-cancer drug (competitive drug), i.e., CD16-NK-92 cells, were injected and used, respectively.

[0184] Referring to FIG. 18, when NK cells containing a TCR according to one embodiment of the present invention (Daan NK cell therapy) were injected, the tumor volume in an animal model was about 200% or less, which was lower than that in the case of treatment with a competitive drug and wild-type NK cells.

[0185] That is, it can mean that the NK cells comprising the TCR according to one embodiment of the present invention are more effective in tumor suppression than the injection of conventional anti-cancer drugs and general immune cells, and in particular, the NK cells comprising the TCR according to one embodiment of the present invention have a statistically significant difference (p<0.001) compared to the injection of systemic immune cells, which can mean that the targeted anti-cancer (immune) effect is significantly improved in tumors containing the EGFR-L858R mutant antigen (HVKITDFGR-A*33:03) (*=p<0.05, ***=p<0.001).

[0186] Although the embodiments of the present invention have been described in detail above with reference to the attached drawings, the present invention is not necessarily limited to such embodiments, and can be implemented in various modifications within the scope of the technical idea of ​​the present invention. Therefore, the embodiments disclosed in the present invention are for the purpose of explaining, not limiting, the technical idea of ​​the present invention, and the scope of the technical idea of ​​the present invention is not limited by such embodiments. Therefore, it should be understood that the above-described embodiments are illustrative and not limiting in all respects. The scope of protection of the present invention should be interpreted by the following claims, and all technical ideas within the scope equivalent thereto should be interpreted as being included in the scope of the present invention.

[0187] [Item 1] A T cell receptor, (i) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAFIGHGGSQGNLIF (SEQ ID NO: 1) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSMQGAMSEQFF (SEQ ID NO: 13) or a variant thereof; or (ii) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAATGTYKYIF (SEQ ID NO: 2) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSPEFARALDNQPQHF (SEQ ID NO: 14) or a variant thereof; or (iii) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAYGGGSEKLVF (SEQ ID NO: 3) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSSATGTQGYTF (SEQ ID NO: 15) or a variant thereof; or (iv) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CALINARLMF (SEQ ID NO: 4) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSFTNTGELFF (SEQ ID NO: 16) or a variant thereof; or (v) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAVNGGSQGNLIF (SEQ ID NO: 5) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSMWQGNGEQYF (SEQ ID NO: 17) or a variant thereof; or (vi) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAMREGYGGATNKLIF (SEQ ID NO: 6) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSVGPGTTSYNEQFF (SEQ ID NO: 18) or a variant thereof; or (vii) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAYNNGDGGSQGNLIF (SEQ ID NO: 7) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CATSRDRSTDTQYF (SEQ ID NO: 19) or a variant thereof; or (viii) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CATDGGSARQLTF (SEQ ID NO: 8) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSLGLSGYTF (SEQ ID NO: 20) or a variant thereof; or (ix) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CATLYNTDKLIF (SEQ ID NO: 9) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSQSMNTEAFF (SEQ ID NO: 21) or a variant thereof; or (x) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAMRGPWRGSSGSARQLTF (SEQ ID NO: 10) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASRTGLSYEQYF (SEQ ID NO: 22) or a variant thereof; or (xi) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CALSVRGFKTSYDKVIF (SEQ ID NO: 11) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSFGSAYNEQFF (SEQ ID NO: 23) or a variant thereof; or (xii) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAVNMMDSSYKLIF (SEQ ID NO: 12) or a variant thereof; and A T cell receptor comprising a complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSFPTARSNTEAFF (SEQ ID NO: 24) or a variant thereof. [Item 2] The T cell receptor 2. The T cell receptor of item 1, capable of binding to an epitope contained within the amino acid sequence of HVKITDFGR (SEQ ID NO: 49) or an MHC-binding form thereof. [Item 3] The epitope is 3. The T cell receptor according to item 2, having binding affinity with at least one of HLA-A*33:03 and HLA-A*31:01. [Item 4] The T cell receptor 2. The T cell receptor of item 1, which targets an EGFRL858R mutation. [Item 5] The TCR α chain variable region It consists of the amino acid sequence of SEQ ID NO: 55, The sequence number 55 is 2. The T cell receptor of item 1, comprising the amino acid sequence of SEQ ID NO:1. [Item 6] The TCR α chain variable region It consists of the amino acid sequence of SEQ ID NO: 57, The sequence number 57 is 2. The T cell receptor of item 1, comprising the amino acid sequence of SEQ ID NO:2. [Item 7] The TCR β chain variable region It consists of the amino acid sequence of SEQ ID NO: 56, The sequence number 56 is 2. The T cell receptor of item 1, comprising the amino acid sequence of SEQ ID NO: 13. [Item 8] The TCR β chain variable region It consists of the amino acid sequence of SEQ ID NO: 58, The sequence number 58 is 2. The T cell receptor of item 1, comprising the amino acid sequence of SEQ ID NO: 14. [Item 9] The T cell receptor 2. The T cell receptor according to item 1, which is a single chain type. [Item 10] The TCR α chain variable region and the TCR β chain variable region 3. The T cell receptor of item 2, linked by a linker sequence. [Item 11] A nucleic acid encoding the T cell receptor according to any one of items 1 to 10. [Item 12] The nucleic acid is At least one nucleic acid sequence of SEQ ID NOs: 51 to 54; or 12. The nucleic acid according to item 11, comprising a nucleic acid sequence having at least 80% identity to at least one of the nucleic acid sequences of SEQ ID NOs: 51 to 54. [Item 13] The sequence numbers 51 and 53 are 13. The nucleic acid of item 12, which is a nucleic acid sequence encoding a TCR alpha chain variable region. [Item 14] The sequence numbers 52 and 54 are 13. The nucleic acid of item 12, which is a nucleic acid sequence encoding a TCR beta chain variable region. [Item 15] The nucleic acid is 12. The nucleic acid of item 11, further comprising Furin, 2A and IRES sequences. [Item 16] A vector comprising the nucleic acid according to any one of items 11 to 15. [Item 17] The vector comprises: 17. The vector according to item 16, which is an expression vector. [Item 18] The expression vector comprises: 18. The vector according to item 17, which is a lentiviral vector. [Item 19] The vector comprises: The nucleic acid sequence of SEQ ID NO:50; or 17. The vector according to item 16, comprising a nucleic acid sequence having at least 80% identity to the nucleic acid sequence of SEQ ID NO: 50. [Item 20] An immune cell comprising the T cell receptor according to any one of items 1 to 10. [Item 21] The immune cells are 21. The immune cell according to item 20, which is an NK-92 cell. [Item 22] 11. A cell therapy drug comprising an immune cell comprising a T cell receptor according to any one of items 1 to 10. [Item 23] The cell therapy described in item 22, further comprising an effector T cell. [Item 24] The cell therapy drug comprises: 23. The cell therapy agent according to item 22, which is a therapeutic agent for a solid tumor.

Claims

[Claim 1] A T cell receptor, (i) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAFIGHGGSQGNLIF (SEQ ID NO: 1) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSMQGAMSEQFF (SEQ ID NO: 13) or a variant thereof; or (ii) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAATGTYKYIF (SEQ ID NO: 2) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSPEFARALDNQPQHF (SEQ ID NO: 14) or a variant thereof; or (iii) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAYGGGSEKLVF (SEQ ID NO: 3) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSSATGTQGYTF (SEQ ID NO: 15) or a variant thereof; or (iv) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CALINARLMF (SEQ ID NO: 4) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSFTNTGELFF (SEQ ID NO: 16) or a variant thereof; or (v) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAVNGGSQGNLIF (SEQ ID NO: 5) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSMWQGNGEQYF (SEQ ID NO: 17) or a variant thereof; or (vi) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) alpha chain variable region comprising the amino acid sequence CAMREGYGGATNKLIF (SEQ ID NO: 6) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSVGPGTTSYNEQFF (SEQ ID NO: 18) or a variant thereof; or (vii) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAYNNGDGGSQGNLIF (SEQ ID NO: 7) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CATSRDRSTDTQYF (SEQ ID NO: 19) or a variant thereof; or (viii) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) alpha chain variable region comprising the amino acid sequence CATDGGSARQLTF (SEQ ID NO: 8) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSLGLSGYTF (SEQ ID NO: 20) or a variant thereof; or (ix) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CATLYNTDKLIF (SEQ ID NO: 9) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSQSMNTEAFF (SEQ ID NO:21) or a variant thereof; or (x) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) alpha chain variable region comprising the amino acid sequence CAMRGPWRGSSSGSARQLTF (SEQ ID NO: 10) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASRTGLSYEQYF (SEQ ID NO: 22) or a variant thereof; or (xi) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) alpha chain variable region comprising the amino acid sequence CALSVRGFKTSYDKVIF (SEQ ID NO: 11) or a variant thereof; and Complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSFGSAYNEQFF (SEQ ID NO: 23) or a variant thereof; or (xii) a complementarity determining region (CDR) 3 of a T cell receptor (TCR) α chain variable region comprising the amino acid sequence CAVNMMDSSYKLIF (SEQ ID NO: 12) or a variant thereof; and A T cell receptor comprising a complementarity determining region (CDR) 3 of a T cell receptor (TCR) beta chain variable region comprising the amino acid sequence CASSFPTARSNTEAFF (SEQ ID NO: 24) or a variant thereof.

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