WO2024010119A1 - Récepteur antigénique chimérique ciblant simultanément le mutant egfr et epha2 - Google Patents

Récepteur antigénique chimérique ciblant simultanément le mutant egfr et epha2 Download PDF

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WO2024010119A1
WO2024010119A1 PCT/KR2022/009890 KR2022009890W WO2024010119A1 WO 2024010119 A1 WO2024010119 A1 WO 2024010119A1 KR 2022009890 W KR2022009890 W KR 2022009890W WO 2024010119 A1 WO2024010119 A1 WO 2024010119A1
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seq
light chain
heavy chain
chain variable
scfv
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PCT/KR2022/009890
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Korean (ko)
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권병세
유미진
김나래
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주식회사 유틸렉스
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention relates to a chimeric antigen receptor that simultaneously targets mutant EGFR and EphA2 expressed in various cancers.
  • Cancer has a high mortality rate worldwide and is the most common cause of death in Western society after cardiovascular disease.
  • brain cancer, colon cancer, breast cancer, and prostate cancer are continuously increasing due to various causes such as the aging of the population, rapid increase in environmental pollutants, and increased intake of high-fat diets.
  • brain cancer has a very poor prognosis, with an average survival period of 14 months after diagnosis.
  • the average survival time has only been extended by 6 months.
  • the standard treatment for brain cancer is surgery, radiation therapy, and chemical treatment, but the probability of cure with these treatments is very low, and they also have the problem of causing serious side effects.
  • CAR-T T cells expressing chimeric antigen receptors (CAR) have been approved as a treatment for blood cancer by showing high anti-cancer efficacy, but the efficacy of CAR-T is reported to be insufficient for solid cancer.
  • CAR-T cell therapy for solid cancer is difficult in selecting target antigens due to the absence of cancer cell-specific antigens.
  • CAR-T cell therapy is being developed targeting antigens overexpressed on solid cancer cells, but many target antigens are also expressed on normal cells present in healthy tissues, so CAR-T cells attack antigens present on normal cells. There is a problem that causes toxicity.
  • solid cancers have high antigenic heterogeneity, and heterogeneity in antigen expression exists between tumor cells even within the tumor tissue of the same patient.
  • the characteristics are very diverse compared to other cancers, so ‘targeted treatment’ targeting a single cancer antigen is almost impossible.
  • a phase 1 clinical trial using EGFRvIII CAR-T targeting EGFRvIII, a mutation of EGFR has been conducted.
  • the present inventors discovered solid tumor-specific cancer antigens, created CAR-T that can simultaneously target two cancer antigens, and developed a CAR-T that is specific for target cells expressing its target antigens.
  • the present invention was completed by confirming its killing activity and excellent therapeutic effect in cancer-inducing animal models.
  • the object of the present invention is to provide a polynucleotide containing a nucleic acid sequence encoding a mutant epidermal growth factor receptor (EGFR)-specific chimeric antigen receptor (CAR) and EphA2 T cell engager. It is done.
  • EGFR epidermal growth factor receptor
  • CAR chimeric antigen receptor
  • Another object of the present invention is to provide a vector containing the polynucleotide according to the present invention.
  • Another object of the present invention is to provide a method for producing CAR-T that secretes EphA2 T cell engager and targets EGFR mutations.
  • Another object of the present invention is to provide transformed T cells expressing the polynucleotide according to the present invention.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer.
  • Another object of the present invention is to provide a method for treating cancer.
  • the present invention includes a nucleic acid sequence encoding a mutant epidermal growth factor receptor (EGFR)-specific chimeric antigen receptor (CAR) and an EphA2 T cell engager.
  • EGFR epidermal growth factor receptor
  • CAR chimeric antigen receptor
  • EphA2 T cell engager EphA2 T cell engager
  • the nucleic acid sequence encoding the EphA2 T cell engager is an scFv that binds to EphA2 (ephrin type-A receptor 2) containing the amino acid sequence shown in SEQ ID NO: 87; and a nucleic acid sequence encoding a scFv that binds to CD3 and includes the amino acid sequence shown in SEQ ID NO: 88,
  • the nucleic acid sequence encoding the mutant EGFR-specific chimeric antigen receptor provides a polynucleotide characterized in that it comprises a nucleic acid sequence encoding a single chain variable fragment (scFv) selected from the following group:
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 1, the light chain CDR2 shown in SEQ ID NO: 2, and the light chain CDR3 shown in SEQ ID NO: 3, the heavy chain CDR1 shown in SEQ ID NO: 4, and the light chain CDR3 shown in SEQ ID NO: 5.
  • a single chain variable fragment (scFv) comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 6;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 11, the light chain CDR2 shown in SEQ ID NO: 12, and the light chain CDR3 shown in SEQ ID NO: 13, the heavy chain CDR1 shown in SEQ ID NO: 14, and the light chain CDR3 shown in SEQ ID NO: 15.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 16;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 21, the light chain CDR2 shown in SEQ ID NO: 22, and the light chain CDR3 shown in SEQ ID NO: 23, the heavy chain CDR1 shown in SEQ ID NO: 24, and the light chain CDR3 shown in SEQ ID NO: 25.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 26;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 31, the light chain CDR2 shown in SEQ ID NO: 32, and the light chain CDR3 shown in SEQ ID NO: 33, the heavy chain CDR1 shown in SEQ ID NO: 34, and the light chain CDR3 shown in SEQ ID NO: 35.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 36;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 41, the light chain CDR2 shown in SEQ ID NO: 42, and the light chain CDR3 shown in SEQ ID NO: 43, the heavy chain CDR1 shown in SEQ ID NO: 44, and the light chain CDR3 shown in SEQ ID NO: 45.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 46;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 51, the light chain CDR2 shown in SEQ ID NO: 52, and the light chain CDR3 shown in SEQ ID NO: 53, the heavy chain CDR1 shown in SEQ ID NO: 54, and the light chain CDR3 shown in SEQ ID NO: 55.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 56.
  • the present invention provides a vector containing the polynucleotide according to the present invention.
  • the present invention secretes an EphA2 T cell engager, which includes the step of infecting a host cell with a vector containing the polynucleotide according to the present invention and EGFR mutation.
  • the present invention provides transformed T cells expressing the polynucleotide according to the present invention.
  • the present invention provides a polynucleotide according to the present invention.
  • it provides a pharmaceutical composition for preventing or treating cancer, comprising transformed T cells according to the present invention.
  • the present invention provides a) a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 1, the light chain CDR2 shown in SEQ ID NO: 2, and the light chain CDR3 shown in SEQ ID NO: 3, the heavy chain CDR1 shown in SEQ ID NO: 4, SEQ ID NO: A single chain variable fragment (scFv) comprising a heavy chain variable region including the heavy chain CDR2 represented by 5 and the heavy chain CDR3 represented by SEQ ID NO: 6;
  • scFv single chain variable fragment
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 11, the light chain CDR2 shown in SEQ ID NO: 12, and the light chain CDR3 shown in SEQ ID NO: 13, the heavy chain CDR1 shown in SEQ ID NO: 14, and the light chain CDR3 shown in SEQ ID NO: 15.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 16;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 21, the light chain CDR2 shown in SEQ ID NO: 22, and the light chain CDR3 shown in SEQ ID NO: 23, the heavy chain CDR1 shown in SEQ ID NO: 24, and the light chain CDR3 shown in SEQ ID NO: 25.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 26;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 31, the light chain CDR2 shown in SEQ ID NO: 32, and the light chain CDR3 shown in SEQ ID NO: 33, the heavy chain CDR1 shown in SEQ ID NO: 34, and the light chain CDR3 shown in SEQ ID NO: 35.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 36;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 41, the light chain CDR2 shown in SEQ ID NO: 42, and the light chain CDR3 shown in SEQ ID NO: 43, the heavy chain CDR1 shown in SEQ ID NO: 44, and the light chain CDR3 shown in SEQ ID NO: 45.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 46;
  • receptor transformed T cells expressing a specific chimeric antigen receptor (CAR); and
  • EphA2 ephrin type-A receptor 2
  • CD3 containing the amino acid sequence shown in SEQ ID NO: 88. It provides a pharmaceutical composition for preventing or treating cancer, including an EphA2 T cell engager.
  • the present invention provides a polynucleotide according to the present invention; or administering the transformed T cells according to the present invention to an individual.
  • the present invention provides a) a light chain variable region comprising a light chain CDR1 represented by SEQ ID NO: 1, a light chain CDR2 represented by SEQ ID NO: 2, and a light chain CDR3 represented by SEQ ID NO: 3, SEQ ID NO: A single chain variable fragment (scFv) comprising a heavy chain variable region including the heavy chain CDR1 represented by 4, the heavy chain CDR2 represented by SEQ ID NO: 5, and the heavy chain CDR3 represented by SEQ ID NO: 6;
  • scFv single chain variable fragment
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 11, the light chain CDR2 shown in SEQ ID NO: 12, and the light chain CDR3 shown in SEQ ID NO: 13, the heavy chain CDR1 shown in SEQ ID NO: 14, and the light chain CDR3 shown in SEQ ID NO: 15.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 16;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 21, the light chain CDR2 shown in SEQ ID NO: 22, and the light chain CDR3 shown in SEQ ID NO: 23, the heavy chain CDR1 shown in SEQ ID NO: 24, and the light chain CDR3 shown in SEQ ID NO: 25.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 26;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 31, the light chain CDR2 shown in SEQ ID NO: 32, and the light chain CDR3 shown in SEQ ID NO: 33, the heavy chain CDR1 shown in SEQ ID NO: 34, and the light chain CDR3 shown in SEQ ID NO: 35.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 36;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 41, the light chain CDR2 shown in SEQ ID NO: 42, and the light chain CDR3 shown in SEQ ID NO: 43, the heavy chain CDR1 shown in SEQ ID NO: 44, and the light chain CDR3 shown in SEQ ID NO: 45.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 46;
  • receptor transformed T cells expressing a specific chimeric antigen receptor (CAR); and
  • EphA2 Ephrin type-A receptor 2
  • EphA2 T cell engager comprising an scFv binding to CD3 and containing the amino acid sequence shown in SEQ ID NO: 88.
  • CAR-T cells that secrete the EphA2 T cell engager according to the present invention and target EGFRvIII show specific killing activity against target cells expressing EGFRvIII and/or EphA2, and the effect is similar to CAR-T cells that target only EGFRvIII. Not only was it confirmed to be superior to cells, but it was also confirmed to have a significantly superior therapeutic effect in an animal model causing brain cancer, which could be used to prevent, improve, or treat various solid cancers, including brain cancer, that express EGFRvIII and/or EphA2. It can be useful for this purpose.
  • Figure 1 shows the production and expression of a T cell engager containing an scFv capable of binding to the EphA2 antigen or a T cell engager containing an scFv capable of binding to the CD19 antigen (A) and confirmation of the production and expression of the T cell engager containing an scFv capable of binding to the EphA2 antigen. This is the result of confirming the function of the T cell engager (B and C).
  • Figure 2 shows the results of confirming CAR expression of CAR-T containing an scFv targeting EGFRvIII.
  • Figure 3 shows the results confirming the synergistic effect of EphA2 T cell engager and EGFRvIII CAR-T, showing the killing ability (A) of CAR-T cells and T cell engager at 6 hours or 24 hours and IFN at 6 hours or 24 hours. This is the result of confirming - ⁇ secretion (B).
  • Figure 4 is a schematic diagram showing the structure of the CAR structure for the production of CAR-T targeting EGFRvIII and secreting the EphA2 T cell engager.
  • This is a schematic diagram showing the structure (B) of the EGFRvIII CAR structure.
  • Figure 5 shows the results of confirming the specific killing ability on target cells of CAR-T cell culture media that secretes EphA2 T cell engager and targets EGFRvIII.
  • Figure 6 shows the results of confirming the target-specific activation and killing ability of EGFRvIII CAR-T secreting EphA2 T cell engager on cells expressing EGFRvIII, showing T cell proliferation (A), IFN- ⁇ secretion (B) and target This is the result of confirming cell killing ability (C).
  • Figure 7 shows the results of confirming the target-specific activation and killing ability of EGFRvIII CAR-T secreting EphA2 T cell engager on cells expressing EphA2, showing T cell proliferation (A), IFN- ⁇ secretion (B), and target This is the result of confirming cell killing ability (C).
  • Figure 8 shows the results for the production of an animal model inducing glioblastoma, confirming the expression of EphA2 in cells for inducing glioblastoma (A), and confirming the expression of the EGFRvIII gene after introducing it into the cells (B) ), This is the result of confirming the expression of luciferase (C) after introducing the EGFRvIII gene into the above cells.
  • Figure 9 shows the results of confirming cancer progression and survival up to 98 days in each animal model after injecting EGFRvIII CAR-T, which secretes EphA2 T cell engager, into an animal model causing glioblastoma.
  • Figure 10 shows the results of confirming the size of the cancer by checking the photon value of each animal model after injecting EGFRvIII CAR-T secreting EphA2 T cell engager into an animal model causing glioblastoma.
  • Figure 11 shows the survival rate (A) and body weight (B) of each experimental group after injection of EGFRvIII CAR-T secreting EphA2 T cell engager into an animal model causing glioblastoma. This is one result.
  • Figure 12 shows the results of confirming the difference in CAR expression according to the linker length between scFvs targeting EGFRvII in EGFRvIII CAR-T secreting EphA2 T cell engager, the expression level of CAR in each experimental group (A), and CAR expression This is the result of confirming the MFI (Mean Fluorescence Intensity) value (B) of these cells after isolating only those cells.
  • Figure 13 shows the results of confirming the growth of CAR-T cells and the concentration of secreted EphA2 T cell engager in the culture medium according to the linker length between scFvs targeting EGFRvII in EGFRvIII CAR-T secreting EphA2 T cell engager.
  • Figures 14 and 15 show cells transduced with EGFRvIII CAR lentivirus containing six types of scFv (430, L1A, H2C, L2, L3A, or L3B scFv) targeting EGFRvIII, which secrete EphA2 T cell engager. This is the result of confirming the degree of activation by cell lines expressing each target antigen.
  • scFv 430, L1A, H2C, L2, L3A, or L3B scFv
  • Figure 16 shows the K562-EGFRvIII cell line (B), in which the EGFRvIII gene was introduced into the K562 cell line (A), which does not express EGFRvIII and EphA2, and the antigen introduced in the K562-EGFRvIII/EphA2 cell line (C), in which the EphA2 gene was introduced.
  • the expression of was confirmed through FACS staining, and is a result for the production of target cells used in the analysis of FIGS. 14 and 15 above.
  • Figure 17 shows the results of confirming CAR expression of EGFRvIII CAR-T cells containing various scFvs targeting EGFRvIII and EGFRvIII CAR-T cells containing the same scFv and secreting the EphA2 T cell engager.
  • Figures 18 to 20 show the results of monitoring the target cell killing activity of EGFRvIII CAR-T cells containing various scFvs targeting EGFRvIII and EGFRvIII CAR-T cells containing the same scFv and secreting EphA2 T cell engager for 46 hours. am.
  • Figure 21 shows the injection of EGFRvIII CAR-T cells containing 430 SL (short linker) scFv or L1A SL scFv and EGFRvIII CAR-T cells containing L1A SL scFv secreting EphA2 T cell engager into a glioblastoma animal model. , This is the result of confirming the degree of cancer progression over time.
  • Figure 22 shows the results confirming the anticancer effect of EGFRvIII CAR-T cells containing 430 SL scFv or L1A SL scFv and EGFRvIII CAR-T cells containing L1A SL scFv secreting EphA2 T cell engager in a glioblastoma animal model. , This is the result of comparing the size of the cancer by measuring the photon value of each experimental group.
  • Figure 23 shows the body weight of the glioblastoma animal model after injecting EGFRvIII CAR-T cells containing 430 SL scFv or L1A SL scFv and EGFRvIII CAR-T cells containing L1A SL scFv secreting the EphA2 T cell engager. This is the result of confirming the impact.
  • the present invention is a polynucleotide comprising a nucleic acid sequence encoding a mutant epidermal growth factor receptor (EGFR)-specific chimeric antigen receptor (CAR) and EphA2 T cell engager,
  • EGFR epidermal growth factor receptor
  • CAR chimeric antigen receptor
  • the nucleic acid sequence encoding the EphA2 T cell engager is an scFv that binds to EphA2 (ephrin type-A receptor 2) containing the amino acid sequence shown in SEQ ID NO: 87; and a nucleic acid sequence encoding a scFv that binds to CD3 and includes the amino acid sequence shown in SEQ ID NO: 88,
  • the nucleic acid sequence encoding the mutant EGFR-specific chimeric antigen receptor provides a polynucleotide characterized in that it comprises a nucleic acid sequence encoding a single chain variable fragment (scFv) selected from the following group:
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 1, the light chain CDR2 shown in SEQ ID NO: 2, and the light chain CDR3 shown in SEQ ID NO: 3, the heavy chain CDR1 shown in SEQ ID NO: 4, and the light chain CDR3 shown in SEQ ID NO: 5.
  • a single chain variable fragment (scFv) comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 6;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 11, the light chain CDR2 shown in SEQ ID NO: 12, and the light chain CDR3 shown in SEQ ID NO: 13, the heavy chain CDR1 shown in SEQ ID NO: 14, and the light chain CDR3 shown in SEQ ID NO: 15.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 16;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 21, the light chain CDR2 shown in SEQ ID NO: 22, and the light chain CDR3 shown in SEQ ID NO: 23, the heavy chain CDR1 shown in SEQ ID NO: 24, and the light chain CDR3 shown in SEQ ID NO: 25.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 26;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 31, the light chain CDR2 shown in SEQ ID NO: 32, and the light chain CDR3 shown in SEQ ID NO: 33, the heavy chain CDR1 shown in SEQ ID NO: 34, and the light chain CDR3 shown in SEQ ID NO: 35.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 36;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 41, the light chain CDR2 shown in SEQ ID NO: 42, and the light chain CDR3 shown in SEQ ID NO: 43, the heavy chain CDR1 shown in SEQ ID NO: 44, and the light chain CDR3 shown in SEQ ID NO: 45.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 46;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 51, the light chain CDR2 shown in SEQ ID NO: 52, and the light chain CDR3 shown in SEQ ID NO: 53, the heavy chain CDR1 shown in SEQ ID NO: 54, and the light chain CDR3 shown in SEQ ID NO: 55.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 56.
  • the present invention specifically targets EGFRvIII, a mutant EGFR, a cancer antigen specifically expressed in solid tumors, and the EGFRvIII has accession number: NM_001346941.1.
  • CAR-T expressing the polynucleotide of the present invention can specifically target EGFRvIII and EphA2 (EPH receptor A2) at the same time.
  • EphA2 is characterized by accession number: NM_001329090.2.
  • the T cell engager is a double antibody in which two scFvs are connected by a linker.
  • One scFv recognizes a cancer antigen (EphA2 antigen in the present invention), and the other scFv recognizes CD3 of T cells. This allows the T cells to kill cancer cells by connecting the two cells in a close distance.
  • the polynucleotide of the present invention includes a nucleic acid sequence encoding an EphA2 T cell engager, and the EphA2 T cell engager may include a target cell targeting site and a T cell activation site. More specifically, the EphA2 T cell engager of the present invention includes an scFv that binds to EphA2 (ephrin type-A receptor 2) containing the amino acid sequence shown in SEQ ID NO: 87; and an scFv binding to CD3 containing the amino acid sequence shown in SEQ ID NO: 88, and can be secreted extracellularly or administered in combination to enhance the activity of CAR-T cells targeting EGFRvIII.
  • EphA2 T cell engager of the present invention includes an scFv that binds to EphA2 (ephrin type-A receptor 2) containing the amino acid sequence shown in SEQ ID NO: 87; and an scFv binding to CD3 containing the amino acid sequence shown in SEQ
  • CAR-T expressing the polynucleotide of the present invention secretes EphA2 T cell engager, and CAR-T targeting EGFRvIII (EGFRvIII CAR-T secreting EphA2 T cell engager, EphA2 T cell engager secreting EGFRvIII CAR- T) means.
  • the a) single chain variable fragment (scFv) is characterized by comprising a light chain variable region represented by SEQ ID NO: 7 and a heavy chain variable region represented by SEQ ID NO: 8.
  • the light chain variable region of a) single chain variable fragment (scFv) may be encoded by the nucleotide sequence represented by SEQ ID NO: 9
  • the heavy chain variable region may be encoded by the nucleotide sequence represented by SEQ ID NO: 10.
  • the b) single chain variable fragment (scFv) is characterized by comprising a light chain variable region represented by SEQ ID NO: 17 and a heavy chain variable region represented by SEQ ID NO: 18.
  • the light chain variable region of the b) single chain variable fragment (scFv) may be encoded by the nucleotide sequence represented by SEQ ID NO: 19
  • the heavy chain variable region may be encoded by the nucleotide sequence represented by SEQ ID NO: 20.
  • the c) single chain variable fragment (scFv) is characterized by comprising a light chain variable region represented by SEQ ID NO: 27 and a heavy chain variable region represented by SEQ ID NO: 28.
  • the light chain variable region of the c) single chain variable fragment (scFv) may be encoded by the nucleotide sequence represented by SEQ ID NO: 29, and the heavy chain variable region may be encoded by the nucleotide sequence represented by SEQ ID NO: 30.
  • the d) single chain variable fragment (scFv) is characterized by comprising a light chain variable region represented by SEQ ID NO: 37 and a heavy chain variable region represented by SEQ ID NO: 38.
  • the light chain variable region of the d) single chain variable fragment (scFv) may be encoded by the nucleotide sequence represented by SEQ ID NO: 39
  • the heavy chain variable region may be encoded by the nucleotide sequence represented by SEQ ID NO: 40.
  • the e) single chain variable fragment (scFv) is characterized by comprising a light chain variable region represented by SEQ ID NO: 47 and a heavy chain variable region represented by SEQ ID NO: 48.
  • the light chain variable region of the e) single chain variable fragment (scFv) may be encoded by the nucleotide sequence represented by SEQ ID NO: 49
  • the heavy chain variable region may be encoded by the nucleotide sequence represented by SEQ ID NO: 50.
  • the f) single chain variable fragment (scFv) is characterized by comprising a light chain variable region represented by SEQ ID NO: 57 and a heavy chain variable region represented by SEQ ID NO: 58.
  • the light chain variable region of f) single chain variable fragment (scFv) may be encoded by the base sequence shown in SEQ ID NO: 59
  • the heavy chain variable region may be encoded by the base sequence shown in SEQ ID NO: 60.
  • the light chain variable region and the heavy chain variable region of the single chain variable fragment (scFv) may be connected by a (GGGGS)m linker, where m is 1 to 10.
  • m may be 1 to 5, and more preferably, m of the linker may be 1 or 3, and may include an amino acid sequence represented by any one selected from the group consisting of SEQ ID NO: 61 and SEQ ID NO: 62. .
  • the mutant EGFR-specific chimeric antigen receptor is a light chain variable region and a heavy chain variable region amino acids linked by a linker comprising an amino acid sequence represented by any one selected from the group consisting of SEQ ID NO: 61 and SEQ ID NO: 62.
  • amino acid represented by any one selected from the group consisting of SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, and SEQ ID NO: 85 The sequence was synthesized by mutating some of the amino acid sequences shown in SEQ ID NO: 63 or SEQ ID NO: 65.
  • the amino acid sequence of the scFv binding to the mutant EGFR containing the LL (long linker) containing the amino acid sequence shown in SEQ ID NO: 61 is as follows: SEQ ID NO: 67 (mutation positions: p.E181K and p.T221I, L1A LL ), SEQ ID NO: 71 (mutation position: p.V97L and p.E181V, designated L2 LL), SEQ ID NO: 75 (mutation position: p.L225P, designated L3A LL), SEQ ID NO: 79 (mutation position: p .D94E, p.V97I and p.E181K, designated L3B LL) or SEQ ID NO: 83 (mutation location: p.E181K and p.S187L, designated H2C LL).
  • amino acid sequence of scFv that binds to mutant EGFR containing SL (short linker) containing the amino acid sequence shown in SEQ ID NO: 62 is as follows: SEQ ID NO: 69 (mutation positions: p.E171K and p.T211I, L1A SL), SEQ ID NO: 73 (mutation position: p.V97L and p.E171V, designated L2 SL), SEQ ID NO: 77 (mutation position: p.L215P, designated L3A SL), SEQ ID NO: 81 (mutation position: p.D94E, p.V97I and p.E171K, designated L3B SL) or SEQ ID NO: 85 (mutation location: p.E171K and p.S177L, designated H2C SL).
  • the single-chain variable fragment (scFv) containing the amino acid sequence represented by SEQ ID NO: 63 is encoded by the base sequence represented by SEQ ID NO: 64;
  • the single chain variable fragment (scFv) containing the amino acid sequence shown in SEQ ID NO: 65 is encoded by the base sequence shown in SEQ ID NO: 66;
  • the single chain variable fragment (scFv) containing the amino acid sequence shown in SEQ ID NO: 67 is encoded by the base sequence shown in SEQ ID NO: 68;
  • the single chain variable fragment (scFv) containing the amino acid sequence shown in SEQ ID NO: 69 is encoded by the base sequence shown in SEQ ID NO: 70;
  • the single chain variable fragment (scFv) containing the amino acid sequence represented by SEQ ID NO: 71 is encoded by the base sequence represented by SEQ ID NO: 72;
  • the single chain variable fragment (scFv) containing the amino acid sequence shown in SEQ ID NO: 73 is encoded by the base
  • the scFv binding to EphA2 (ephrin type-A receptor 2) containing the amino acid sequence shown in SEQ ID NO: 87 and the scFv binding to CD3 containing the amino acid sequence shown in SEQ ID NO: 88 are characterized in that they are connected by a linker. do.
  • the linker may include the amino acid sequence represented by SEQ ID NO: 62.
  • the nucleic acid sequence encoding the chimeric antigen receptor specific for the mutant EGFR, the nucleic acid sequence encoding the scFv binding to EphA2, and the scFv binding to CD3 are sequentially linked.
  • SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 57, SEQ ID NO: No. 58, SEQ ID No. 63, Seq. it is clear to those skilled in the art that any one or more amino acid sequences selected from the group consisting of SEQ ID NO: 87 and SEQ ID NO: 88 will be limited to including mutations in the amino acid sequence that exhibit equivalent biological activity.
  • amino acid mutations are made based on the relative similarity of amino acid side chain substitutions, such as hydrophobicity, hydrophilicity, charge, size, etc.
  • Analysis of the size, shape and type of amino acid side chain substitutions shows that arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan, and tyrosine can be said to be biologically equivalent in function.
  • the hydrophobic index of the amino acid may be considered.
  • Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); leucine (+3.8); phenylalanine (+2.8); Cysteine/Cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); histidine (-3.2); glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); and arginine (-4.5).
  • the hydrophobic amino acid index is very important in imparting interactive biological functions to proteins or peptides. It is a known fact that similar biological activity can be maintained only when substituted with an amino acid having a similar hydrophobic index.
  • substitution is made between amino acids showing a difference in the hydrophobicity index, preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
  • hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Asphaltate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); threonine (-0.4); Proline (-0.5 ⁇ 1); Alanine (-0.5); histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); leucine (-1.8); isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).
  • substitution is made between amino acids showing a difference in hydrophilicity value, preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
  • Amino acid exchanges in proteins or peptides that do not overall alter the activity of the molecule are known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979).
  • the most common exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
  • SEQ ID NO: 7 SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 47 of the present invention
  • SEQ ID NO: 48 SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO:
  • Any one or more amino acid sequences selected from the group consisting of SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, and SEQ ID NO: 88 are interpreted to also include sequences showing substantial identity with the sequences listed in the sequence list.
  • amino acid sequence represented by any one selected from the group consisting of SEQ ID NO: 61 and SEQ ID NO: 62 may also include mutations in the amino acid sequence that exhibit equivalent biological activity for the same reason as above.
  • the present invention relates to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84 and SEQ ID NO: 86 It may contain one or more base sequences and functional equivalents.
  • the “functional equivalent” refers to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, Any one or more base sequences selected from the group consisting of SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, and SEQ ID NO: 86, and at least 70% or more, preferably 80% or more, more preferably 90% or more, Even more preferably, those having a sequence homology of 95% or more include SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:
  • SEQ ID NO: 50 refers to a polynucleotide that exhibits substantially the same physiological activity as the polynucleotide represented by one or more base sequences selected from the group consisting of No. 84 and SEQ ID No. 86.
  • the “% sequence homology” for a polynucleotide is determined by comparing a comparison region with two optimally aligned sequences, where a portion of the polynucleotide sequence in the comparison region is a reference sequence (additions or deletions) for the optimal alignment of the two sequences. may contain additions or deletions (i.e. gaps) compared to those that do not contain .
  • nucleic acid sequence encoding the mutant EGFR-specific chimeric antigen receptor of the present invention may further include a nucleic acid sequence encoding one or more types selected from the group consisting of a signal peptide, a stem domain, a transmembrane domain, and an intracellular signaling domain. there is.
  • polynucleotide may further include a self-cleavage domain.
  • the nucleic acid sequence encoding the mutant EGFR-specific chimeric antigen receptor, the nucleic acid sequence encoding the self-cleavage domain, and the nucleic acid sequence encoding the EphA2 T cell engager may be sequentially linked.
  • the signal peptide serves to send the nascent protein to the endoplasmic reticulum
  • any polypeptide known to have the same or similar function as the above can be used without limitation regardless of whether it is from a natural or synthetic source. It is not limited thereto, but may be one or more selected from the group consisting of CD8, CD28, GM-CSF, CD4, and CD137. In the present invention, a signal peptide derived from CD8 was used.
  • the term “stem domain” refers to any polypeptide that functions to link the transmembrane domain to the scFv of the present invention. If the polypeptide is known to have the same or similar function as the above, it may be from a natural or synthetic source. It can be used without limitation regardless of origin. It is not limited thereto, but may be one selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, and CD8 hinge. One embodiment of the present invention In the example, the CD8 hinge domain was used.
  • transmembrane domain refers to any polypeptide that allows the CAR according to the present invention to be expressed on the surface membrane of a cell.
  • Suitable transmembrane domains for CARs disclosed herein include (a) the ability to be expressed on the surface of cells, such as immune cells, such as, but not limited to, lymphoid cells or natural killer (NK) cells, and (b) has the ability to interact with the scFv and intracellular signaling domains according to the invention to direct the cellular response of immune cells against predefined target cells.
  • the transmembrane domain can also be used without limitation, regardless of whether it is from a natural or synthetic source, as long as it is a polypeptide known to have the same or similar function as the above.
  • Fc ⁇ R Fc ⁇ R
  • ICOS CD278
  • 4-1BB CD137
  • OX40 CD134
  • CD27, CD28, IL-2R ⁇ , CD40, DAP10 MHC class I molecule, TNF receptor protein, Immunoglobulin-like protein , cytokine receptor, integrin, SLAM protein, activated NK cell receptor, BTLA, Toll ligand receptor, CD2, CD7, CD30, CDS, ICAM-1, B7-H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 ⁇ , CD8 ⁇ , ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a , LFA-1, ITGAM, CD103,
  • intracellular signaling domain refers to the portion of a protein that transmits effector signal function signals and instructs cells to perform specialized functions. This means that after the scFv according to the present invention binds to the target, It is responsible for intracellular signaling and causes T cell activation.Intracellular signaling domain.
  • any polypeptide known to have the same or similar function as the above can be used without limitation, regardless of whether it is from a natural or synthetic source.
  • the intracellular transduction domains include CD3 ⁇ , Fc ⁇ R, ICOS (CD278), 4-1BB (CD137), OX40 (CD134), CD27, CD28, IL-2R ⁇ , IL-15R- ⁇ , MyD88, DAP10 , DAP12, MHC class I molecule, TNF receptor protein, Immunoglobulin-like protein, cytokine receptor, integrin, SLAM protein, activated NK cell receptor, BTLA, Toll ligand receptor, CD2, CD7, CD30, CD40, CDS, ICAM-1 , B7-H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA4, VLA1, CD49a, IA4 , CD49D, ITGA6,
  • the term “self-cleavage domain” refers to a cleavage site for producing multiple proteins from the same transcript.
  • the self-cleavage domain includes EGFRvIII CAR (signal peptide, scFv binding to EGFRvIII, stem domain, transmembrane domain, intracellular signaling domain) and EphA2 T cell engager (scFv binding to EphA2, linker, CD3 scFv that binds to) to express each protein.
  • the self-cleavage domain is characterized in that it is a 2A peptide, and the 2A peptide is Thoseaasigna virus 2A (T2A), porcine teschovirus-1 2A (P2A), and equine rhinitis A virus (rhinitis A). virus) 2A (E2A) and foot-and-mouth disease virus (F2A), but is not limited thereto.
  • T2A was used.
  • the polynucleotide of the present invention may further include a tag peptide for confirming or purifying the expression of the scFv targeting EGFRvIII, the scFv binding to EphA2, and the scFv binding to CD3 according to the present invention.
  • a tag peptide for confirming or purifying the expression of the scFv targeting EGFRvIII, the scFv binding to EphA2, and the scFv binding to CD3 according to the present invention.
  • Any known tag peptide can be used without limitation, and in one embodiment of the present invention, Flag peptide (DYKDDDDK) (SEQ ID NO: 89), HIS peptide (HHHHHH) (SEQ ID NO: 90), Myc peptide (EQKLISEEDL) (SEQ ID NO: 91) and HiBiT peptide (VSGWRLFKKIS) (SEQ ID NO: 92) were used.
  • the present invention provides a vector containing the polynucleotide according to the present invention.
  • the present invention provides a method for producing a CAR-T that secretes an EphA2 T cell engager and targets an EGFR mutation, comprising the step of infecting a host cell with a vector containing the polynucleotide according to the present invention. provides.
  • the "vector” refers to a gene construct containing the nucleotide sequence of a gene operably linked to a suitable regulatory sequence to enable expression of the target gene in a suitable host, wherein the regulatory sequence initiates transcription. It may include a promoter, an optional operator sequence to control such transcription, and sequences to control termination of transcription and translation.
  • the vector of the present invention is not particularly limited as long as it can replicate within cells, and any vector known in the art can be used, for example, plasmid, cosmid, phage particle, or viral vector.
  • the "recombinant vector” is used as an expression vector for a target protein that can express the target protein with high efficiency in an appropriate host cell when the gene encoding the target protein to be expressed is operably linked.
  • the recombinant vector may be capable of expression in host cells.
  • the host cell may preferably be a eukaryotic cell, and expression control sequences such as promoters, terminators, enhancers, etc., sequences for membrane targeting or secretion, etc. are appropriately selected depending on the type of host cell. and can be combined in various ways depending on the purpose.
  • the “infection” may mean “transformation.”
  • transformation means introducing DNA into a host so that the DNA can be replicated as an extrachromosomal factor or through completion of chromosomal integration.
  • Host cells of the invention may be prokaryotic or eukaryotic cells. Additionally, hosts with high efficiency of introducing DNA and high expression efficiency of introduced DNA are usually used. Known eukaryotic and prokaryotic hosts such as Escherichia coli, Pseudomonas, Bacillus, Streptomyces, fungi and yeast, insect cells such as Spodoptera frugiperda (SF9), animal cells such as CHO and mouse cells, COS 1, COS 7, African green monkey cells such as BSC 1, BSC 40 and BMT 10, and tissue cultured human cells are examples of host cells that can be used. In one embodiment of the present invention, 293T cells were used as host cells. Of course, it should be understood that not all vectors and expression control sequences are equally functional in expressing the DNA sequence of the present invention. Likewise, not all hosts perform equally well for the same expression system.
  • those skilled in the art can make an appropriate selection among various vectors, expression control sequences, and hosts without excessive experimental burden and without departing from the scope of the present invention.
  • the host when choosing a vector, the host must be considered, because the vector must replicate within it.
  • the copy number of the vector, the ability to control copy number and the expression of other proteins encoded by the vector, such as antibiotic markers, should also be considered.
  • various factors must be considered. For example, the relative strength of the sequences, their controllability and their compatibility with the DNA sequences of the invention should be considered, especially in relation to possible secondary structures.
  • the single cell host must be prepared by the selected vector, the toxicity of the product encoded by the DNA sequence of the present invention, the secretion characteristics, the ability to correctly fold the protein, the culture and fermentation requirements, and the product encoded by the DNA sequence of the present invention from the host. It must be selected taking into account factors such as ease of purification. Within these parameters, one skilled in the art can select various vector/expression control sequence/host combinations capable of expressing the DNA sequence of the present invention in fermentation or large-scale animal culture.
  • the present invention provides transformed T cells expressing the polynucleotide according to the present invention.
  • the T cells are characterized by secreting EphA2 T cell engager and targeting EGFR mutations.
  • the T cells may be alpha beta T cells, gamma delta T cells, or NKT cells.
  • the T cells may be allogeneic T cells, autologous T cells, engineered autologous T cells (eACT), or tumor-infiltrating lymphocytes (TIL).
  • EGFRvIII CAR-T cells secreting the EphA2 T cell engager of the present invention have excellent killing ability by specifically reacting to EphA2 and EGFRvIII, respectively.
  • its effect was significantly superior to that of CAR-T cells targeting only EGFRvIII, and it also showed a significantly better therapeutic effect in animal models that caused brain cancer.
  • the present invention provides a polynucleotide according to the present invention; Alternatively, it provides a pharmaceutical composition for preventing or treating cancer, comprising transformed T cells according to the present invention.
  • the present invention provides a) a light chain variable region comprising the light chain CDR1 represented by SEQ ID NO: 1, the light chain CDR2 represented by SEQ ID NO: 2, and the light chain CDR3 represented by SEQ ID NO: 3, the heavy chain CDR1 represented by SEQ ID NO: 4, SEQ ID NO: A single chain variable fragment (scFv) comprising a heavy chain variable region including the heavy chain CDR2 represented by 5 and the heavy chain CDR3 represented by SEQ ID NO: 6;
  • scFv single chain variable fragment
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 11, the light chain CDR2 shown in SEQ ID NO: 12, and the light chain CDR3 shown in SEQ ID NO: 13, the heavy chain CDR1 shown in SEQ ID NO: 14, and the light chain CDR3 shown in SEQ ID NO: 15.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 16;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 21, the light chain CDR2 shown in SEQ ID NO: 22, and the light chain CDR3 shown in SEQ ID NO: 23, the heavy chain CDR1 shown in SEQ ID NO: 24, and the light chain CDR3 shown in SEQ ID NO: 25.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 26;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 31, the light chain CDR2 shown in SEQ ID NO: 32, and the light chain CDR3 shown in SEQ ID NO: 33, the heavy chain CDR1 shown in SEQ ID NO: 34, and the light chain CDR3 shown in SEQ ID NO: 35.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 36;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 41, the light chain CDR2 shown in SEQ ID NO: 42, and the light chain CDR3 shown in SEQ ID NO: 43, the heavy chain CDR1 shown in SEQ ID NO: 44, and the light chain CDR3 shown in SEQ ID NO: 45.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 46;
  • receptor transformed T cells expressing a specific chimeric antigen receptor (CAR); and
  • EphA2 ephrin type-A receptor 2
  • CD3 containing the amino acid sequence shown in SEQ ID NO: 88. It provides a pharmaceutical composition for preventing or treating cancer, including an EphA2 T cell engager.
  • the cancer is characterized as a solid cancer, and the solid cancer is an abnormal tissue mass that does not contain a cyst or fluid area and may be malignant or benign. Preferably, it is a solid tumor expressing EGFRvIII and/or EphA2. More preferably, the cancer may be one or more types selected from the group consisting of brain cancer, breast cancer, lung cancer, squamous cell cancer, stomach cancer, colon cancer, head and neck cancer, pancreatic cancer, uterine cancer, ovarian cancer, bladder cancer, and anal cancer, but is limited thereto. That is not the case.
  • carcinomas expressing EGFRvIII and/or EphA2 may become more diverse than currently known in the future.
  • carcinomas that do not express EGFRvIII and/or EphA2 they are engineered to express EGFRvIII and/or EphA2 using known technologies (Ex, viral transporters) that enable the expression of EGFRvIII and/or EphA2, and then treated with the polypeptide according to the present invention.
  • Nucleotides, transformed T cells according to the present invention or transformed T cells expressing a mutant EGFR-specific CAR and EphA2 T cell engager can be applied.
  • the pharmaceutical composition may be in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition may be intended for human subjects.
  • the pharmaceutical composition is not limited to these, but can be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, external preparations, suppositories, and sterile injection solutions according to conventional methods.
  • the composition of the present invention may preferably be formulated for intravenous administration.
  • the pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. for oral administration.
  • buffers, preservatives, and analgesics can be used.
  • Topics, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives, etc. can be used.
  • the dosage form of the pharmaceutical composition according to the present invention can be prepared in various ways by mixing it with a pharmaceutically acceptable carrier as described above.
  • oral administration it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be manufactured in the form of unit dosage ampoules or multiple dosage forms. there is.
  • examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil may be used.
  • fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.
  • the route of administration of the pharmaceutical composition according to the present invention is not limited to these, but is oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, and topical. , sublingual or rectal.
  • parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • the pharmaceutical composition according to the present invention can also be administered in the form of a suppository for rectal administration.
  • the pharmaceutical composition according to the present invention is influenced by various factors, including the activity of the specific active ingredient used, age, body weight, general health, gender, diet, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated.
  • the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route and period, but may be appropriately selected by a person skilled in the art.
  • the exact amount of the composition of the present invention to be administered can be determined by the doctor, and the doctor may determine the age of the patient (subject). , taking into account individual differences in body weight, tumor size, extent of infection or metastasis, and disability.
  • the pharmaceutical composition comprising the transformed T cells of the present invention is administered at a dose of 10 4 to 10 9 cells/kg of body weight, preferably 10 5 to 10 6 cells/kg of body weight (within this range). inclusive of all integer values).
  • the pharmaceutical composition comprising the transformed T cells of the present invention can be administered multiple times in such doses and can be administered using injection techniques well known in immunotherapy.
  • the optimal dosage and treatment regimen for a particular patient can be readily determined by those skilled in the medical arts through monitoring the patient's disease signs and adjusting the therapy accordingly.
  • Administering the composition to a subject can be performed in any convenient manner, including nebulization, injection, swallowing, infusion, implantation, or implantation.
  • the compositions described herein can be administered to a patient by subcutaneous, intradermal, intratumoral, intranodal, intraspinal, intramuscular, intravenous (i.v.) injection or intraperitoneally.
  • the pharmaceutical composition of the present invention can be administered to a patient by intradermal or subcutaneous injection, preferably i.v. It can be administered by injection.
  • the pharmaceutical composition according to the invention can be injected directly into a tumor, lymph node or site of infection.
  • the present invention provides a polynucleotide according to the present invention; or administering the transformed T cells according to the present invention to an individual.
  • the present invention provides a) a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 1, the light chain CDR2 shown in SEQ ID NO: 2, and the light chain CDR3 shown in SEQ ID NO: 3, the heavy chain CDR1 shown in SEQ ID NO: 4, SEQ ID NO: A single chain variable fragment (scFv) comprising a heavy chain variable region including the heavy chain CDR2 represented by 5 and the heavy chain CDR3 represented by SEQ ID NO: 6;
  • scFv single chain variable fragment
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 11, the light chain CDR2 shown in SEQ ID NO: 12, and the light chain CDR3 shown in SEQ ID NO: 13, the heavy chain CDR1 shown in SEQ ID NO: 14, and the light chain CDR3 shown in SEQ ID NO: 15.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 16;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 21, the light chain CDR2 shown in SEQ ID NO: 22, and the light chain CDR3 shown in SEQ ID NO: 23, the heavy chain CDR1 shown in SEQ ID NO: 24, and the light chain CDR3 shown in SEQ ID NO: 25.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 26;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 31, the light chain CDR2 shown in SEQ ID NO: 32, and the light chain CDR3 shown in SEQ ID NO: 33, the heavy chain CDR1 shown in SEQ ID NO: 34, and the light chain CDR3 shown in SEQ ID NO: 35.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 36;
  • a light chain variable region comprising the light chain CDR1 shown in SEQ ID NO: 41, the light chain CDR2 shown in SEQ ID NO: 42, and the light chain CDR3 shown in SEQ ID NO: 43, the heavy chain CDR1 shown in SEQ ID NO: 44, and the light chain CDR3 shown in SEQ ID NO: 45.
  • An scFv comprising a heavy chain variable region including heavy chain CDR2 and heavy chain CDR3 represented by SEQ ID NO: 46;
  • receptor transformed T cells expressing a specific chimeric antigen receptor (CAR); and
  • EphA2 Ephrin type-A receptor 2
  • EphA2 T cell engager comprising an scFv binding to CD3 and containing the amino acid sequence shown in SEQ ID NO: 88.
  • the subject is preferably a mammal, including a human, and includes patients in need of cancer treatment, patients who are being treated, patients who have received treatment, and patients in need of treatment, and who have undergone surgical surgery for cancer treatment. Patients who underwent surgery may also be included.
  • the cancer is characterized as a solid cancer, and the solid cancer is an abnormal tissue mass that does not contain a cyst or fluid area and may be malignant or benign. Preferably, it is a solid tumor expressing EGFRvIII and/or EphA2. More preferably, the cancer may be one or more types selected from the group consisting of brain cancer, breast cancer, lung cancer, squamous cell cancer, stomach cancer, colon cancer, head and neck cancer, pancreatic cancer, uterine cancer, ovarian cancer, bladder cancer, and anal cancer, but is limited thereto. That is not the case.
  • the cells activated and expanded using the methods described herein or other methods known in the art to expand the transformed T cells of the invention to therapeutic levels are administered to a patient using any of the following methods: It may be administered in combination (e.g., simultaneously or subsequently) with a related treatment form.
  • the polynucleotide according to the present invention, the transformed T cell according to the present invention, the transformed T cell expressing a mutant EGFR-specific CAR, and the EphA2 T cell engager or the pharmaceutical composition of the present invention are used as chemotherapy, radiation, and anticancer agents. It can be used in combination with treatment methods for treating solid tumors, such as immunotherapy.
  • a gene sequence was first synthesized in which an scFv capable of binding to the EphA2 antigen and an scFv capable of binding to the CD3 antigen were connected by a linker. This was cloned into the pcDNA3.3 vector and used as an expression vector.
  • a gene sequence was synthesized in which an scFv that can bind to the CD19 antigen instead of the EphA2 antigen and an scFv that can bind to the CD3 antigen were connected by a linker. As described above, it was cloned into the pcDNA3.3 vector.
  • HIS peptide HHHHHH
  • Myc peptide EQKLISEEDL
  • SEQ ID NO: 91 HIS peptide
  • His peptide and HiBiT peptide SEQ ID NO: 92
  • GSSGGSSG SEQ ID NO: 93
  • the two expression vectors for producing the EphA2 T cell engager and CD19 T cell engager prepared in Example 1 were transfected into Expi293F cells by a method known in the art. After 5-7 days, the culture medium was collected and purified using a Ni-NTA affinity column. Afterwards, the specific expression of these proteins was confirmed by performing western blot according to a method known in the art using an anti-His antibody (purchased from Biolegend), as shown in A in Figure 1. .
  • NFAT Nuclear factor of activated T-cells reporter
  • luc Proliferative cascade
  • BPS Bioscience NFAT reporter
  • luciferase activity increases when the NFAT transcription factor is activated by an external stimulus. Therefore, it can be quantified by measuring luminescence (RLU) after adding the luciferase substrate.
  • NFAT reporter (luc)-Jurkat cells effector cells, 2 ⁇ 10 4 cells
  • U-87MG ATCC, target cells, 2 ⁇ 10 4 cells
  • EphA2 T cell engager quantified using His tag ELISA (Genescript) was added at various concentrations and cultured for 4 hours.
  • luciferase substrate Promega was added and the luciferase activity of NFAT reporter (luc)-Jurkat cells was measured.
  • T cells effector cells, 1 ⁇ 10 4 cells
  • 293 cells 293 cells overexpressing the EphA2 antigen
  • various concentrations of EphA2 T cell engager were mixed and cultured for 24 hours.
  • the T cell killing ability mediated by the EphA2 T cell engager was investigated by measuring the luciferase activity of the target cells.
  • 293 cells overexpressing the EphA2 antigen were established as follows.
  • the EphA2 gene expression vector purchased from Sino Biological was introduced into 293 cells purchased from ATCC using the SF cell line Afterwards, the cells were subcultured using medium supplemented with 300 ⁇ g/mL of hygromycin, and only cells that highly expressed the EphA2 antigen were selected using FACS.
  • the 293-EphA2 cell line established in this way was transduced with a lentivirus containing a luciferase gene to express luciferase and used for experiments.
  • the specific target cell killing ability by the EphA2 T cell engager was calculated according to Equation 1 below.
  • Target cell killing ability ⁇ (RLU (relatively light unit) value when co-culturing Target cells + T cells) - (RLU value when co-culturing Target cells + T cells + Engager) ⁇ /(RLU when co-culturing Target cells + T cells value)
  • the EphA2 T cell engager was able to kill target cells in proportion to its concentration.
  • CD19 T cell engager a negative control, it was unable to kill target cells regardless of concentration.
  • GGGGS is composed of GGGGSGGGGSGGGGS (SEQ ID NO: 61) repeated three times
  • SL short linker
  • an scFv with some amino acid sequences mutated was also produced based on the wild type scFv sequence (SEQ ID NO: 63 (named 430 LL) and SEQ ID NO: 65 (named 430 SL)).
  • SEQ ID NO: 67 mutant positions: p.E181K and p.T221I, named L1A LL
  • SEQ ID NO: 71 mutant positions: p.V97L and p.E181V, (named L2 LL)
  • SEQ ID NO: 75 mutant position: p.L225P, designated L3A LL
  • SEQ ID NO: 79 mutant position: p.D94E, p.V97I and p.E181K, designated L3B LL
  • SEQ ID NO: 83 mutantation positions: p.E181K and p.S187L, designated H2C LL.
  • SEQ ID NO: 69 mutant positions: p.E171K and p.T211I, named L1A SL
  • SEQ ID NO: 73 mutant positions: p.V97L and p.E171V, L2
  • SEQ ID NO. 77 mutant position: p.L215P, designated L3A SL
  • SEQ ID NO. 81 mutant position: p.D94E, p.V97I and p.E171K, designated L3B SL
  • SEQ ID NO. 85 Mutation locations: p.E171K and p.S177L, designated H2C SL.
  • a CAR structure was produced to produce CAR-T that can kill cancer cells by targeting EGFRvIII.
  • CD8 signal sequence signal sequence, signal seg
  • hinge hinge
  • transmembrane domain TM
  • TM transmembrane domain
  • the lentivector prepared in Example 3 was transfected into Lenti- It was produced by transfection.
  • the produced lentivirus was concentrated, the titer was measured, and then stored at -80°C until use.
  • PBMC peripheral blood mononuclear cells
  • cells (293-EGFRvIII/EphA2) overexpressing the EphA2 gene were established in 293 cells (293-EGFRvIII, purchased from Cether) overexpressing the EGFRvIII antigen, and luciferase was introduced into these cells to kill target cells. It was possible to measure by luminescence.
  • the EphA2 gene expression vector for overexpressing the EphA2 gene in 293-EGFRvIII cells was purchased from Sino Biological, and this plasmid was introduced into 293-EGFRvIII cells with a Lonza 4D Nucleofector device using the SF cell line Cells expressing EphA2 were selected by subculturing them in medium supplemented with 300 ⁇ g/mL of hygromycin.
  • the 293-EGFRvIII/EphA2 cell line established in this way was transduced with a lentivirus containing a luciferase gene to express luciferase and used for experiments.
  • EGFRvIII CAR-T cells effector cells, 2 ⁇ 10 4 cells
  • target cells luciferase expression, 293/EGFRvIII/ EphA2, 2 ⁇ 10 4 cells
  • target cells luciferase expression, 293/EGFRvIII/ EphA2, 2 ⁇ 10 4 cells
  • the killing activity of CAR-T cells and T cell engagers was confirmed by measuring the luciferase activity of the target cells, and is shown in A in Figure 3.
  • the co-culture medium was collected and the concentration of interferon- ⁇ (IFN- ⁇ ) secreted when T cells were activated was measured using an ELISA kit (Biolegend), and the results are shown in B in Figure 3.
  • IFN- ⁇ interferon- ⁇
  • Example 5 it was confirmed that a synergistic effect occurred in T cell activation or target cell killing when EphA2 T cell engager and EGFRvIII CAR-T were treated together at a suboptimum concentration, so the EphA2 T cell engager was secreted. EGFRvIII CAR-T was produced.
  • T2A EGRGSLL TCGDVEENPGP
  • EphA2 T of Example 1 are added to the C-terminus (C-terminus of CD3 ⁇ ) of the EGFRvIII CAR construct in A in FIG. 4.
  • a gene fused to the cell engager sequence was designed and synthesized. Lentivirus was produced using the gene in the same manner as in Example 3.
  • EphA2 T cell engager secreting EGFRvIII CAR-T cells were produced in the same manner as in Example 4.
  • EGFRvIII CAR-T cells secreting CD19 T cell engager were produced using the same method as CD19 T cell engager.
  • EGFRvIII CAR-T cells secreting the EphA2 T cell engager according to the present invention are activated by specifically reacting to each target when they encounter the target cells of the T cell engager and the target cells of CAR-T.
  • EGFRvIII CAR-T cells secreting EphA2 T cell engager were stained with anti-Flag-PE or anti-Flag-APC (L5, Biolegend) to confirm the level of CAR expressed on the surface, and then Tag-it violet cell proliferation. and labeled with Violet cell proliferation and tracking dye (Biolegend).
  • CAR-T In the case of CAR-T, it is quickly activated by target cells and kills the target cells, whereas in the case of T cell engager, it was judged that it takes time to reach a certain concentration to be secreted and effective. Therefore, it was inferred that the ability to kill target cells was delayed compared to CAR-T. Therefore, although there is a difference in the time required for CAR-T and T cell engager to act, EGFRvIII CAR-T cells secreting the EphA2 T cell engager according to the present invention can be activated by reacting specifically with EphA2 and EGFRvIII. It was confirmed that target cells can be killed.
  • CAR-T cells targeting EGFRvIII according to the present invention which had confirmed target cell killing ability in vitro, was confirmed in vivo.
  • an orthotopic animal model was first established using a glioblastoma cell line.
  • a glioblastoma cell line U-87MG cells, a cell line widely used in glioblastoma research, were used.
  • U-87MG cells were stained with anti-EphA2 antibody (371805, R&D Systems) and secondary antibody, anti-mouse IgG-APC (poly 4053, biolegend), and expressed on the surface.
  • the level of antigen was confirmed by FACS.
  • U-87MG-Luc2 cell line in which firefly luciferase was introduced, was obtained from ATCC, and the EGFRvIII gene was introduced into this cell line to establish a stable cell line (hereinafter referred to as U87-EGFRvIII).
  • the A19002A antibody was able to well detect two proteins of different sizes, EGFR and EGFRvIII, and the L8A4 antibody showed that it specifically recognized only the EGFRvIII protein.
  • the U87-EGFRvIII cell line was detected at the expected size by both antibodies, confirming that EGFRvIII was expressed (A in Figure 9, lane 3).
  • the cell line was cultured and proliferated, collected, 0.2 It was injected intracranially (IC). After several days, 150 mg/kg of D-Luciferin (PerkinElmer), a luciferase substrate, was injected into the mouse abdominal cavity, and the animal was photographed using an IVIS (In vivo optical imaging system) device. The growth of cancer was monitored by measuring the degree of luminescence.
  • IC intracranially
  • the size of the cancer present in each animal model was quantified by photon value using an IVIS device. According to the quantified photon values, animal models were randomly assigned, 5 animals per group. CAR-T cells in culture were counted and stained with anti-Flag-PE (L5, Biolegend) to confirm the level of CAR expression. Depending on the CAR expression rate, CAR + cells were collected at 0.1 ⁇ 10 6 or 0.5 ⁇ 10 6 cells per animal. After washing, it was suspended in 100 ⁇ L of 5% HSA and injected into animals by IV.
  • EGFRvIII CAR-T cells secreting CD19 T cell engager were injected as a control group. Afterwards, changes in cancer size were monitored by measuring the photon value of each animal model using an IVIS device every week. Additionally, the animal's body weight was measured weekly.
  • Engager-secreting CAR-T cells (LL & SL) that secrete the same EphA2 T cell engager but have different linker lengths of the scFv that recognizes EGFRvIII, differences in efficacy according to linker length were confirmed.
  • HiBiT was tagged instead of the existing myc so that even a very small amount of engager could be detected.
  • Flag positive cells were isolated using MojoSort Human anti-APC nanobeads (Biolegend). As a result, as shown in B in FIG. 12, CAR+ cells after separation were more than 97%. In addition, as a result of checking the MFI of the above cells, it was confirmed that 430SL CAR-T cells secreting EphA2 T cell engager were higher than 430LL CAR-T cells secreting EphA2 T cell engager (1307 vs. 960).
  • scFv containing SL was used.
  • lentivirus was produced in the same manner as described in Example 4 above, and NFAT reporter (luc)-Jurkat cells were infected using this to induce CAR expression.
  • NFAT reporter (luc)-Jurkat cells in which CAR expression was confirmed by staining with anti-Flag-APC (L5, Biolegend), were used as effector cells (2 ⁇ 10 4 cells), and various ratios of target cells (K562 ( Chronic myeloid leukemia cell lines), K562-EGFRvIII, K562-EGFRvIII/EphA2) were mixed and co-cultured for 4 hours.
  • the luciferase substrate Promega was added to measure the luciferase activity of the effector cells to confirm the activity of CAR and T cell engager for each antigen.
  • NFAT reporter (luc)-Jurkat cells which did not express CAR, were used as effector cells and reacted with target cells expressing the target antigen, luciferase activity was not confirmed. In general, it was confirmed that NFAT reporter (luc)-Jurkat cells with high CAR expression had high luciferase activity.
  • K562 cells used as target cells were purchased from ATCC.
  • K562 cells were transduced using a lentivirus simultaneously expressing EGFRvIII antigen and GFP, and only cells expressing GFP were selected by FACS.
  • the K562-EGFRvIII cell line was established by subculturing the cells in medium supplemented with 2 ⁇ g/mL of blasticidin to select cells expressing EGFRvIII.
  • EphA2 gene expression vector (purchased from Sino Biological) was introduced into the K562-EGFRvIII cell line established above using the SF cell line It was established by subculturing using the added medium and selecting cells expressing EphA2.
  • anti-EGFRvIII L8A4, Absolute Antibody
  • anti-EphA2 371805, R & D Systems
  • anti-mouse IgG-APC poly 4053, After staining with Biolegend, expression was confirmed by FACS.
  • Example 12 Monitoring target cell killing activity between EGFRvIII CAR-T cells containing various mutant scFvs and EGFRvIII CAR-T cells containing the same scFv and secreting EphA2 T cell engager
  • EGFRvIII CAR-T cells that secrete EphA2 T cell engager and contain various mutant scFvs have better cancer cell killing ability than EGFRvIII CAR-T cells that contain mutant scFv and do not secrete EphA2 T cell engager.
  • CAR expression in these cells was confirmed by staining with anti-Flag-PE (L5, Biolegend) (see Figure 17).
  • CAR-T cells whose expression was confirmed were used as effector cells, and target cells (U87-EGFRvIII, DKMG-EGFRvIII, or 293-EGFRvIII/EphA2) that express antigens EGFRvIII and EphA2 and simultaneously express GFP were used in various ratios. Mixed.
  • target cells U87-EGFRvIII, DKMG-EGFRvIII, or 293-EGFRvIII/EphA2
  • target cells U87-EGFRvIII, DKMG-EGFRvIII, or 293-EGFRvIII/EphA2 that express antigens EGFRvIII and EphA2 and simultaneously express GFP were used in various ratios.
  • target cells U87-EGFRvIII, DKMG-EGFRvIII, or 293-EGFRvIII/EphA2 that express antigens EGFR
  • U87-EGFRvIII, DKMG-EGFRvIII, and 293-EGFRvIII/EphA2 used as target cells U87-EGFRvIII and 293-EGFRvIII/EphA2 were produced in the same manner as described in Examples 5 and 9 above.
  • DKMG-EGFRvIII a DKMG cell line that overexpressed EGFRvIII, was purchased from Cether and used. These target cells were transduced with a lentivirus containing the GFP gene, expressed GFP, and used for experiments.
  • Example 13 Comparison of the effects of EGFRvIII CAR-T cells containing 430 SL scFv or L1A SL scFv and secreting EphA2 T cell engager and EGFRvIII CAR-T cells containing L1A SL scFv in a glioblastoma animal model
  • EGFRvIII CAR-T cells containing 430 LL scFv and EGFRvIII CAR-T cells containing 430 LL scFv and secreting EphA2 T cell engager were used to produce EGFRvIII CAR-T compared to EGFRvIII CAR-T. It was confirmed that the in vivo efficacy of T cells was superior. In particular, in Example 10, it was confirmed that the length of the scFv linker affects the growth of CAR-T cells and also affects the amount of T cell engager secreted by CAR-T. EphA2 T cells containing an scFv with SL The in vivo efficacy of EGFRvIII CAR-T cells secreting engager was confirmed.
  • EGFRvIII CAR-T cells secreting EphA2 T cell engager containing L1A SL scFv were used, which are representative of various EGFRvIII CAR-T cells secreting EphA2 T cell engager.
  • EGFRvIII CAR-T cells containing 430 SL scFv and secreting EphA2 T cell engager and EGFRvIII CAR-T cells containing L1A SL scFv that do not secrete EphA2 T cell engager were used.
  • the size of the cancer present in each animal was quantified by photon value using an IVIS device, and the animal models were randomly assigned to 5 animals per experimental group according to the photon value.
  • CAR-T cells in culture were counted and stained with anti-Flag-PE (L5, Biolegend) to confirm the level of CAR expression.
  • CAR + cells were collected and washed at 0.1 ⁇ 10 6 or 0.05 ⁇ 10 6 cells per animal, then suspended in 100 ⁇ L of 5% HSA and injected into the animal by IV. Afterwards, changes in cancer size were monitored by measuring the photon value of each animal model using an IVIS device every week. Additionally, the body weight of the animal model was measured weekly.
  • the present invention manufactures EGFRvIII CAR-T cells secreting EphA2 T cell engager and then confirms its activity.
  • EGFRvIII CAR-T cells secreting EphA2 T cell engager according to the present invention are EGFRvIII and/or EphA2 cells. Not only did it show specific killing activity against target cells expressing and confirm that its effect was superior to CAR-T cells targeting only EGFRvIII, but it was also confirmed that it showed a significantly superior therapeutic effect in an animal model causing brain cancer. did.

Abstract

La présente invention concerne des cellules CAR-T qui sécrètent l'activateur de cellules T EphA2 et ciblent EGFRvIII, qui est un mutant EGFR, et selon la présente invention, des cellules CAR-T EGFRvIII qui sécrètent l'activateur de cellules T EphA2 présentent une activité destructrice spécifique contre des cellules cibles exprimant EGFRvIII et/ou EphA2, et non seulement un effet supérieur à celui des cellules CAR-T ciblant uniquement l'EGFRvIII a été confirmé, mais un effet thérapeutique nettement supérieur a également été confirmé dans un modèle animal de cancer cérébral induit, et la présente invention peut ainsi être utilisée de manière efficace pour prévenir, atténuer ou traiter divers cancers solides, y compris le cancer du cerveau, exprimant EGFRvIII et/ou EphA2.
PCT/KR2022/009890 2022-07-07 2022-07-07 Récepteur antigénique chimérique ciblant simultanément le mutant egfr et epha2 WO2024010119A1 (fr)

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