Safe and effective gene and cell therapy system

Abstract

The present invention relates to a gene and cell therapy system in which a chimeric antigen receptor (CAR) is co-expressed with IL-12, or with both IL-12 and a CTLA4-CD28 fusion protein (CTC28), and to uses thereof. According to the present invention, when CAR-T cells are administered by carrying IL-12, or both IL-12 and CTC28, without lymphodepletion, the migration into normal tissues is restricted while the CAR-T cells exhibit a potent anti-tumor effect. Accordingly, the present invention can be effectively used in immunotherapy for cancer treatment.

Description

[0001] Specification Title of the invention: Safe and effective gene cell therapy system Technical field The present invention relates to a safe and effective gene cell therapy system, and more particularly, to a gene cell therapy system in which a chimeric antigen receptor is expressed together with IL-12, or IL-12 and CTLA4-CD28 fusion protein, and uses thereof. Background art

[0002] CAR-T cell therapy targeting hematological malignancies such as CD19 and BCMA (B cell maturation antigen) has received FDA approval for use to date, with six therapies having shown dramatic efficacy in clinical trials. However, this efficacy is currently limited to hematological malignancies, and has not yet shown significant efficacy in solid tumors, which account for more than 90% of all cancers. Therefore, a treatment strategy for universal use of CAR-T cells in solid tumors is needed. One obstacle to CAR-T cell therapy for solid tumors is that CAR-T cell activity is reduced due to the immunosuppressive tumor microenvironment of solid tumors, so CAR-T cell activity must be increased. On the other hand, since most solid tumor antigens that are targeted by CAR-T cells are expressed in small amounts in normal tissues, there is a problem that increasing CAR-T cell activity increases normal tissue toxicity (on-target off-tumor toxicity) of CAR-T cells (Nat. Rev. Clin. Oncol. 17, 147—167 (2020)). Therefore, the contradiction of increasing CAR-T cell activity without increasing normal tissue toxicity must be resolved. A representative existing strategy for preventing normal tissue toxicity is the strategy of using low affinity CAR-T cells. Low affinity CAR-T cells require contact with many antigens for activation, so they do not react to small amounts of antigens on normal tissues, but only react to large amounts of antigens on tumor cells. Therefore, they can exhibit toxicity only to tumors without toxicity to normal tissues. However, this strategy has the disadvantage that the antitumor efficacy may not be sufficient when antigen expression within the tumor is not uniform or when some tumor cells show low antigen expression.Another strategy known is the logic gated CAR-T cell strategy, which involves having CAR-T cells express CAR molecules for two different tumor antigens and requiring simultaneous recognition of these two tumor antigens for activation. However, this strategy presents a complication in that it requires the expression of two CAR molecules in a single cell. Therefore, the development of CAR-T cells that can treat solid tumors is still necessary. A novel strategy that has not been attempted so far may be one that inhibits CAR-T cell trafficking to normal tissues. Existing CAR-T cell therapy typically involves lymphodepletion, which removes lymphocytes such as T cells in the body, before CAR-T cell administration. This lymphodepletion is performed through low-dose chemotherapy or total body irradiation. Lymphodepletion has the advantage of allowing the administered CAR-T cells to proliferate (homeostatic proliferation) in the patient's body without competition with other T cells because it removes T cells existing in the body. However, since normal tissues throughout the body are exposed to chemotherapy or radiation, there is a problem of inducing moderate inflammation in the normal tissues. Since it is well known that T cells migrate to areas of inflammation, this systemic inflammation has the effect of recruiting the administered CAR-T cells to normal tissues throughout the body, thereby promoting normal tissue toxicity, which allows the CAR-T cells to recognize and attack low-concentration tumor antigens in normal tissues. However, when CAR-T cells are administered without lymphodepletion, there is a problem that the therapeutic efficacy is lowered because the proliferation of CAR-T cells is limited.The present inventors have developed a T cell proliferation receptor called CTLA4-CD28 chimera (CTC28) through previous research (Korean Patent No. 10-1471647). In addition, in previous research, when producing CAR-T cells that secrete IL-12, a cytokine that activates T cells, it has been reported that IL-12 secreted by CAR-T cells promotes effector cell differentiation of CAR-T cells, thereby increasing the efficacy of CAR-T cells without lymphodepletion. However, the effect may be partial (Clin. Cancer Res. 18(6):1672-83 (2012)). However, in the present invention, when CAR-T cells loaded with IL-12 or CTC28 {IL-12} are administered without lymphodepletion, the migration of CAR-T cells to normal tissues is limited, while the CAR-T cells exhibit a strong anti-tumor effect only in tumors, thereby completing the present invention. Detailed Description of the Invention Technical Problem An object of the present invention is to provide a gene cell therapy system in which a chimeric antigen receptor is expressed together with IL-12, or IL-12 and CTLA4-CD28 fusion protein. Specifically, an object of the present invention is to provide a gene cell therapy system comprising (i) a first gene encoding a chimeric antigen receptor (CAR) including an antigen binding domain, an extracellular domain, a transmembrane domain, and an intracellular domain, and a second gene encoding IL-12; Or (ii) a third gene encoding a fusion protein (CTLA4-CD28 fusion protein; CTC28) comprising a CTLA4 (Cytotoxic T Lymphocyte Antigen-4) protein or a domain thereof, and CD28 or a domain thereof, is provided. Another object of the present invention is to provide a vector comprising the construct, and an immune cell into which the construct or vector has been introduced.Another object of the present invention is to provide an immune cell comprising a gene encoding the chimeric antigen receptor and a gene encoding IL-12, or a gene encoding a CTLA4-CD28 fusion protein together therewith. Another object of the present invention is to provide an immune cell expressing the chimeric antigen receptor and IL-12, or a CTLA4-CD28 fusion protein together therewith. Another object of the present invention is to provide a composition for treating cancer comprising the construct, the vector, or the immune cell, a method for treating cancer using the immune cell, a use of the immune cell for treating cancer, and a use of the immune cell for preparing a medicament for treating cancer. Technical solution In order to achieve the above object, the present invention provides a chimeric antigen receptor (CAR) comprising (i) a first gene encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain, an extracellular domain, a transmembrane domain, and an intracellular domain, and a second gene encoding IL-12; Or (ii) a third gene encoding a fusion protein comprising a CTLA4 (Cytotoxic T Lymphocyte Antigen-4) protein or a domain thereof, and CD28 or a domain thereof (CTLA4-CD28 fusion protein). In one embodiment, the construct may comprise (a) a first gene encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain, an extracellular domain, a transmembrane domain, and an intracellular domain; and (b) a second gene encoding IL-12.In another embodiment, the construct can comprise (a) a first gene encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain, an extracellular domain, a transmembrane domain, and an intracellular domain; (b) a second gene encoding IL-12; and (c) a third gene encoding a fusion protein comprising a CTLA4 protein or a domain thereof, and CD28 or a domain thereof (CTLA4-CD28 fusion protein). As used herein, the term "antigen binding domain" means a domain that directly binds to an antigen, such as an antibody or an antigen binding fragment thereof. As used herein, the term "extracellular domain (EC)" means a domain that is exposed outside the cell and connects the transmembrane domain and the antigen binding domain. As used herein, the term "transmembrane domain (TM)" means a domain that connects the extracellular domain and the intracellular domain and is located in the cell membrane. In this specification, the term "intracellular domain" is also called a cytoplasmic domain, and refers to a domain located inside the cell membrane of a cell, i.e., in the cytoplasm. In this specification, the term "intracellular signaling domain" refers to a portion located inside the cell membrane of an immune cell, i.e., in the intracellular domain, and refers to a site that transmits a signal within a cell when an antigen binding domain linked to an extracellular domain binds to a target antigen. In the present invention, the antigen binding domain may be, but is not limited to, an antibody, an antigen binding fragment thereof, a ligand protein that binds to an antigen, or a domain thereof. In one specific embodiment, the antigen binding domain may include, but is not limited to, an antibody or an antigen binding fragment thereof that specifically binds to one or more antigens selected from the group consisting of:

[0003] 4-1BB, BCMA, BAFF, B7-H3, B7-H6, CA9, CTAG1B, CEA, cyclin, cyclin A2, cyclin Bl, CCL-1, CCR4, CD3, CD4, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD52, CD58, CD62, CD79A, CD79B, CD80, CD123, CD133, CD138, CD171, CSPG4, CLDN18, CLDN18.2, CLDN6, CTLA-4, c-Met, DLL3, EGFR, tEGFR, EGFRvIII, EPG-2, EPG-40, ephrin B2, EPHA2, estrogen receptor, Fc receptor, FCRL5, FGF23, FBP, F0LR1, F0LR2, GD2, ganglioside GD3, gplOO, GPC3, GPCR5D, GM- CSF, Her2 / neu, Her3, Her4, erbB dimers, HMW— MAA, HBsAg, HLA- Al, HLA-A2, IL-22Ra, IL— 13Ra2, ICOS, IGF-1 receptor, integrin avP6, interferon receptor, IFNx, IL-2R, IL-4R, IL-5R, IL-6R, IL-17RA, IL-31R, IL-36R, kdr, Ll-CAM, LI— CE7 epitope of CAM, LRRC8A, Lewis Y, LAG3, MAGEA1, MAGEA3, MAGEA6, MAGEA1O, MSLN, CMV, MUC1, NKG2D ligand, MART-1, NGF, NCAM, NRP-1, NRP-2, carcinoembryonic antigen, PD-L1, FRAME, progesterone receptor, prostate-specific antigen, PSCA, PSMA, RANKL, R0R1, SLAMF7, survivin, TPBG, TAG72, TRP1, TRP2, and Wilms' tumor 1 (WT1). In one embodiment,The antigen binding domain may include an antibody or an antigen binding fragment thereof that specifically binds to c-Met or B7-H3. Specifically, the antigen binding domain may include an antibody or an antigen binding fragment thereof that specifically binds to c-Met, and may include, for example, a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 6; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 7, a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 8, and a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 9, but is not limited thereto. In addition, the antigen binding domain may include an antibody or an antigen binding fragment thereof that specifically binds to B7-H3, for example, a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 36, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 41; or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 45, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 46, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 47; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 48, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 49, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 50; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 54,A heavy chain variable region comprising a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 55 and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 56; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 57, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 59; or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 101, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 102, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 103; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 104, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 105, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 106; A heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 110, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 111, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 112; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 113, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 114, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 115; or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 119, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 120, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 121; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 122, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 123, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 124, but is not limited thereto. In one embodiment,The antibody or antigen-binding fragment thereof may be selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a full length antibody, an Fv, an scFv, a Fab, a Fab', an F(ab')2 and a nanobody comprising the heavy chain variable region and the light chain variable region described above, but is not limited thereto. Specifically, the antibody or antigen-binding fragment thereof may be a humanized antibody. The term "humanized antibody" as used herein refers to a chimeric immunoglobulin, an immunoglobulin chain or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequence of an antibody) containing minimal sequence derived from a non-human immunoglobulin of a non-human (e.g., murine) antibody. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody), such as mouse, rat or rabbit, having the desired specificity, affinity and capacity. In some cases, residues from the Fv framework region (FR) of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody may comprise residues that are not found in either the recipient antibody or in the imported CDR or framework sequences. Such modifications are made to further improve and optimize antibody performance. Generally, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to CDR regions of a non-human immunoglobulin,All or substantially all of the FR region has a sequence of the FR region of a human immunoglobulin. The humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc region) sequence or substantially a human immunoglobulin constant region (Fc region) sequence. In one specific embodiment, the antigen-binding fragment refers to a fragment having an antigen-binding function, and may be, but is not limited to, a single-chain variable fragment of an antibody; scFv, (SCFV)2, FV, Fab, Fab', F(ab')2, a nanobody, or a combination thereof.

[0004] A “single-chain Fv” or “scFv” antibody fragment comprises the VH and VL domains of an antibody, which are present within a single polypeptide chain. The Fv polypeptide may additionally comprise a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding.

[0005] An “Fv” fragment is an antibody fragment that contains a complete antibody recognition and binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain, tightly and virtually covalently associated, for example, in scFv.

[0006] A “Fab” fragment contains the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. An “F(ab')2” antibody fragment typically comprises a pair of Fab fragments covalently linked near their carboxy termini by hinge cysteines between them.

[0007] A “nanobody” is a fragment containing a monomeric variable antibody domain. It is mainly composed of low-molecular-weight fragments derived from antibody domains such as camelids that exhibit target specificity with only a monomeric heavy chain. In one specific example, the chimeric antigen receptor may additionally include a signal peptide (SP) at the N-terminus of the antigen binding domain, but is not limited thereto. In the present invention, the signal peptide may be derived from a molecule selected from the group consisting of CD8a, GM-CSF receptor a, Ig-kappa, and IgG1 heavy chains, but is not limited thereto. Specifically, a CD8a signal peptide may be used as the signal peptide, and the CD8a signal peptide may include an amino acid sequence represented by SEQ ID NO: 60, a portion thereof, or an amino acid sequence having at least 64%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In the present invention, the extracellular domain may be an extracellular domain derived from IgG1, IgG2, IgG4, IgD, CD8, or CD28, but is not limited thereto. In one embodiment, the extracellular domain may be a CD8-derived extracellular domain. It may include all or a portion of the CD8-derived extracellular domain, and specifically, the CD8 may be a human CD8, but is not limited thereto. The CD8-derived extracellular domain may comprise a human CD8-derived extracellular domain, and may specifically include, but is not limited to, an amino acid sequence having at least 64%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or a portion thereof, or an amino acid sequence having at least 64%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.In the present invention, the transmembrane domain may be a transmembrane domain derived from a protein selected from the group consisting of T cell receptor (TCR) a chain, TCRP chain, TCRx chain, TCR5 chain, CD3 zeta (e), CD3 epsilon (e), CD4, CD5, CD8, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, OX40 (CD134), 4-lBB (CD137), ICOS, and CD154, or a transmembrane domain derived from killer immunoglobulin-like receptor (KIR), but is not limited thereto. In one embodiment, the transmembrane domain may be a CD8-derived transmembrane domain. The CD8-derived transmembrane domain may comprise all or part of a CD8-derived transmembrane domain, and specifically, the CD8 may be human CD8, but is not limited thereto. The CD8-derived transmembrane domain may comprise a human CD8-derived transmembrane domain, and specifically, may comprise an amino acid sequence of SEQ ID NO: 64, a part thereof, or an amino acid sequence having at least 64%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, but is not limited thereto. In the present invention, the chimeric antibody receptor may further comprise a hinge domain. The hinge domain may be composed of any oligopeptide or polypeptide, and may comprise 1 to 100 amino acid residues, specifically, 10 to 70 amino acid residues. In the present invention, the intracellular domain may include an intracellular signaling domain and / or a costimulatory domain.The intracellular signaling domain may be one or more intracellular signaling domains selected from the group consisting of CD3 zeta (x), CD3 gamma (x), CD3 delta (6), CD3 epsilon (e), FcR gamma, FcR beta, CD5, CD22, CD79a, CD79b and CD66d, but is not limited thereto, and more specifically, may be CD3 zeta (&). In one embodiment, the intracellular signaling domain of CD3 zeta (&) according to the present invention may have an amino acid sequence including the amino acid sequence of SEQ ID NO: 66, but is not limited thereto. In addition, the intracellular domain according to the present invention may additionally include a costimulatory domain, but is not limited thereto. The costimulatory domain according to the present invention may be one or more costimulatory domains selected from the group consisting of CD2, CD7, CD27, CD28, CD30, CD40, 4-lBB (CD137), OX40 (CD134), ICOS, LFA-1, GITR, MyD88, DAP1, PD-1, LIGHT, NKG2C and B7-H3, but is not limited thereto. Specifically, the costimulatory domain may be a 4-1BB costimulatory domain. In one embodiment, the 4-1BB costimulatory domain according to the present invention may have an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 65, but is not limited thereto. In one embodiment, the intracellular domain according to the present invention may include a CD3 zeta (&) intracellular signaling domain and a 4-1BB costimulatory domain, but is not limited thereto. Specifically, the intracellular signaling domain according to the present invention may include a CD3 zeta intracellular signaling domain comprising an amino acid sequence of SEQ ID NO: 66 and a costimulatory domain of 4-1BB comprising an amino acid sequence of SEQ ID NO: 65.In particular, the chimeric antigen receptor according to the present invention may include one or more intracellular signaling domains and one or more costimulatory domains. When the chimeric antigen receptor according to the present invention includes one or more intracellular signaling domains and one or more costimulatory domains, the one or more costimulatory domains and the one or more intracellular signaling domains may be connected in series with each other. In this case, each domain may be directly connected, or may be connected via an oligopeptide linker or polypeptide linker consisting of 2 to 10 amino acid residues, and specifically, a glycine-serine continuous sequence may be exemplified as such a linker sequence. In the present invention, the chimeric antigen receptor may additionally include a T cell immune function promoting factor, and the T cell immune function promoting factor may include, but is not limited to, IL-7 (interleukin 7), IL-12, IL-15, IL-18, IL-21, or CCL19. With regard to factors promoting immune function of T cells, reference may be made to WO 2016 / 056228 A. In the present invention, the chimeric antigen receptor may further comprise an interleukin receptor chain comprising a JAK binding motif and a STAT 3 / 5 association motif, such as, but not limited to, IL-2RP. In this regard, reference may be made to WO 2016 / 127257 A.

[0008] The first-generation CAR included an extracellular domain containing an antigen recognition site specifically expressed in cancer cells, a transmembrane domain, and an intracellular signaling domain, and used only CD3 as the signaling domain, but had a minimal therapeutic effect on cancer and a short duration of action. This first-generation CAR is specifically described in U.S. Patent No. 6,319,494, which is incorporated herein by reference. To improve responsiveness to immune cells, a second-generation CAR was manufactured that combined a costimulatory domain (CD28 or CD137 / 4-1BB) and CD3, and the number of CAR-containing immune cells remaining in the body significantly increased compared to the first-generation CAR. While the second-generation CAR used one costimulatory domain, the third-generation CAR used two or more costimulatory domains. To achieve expansion and persistence of immune cells containing CARs in vivo, the co-stimulatory domain can be combined with 4-IBB, CD28, or 0X40. Second generation CARs are specifically described in U.S. Patent Nos. 7,741,465, 7,446,190, or 9,212,229, and third generation CARs are specifically described in U.S. Patent No. 8,822,647, which are incorporated herein by reference.

[0009] The fourth generation CARs may further comprise an additional gene encoding a cytokine, such as IL-12 or IL-15, to enable additional expression of the cytokine-based immunoprotein, and the fifth generation CARs may further comprise an interleukin receptor chain, such as IL-2RP, for immune cell enhancement. The fourth generation CAR is specifically described in U.S. Patent No. 10,316,102, and the fifth generation CAR is specifically described in U.S. Patent No. 10,336,810, which are incorporated herein by reference. In one embodiment, the chimeric antigen receptor according to the present invention may comprise, but is not limited to, an antigen binding domain comprising an antibody or antigen binding fragment thereof that specifically binds to c-Met or B7-H3, a CD8-derived extracellular domain, a CD8-derived transmembrane domain, a 4-1BB costimulatory domain, and a CD3 & intracellular signaling domain. Specifically, in the present invention, the chimeric antigen receptor may include, but is not limited to, an anti-B7-H3 antigen binding domain comprising an antibody or antigen-binding fragment thereof that specifically binds to c-Met or B7-H3; a CD8-derived extracellular domain comprising the amino acid sequence of SEQ ID NO: 63; a CD8-derived transmembrane domain comprising the amino acid sequence of SEQ ID NO: 64; a 4-1BB costimulatory domain comprising the amino acid sequence of SEQ ID NO: 65; and a CD3 zeta (&) intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 66. For example, the chimeric antigen receptor according to the present invention may include the amino acid sequence of SEQ ID NO: 11, 61, 68, 75, 91, 126, 133 or 140.

[0010] IL-12 is a representative cytokine that enhances T cell function and is originally secreted mainly by antigen-presenting cells such as dendritic cells and macrophages. Secreted IL-12 transmits T cell activation and effector differentiation signals through the IL-12 receptor on the T cell surface. IL-12 exists in the form of a heterodimer composed of p40 and p35 proteins. In the present invention, the second gene encoding IL-12 may include a first coding sequence encoding IL-12 p40 and a second coding sequence encoding IL-12 p35, but is not limited thereto. In one embodiment, the first coding sequence encoding IL-12 p40 and the second coding sequence encoding IL-12 p35 may be linked via a linker. The IL-12 may be a single-chain IL-12, but is not limited thereto. In another example, IL-12 can be a heteropolymer of non-covalently complexed p40 and p35. In one embodiment, IL-12 can comprise, but is not limited to, the amino acid sequence of SEQ ID NO: 29 or 86; or an amino acid sequence having at least 90% identity thereto. In the present invention, the CTLA4-CD28 fusion protein can comprise, but is not limited to, the extracellular domain of CTLA4 and the intracellular domain of CD28. In one specific embodiment, the CTLA4-CD28 fusion protein can be fused by, but is not limited to, the transmembrane domain of CTLA4 or CD28. In one specific example, the CTLA4-CD28 fusion protein may comprise, but is not limited to, the extracellular domain of CTLA4 - the transmembrane domain of CTLA4 - the intracellular domain of CD28 or the extracellular domain of CTLA4 - the transmembrane domain of CD28 - the intracellular domain of CD28.Specifically, a CTLA4 molecule is a molecule comprising an extracellular domain of cytotoxic T-lymphocyte associated protein 4 (CTP4). The extracellular domain of CTLA4 comprises a portion of the CTLA4 protein that recognizes and binds at least one B7CCD80 / 86 antigen, such as the B7 antigen expressed on B cells and antigen presenting cells (APCs). The extracellular domain may also comprise a fragment or derivative of CTLA4 that binds to the B7 antigen. The CTLA4 extracellular domain can recognize and bind CD80 (B7-1) and / or CD86 (B7-2). The extracellular domain may comprise a fragment or derivative of CTLA4 that binds to CD80 and / or CD86. In one specific embodiment, the fusion protein may comprise, but is not limited to, the extracellular domain of CTLA4 - the transmembrane domain of CTLA4 - the intracellular domain of CD28 or the extracellular domain of CTLA4 - the transmembrane domain of CD28 - the intracellular domain of CD28. In one embodiment, the CTLA4-CD28 fusion protein may comprise, but is not limited to, the amino acid sequence of SEQ ID NO: 24 or 81; or an amino acid sequence having at least 90% identity thereto. In one specific embodiment, the construct according to the present invention may comprise a first gene encoding a chimeric antigen receptor (CAR) and a second gene encoding IL-12, wherein the IL-12 may comprise, but is not limited to, the amino acid sequence of SEQ ID NO: 29 or 86; or an amino acid sequence having at least 90% identity thereto.In one embodiment, a construct according to the present invention may comprise a first gene encoding a chimeric antigen receptor (CAR), a second gene encoding IL-12, and a third gene encoding a CTLA4-CD28 fusion protein, wherein IL-12 comprises an amino acid sequence of SEQ ID NO: 29 or 86; or an amino acid sequence having at least 90% identity thereto.

[0011] The CTLA4-CD28 fusion protein may comprise, but is not limited to, the amino acid sequence of SEQ ID NO: 24 or 81; or an amino acid sequence having at least 90% identity thereto. In one embodiment, in the construct according to the present invention, the first gene encoding the chimeric antigen receptor, the second gene encoding IL-12, and / or the third gene encoding the CTLA4-CD28 fusion protein may be linked to each other by, but is not limited to, a 2A peptide sequence or an IRES sequence. The 2A peptide sequence may be, for example, a T2A peptide, a P2A peptide, an E2A peptide, or an F2A peptide sequence. In one specific embodiment, the construct may further comprise a promoter sequence, and the first gene, the second gene, and the third gene may be operably linked to the promoter sequence, but is not limited thereto. In one embodiment, the first gene, the second gene, and the third gene may be operably linked to independent promoters. In another embodiment, two of the first gene, the second gene, and the third gene may be operably linked to one promoter, and the remaining gene may be operably linked to a separate promoter. The term "operably linked" as used herein means that the first gene, the second gene, and the third gene are operatively linked to each of a promoter or to one or two promoters such that nucleic acid sequences having promoter activity initiate and mediate transcription of the first gene, the second gene, or the third gene. The operably linked gene may be prepared using genetic recombination techniques known in the art. Another aspect provides a vector comprising the construct. In one embodiment, the vector may be, but is not limited to, a viral vector or a non-viral vector.The above viral or non-viral vector can be used without limitation as long as it can transduce or transfect animal cells, particularly T cells, by infection. In one specific example, the viral vector may be any one selected from the group consisting of retrovirus, lentivirus, adenovirus, herpes virus, adeno-associated virus, and vaccinia virus, but is not limited thereto. The non-viral vector preferably uses a transposon system (Hackett et al., US 6,489,458 B), but is not limited thereto, and it is obvious to those skilled in the art that any non-viral vector that can be commonly used and is suitable for the purpose of the present invention can be used. Even if the present application describes "comprising a gene sequence / amino acid sequence of a specific sequence number" or "having a gene sequence / amino acid sequence of a specific sequence number", it is obvious that a gene sequence / amino acid sequence in which some sequences are deleted, modified, substituted or added can also be used in the present application if it has the same or corresponding function as that composed of the gene sequence / amino acid sequence of the corresponding sequence number. In addition, the gene sequence and the base sequence can be used interchangeably in the present application. For example, if it has the same or corresponding function as the chimeric antigen receptor, IL-12, the CTLA4-CD28 chimeric protein, or the construct, it is obvious that a meaningless sequence is added within or at the end of the sequence of the corresponding sequence number, or a part of the sequence within or at the end of the sequence of the corresponding sequence number is deleted, and this also falls within the scope of the present application.Homology and identity refer to the degree to which two given base sequences are related and can be expressed as a percentage. The terms homology and identity are often used interchangeably. Whether any two sequences are homologous or identical can be determined using a well-known computer algorithm such as the "FASTA" program using default parameters, for example, as described by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) can be determined using the GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLAST?, BLASTN, FASTA (Atschul, [S.] [F„ ] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA / . ] (1988) SIAM J Applied Math 48:1073). For example, sequence homology or identity can be determined using BLAST of the National Center for Biotechnology Information database, or ClustalW.In another aspect, the present invention relates to an immune cell into which the construct or a vector comprising the construct is introduced. In addition, the present invention relates to an immune cell comprising a protein expressed from the construct or a vector comprising the construct. In addition, the present invention relates to an immune cell comprising (i) a first gene encoding a chimeric antigen receptor comprising an antigen binding domain, an extracellular domain, a transmembrane domain, and an intracellular domain, and a second gene encoding IL-12; or together with (ii) a third gene encoding a fusion protein comprising a CTLA4 protein or a domain thereof, and CD28 or a domain thereof (CTLA4-CD28 fusion protein). In the above, the antigen binding domain, the extracellular domain, the transmembrane domain, the intracellular domain, IL-12, the CTLA4 protein or a domain thereof, and the CD28 or a domain thereof are as described above. The descriptions of the first gene, the second gene, and the third gene are as described above. In one embodiment, the chimeric antigen receptor may comprise, but is not limited to, an antigen binding domain comprising an antibody or antigen binding fragment thereof that specifically binds to c-Met or B7-H3, a CD8-derived extracellular domain, a CD8-derived transmembrane domain, a 4-1BB costimulatory domain, and a CD3 & intracellular signaling domain. The present invention also relates to an immune cell that expresses (i) a chimeric antigen receptor comprising an antigen binding domain, an extracellular domain, a transmembrane domain, and an intracellular domain, and IL-12; or together with (ii) a fusion protein comprising CTLA4 protein or a domain thereof, and CD28 or a domain thereof (CTLA4-CD28 fusion protein).In the above, the antigen binding domain, the extracellular domain, the transmembrane domain, the intracellular domain, the IL-12, the CTLA4 protein or a domain thereof, and the CD28 or a domain thereof are as described above. In one embodiment, the chimeric antigen receptor may include an antigen binding domain comprising an antibody or an antigen-binding fragment thereof that specifically binds to c-Met or B7-H3, a CD8-derived extracellular domain, a CD8-derived transmembrane domain, a 4-1BB costimulatory domain, and a CD3 & intracellular signaling domain, but is not limited thereto. In the present invention, the immune cell may be, but is not limited to, a T cell, an NK cell, an NKT cell, or a macrophage, and specifically, may be a T cell. The immune cell expressing the chimeric antigen receptor according to the present invention may be a CAR-T cell, a CAR-NK cell (Chimeric Antigen Receptor Natural Killer Cell), a CAR-NKT cell (Chimeric Antigen Receptor Natural killer T Cell), or a CAR-macrophage (Chimeric Antigen Receptor Macrophage). In the present invention, the T cell may be selected from the group consisting of a CD4 positive T cell; a CD8 positive cytotoxic T lymphocyte (CTL); a CD4 and CD8 double negative T support cell; a gamma-delta T cell; a tumor infiltrating lymphocyte (TIL) and a T cell isolated from a peripheral blood mononuclear cell (PBMC). In another aspect, the present invention provides a method for producing a chimeric antigen receptor, comprising: the construct; a vector comprising the construct; an immune cell into which the construct or the vector has been introduced; Or, it relates to a composition comprising the above immune cells.Furthermore, the present invention relates to a pharmaceutical composition for treating cancer, comprising the construct; a vector comprising the construct; an immune cell into which the construct or the vector has been introduced; or the immune cell, and a pharmaceutically acceptable carrier. In the present invention, “cancer” and “tumor” are used interchangeably and refer to or mean a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Cancers that can be treated with the CAR of the present invention include vascularized tumors as well as tumors that are not vascularized or are not yet substantially vascularized. The cancer may include non-solid tumors (e.g., hematological tumors, such as leukemia and lymphoma) or may include solid tumors. Types of cancers that can be treated with the CAR of the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and childhood tumors / cancers are also included. Blood cancers are cancers of the blood or bone marrow. Examples of blood (or hematopoietic) cancers include leukemias, including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, and myeloblastic, prolymphocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemia (e.g., chronic lymphocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia. A solid tumor is an abnormal mass of tissue that usually does not contain a cyst or fluid area. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells that form them (e.g., sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, squamous cell carcinoma, rhabdomyosarcoma, rectal carcinoma, lymphoid malignancies, colon cancer, stomach cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, laryngopharyngeal cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver tumor, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, And CNS tumors (e.g., gliomas (e.g., brainstem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, germ cell tumors, medulloblastomas, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases). The therapeutic composition of the present invention is a composition for preventing or treating cancer, and the term “prevention” of the present invention means any act of inhibiting or delaying the progression of cancer by administering the composition of the present invention, and “treatment” means inhibiting the development of cancer, alleviating or eliminating symptoms. A pharmaceutical composition comprising an immune cell expressing a chimeric antigen receptor according to the present invention may additionally include a pharmaceutically acceptable excipient.Examples of such excipients include, but are not limited to, surfactants, specifically nonionic surfactants of the polysorbate series; buffers such as neutral buffered saline, human salt buffered saline; sugars or sugar alcohols such as glucose, mannose, sucrose, dextran, mannitol; amino acids or proteins or polypeptides such as glycine, histidine; antioxidants; chelating agents such as EDTA or glutathione; penetrants; adjuvants; and preservatives. The compositions of the present invention can be formulated using methods known in the art so as to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal other than a human. The formulations can be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powders. In one embodiment, the pharmaceutical composition according to the present invention can be administered to a subject without lymphodepletion, so that CAR-T cells can be safely used without concern that they will induce inflammation in normal tissues. In another aspect, the present invention relates to a method for treating cancer, comprising administering to a subject an immune cell into which the construct or a vector comprising the construct has been introduced. In addition, the present invention relates to a method for treating cancer, comprising administering to a subject an immune cell comprising a protein expressed from the construct or a vector comprising the construct. In addition, the present invention relates to a method for treating cancer, comprising administering to a subject an immune cell comprising (i) a first gene encoding a chimeric antigen receptor comprising an antigen binding domain, an extracellular domain, a transmembrane domain, and an intracellular domain, and a second gene encoding IL-12; or together (ii) a third gene encoding a fusion protein comprising a CTLA4 protein or a domain thereof, and CD28 or a domain thereof (CTLA4-CD28 fusion protein).The present invention also relates to a method for treating cancer, comprising administering to a subject an immune cell that expresses (i) a chimeric antigen receptor comprising an antigen binding domain, an extracellular domain, a transmembrane domain, and an intracellular domain, and IL-12; or together with (ii) a fusion protein comprising CTLA4 protein or a domain thereof, and CD28 or a domain thereof (CTLA4-CD28 fusion protein). The present invention also relates to the use of the construct, the vector, or the immune cell for treating cancer. The present invention also relates to the use of the construct, the vector, or the immune cell for the manufacture of a medicament for treating cancer. The subject may be a mammal having a tumor, and may be specifically, but is not limited to, a human. The immune cells expressing the chimeric antigen receptor according to the present invention or the composition containing the same may be administered by, but not limited to, infusion, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intrarectal administration, topical administration, intranasal injection, etc. The dosage of the active ingredient may be appropriately selected depending on various factors such as the route of administration, the patient's age, sex, weight, and the severity of the patient, and the therapeutic composition according to the present invention may be administered in combination with a known compound having an effect of preventing, improving, or treating cancer symptoms.Effect of the invention According to one aspect of the present invention, when CAR-T cells simultaneously loaded with IL-12 or CTC284 IL- 12 are administered without lymphodepletion, the CAR-T cells are effective only in tumors while the migration to normal tissues is limited, thereby realizing a safe and effective CAR-T cell. Brief description of the drawings Figure 1 shows the structure of a cMet scFv antibody (VL, light chain variable region; VH, heavy chain variable region; CH2, heavy chain constant region 2; CH3, heavy chain constant region 3; His, 6x histidine tag; HA, HA peptide tag). Figure 2 shows the results of testing the cMet binding of a cMet scFv antibody. Figure 3 is a schematic diagram showing the structure of a mouse cMet CAR (PGK, PGK promoter; m, mouse; h, human; EC, extracellular domain; TM, transmembrane domain; cyt, cytoplasmic domain). Fig. 4 is a graph showing the CAR expression rate in mouse cMet CAR T cells (blue, untransduced T cells; red, cMet CAR T cells) (the numbers in the graph indicate the proportion (%) of CAR-positive cells). Fig. 5 is a graph showing the expression rate of cell surface cMet in E07, a breast cancer cell line derived from C57BL / 6 (B6) mice, and 4T1, a breast cancer cell line derived from Balb / c mice (gray, unstained group; blue, negative control antibody; red, anti-cMet antibody). Fig. 6 is a graph showing the results of evaluating the in vitro tumor killing ability and CAR-T cell activation ability (IFN-x secretion ability) of mouse cMet CAR-T cells against mouse tumors (E0771, B6 mouse breast cancer cells; 4T1, BALB / c mouse breast cancer cells) (Untransduced T, untransduced T cells; cMet, cMet CAR T cells).Figure 7 is a graph showing the results of evaluating the in vivo efficacy and toxicity (body weight loss) of mouse cMet CAR-T cells (IR, irradiation; cMet, cMet CAR T cells). Figure 8 is a schematic diagram showing the structure of cMet-CTC28 CAR (PGK, PGK promoter; m, mouse; EC, extracellular domain; TM, transmembrane domain; cyt, cytoplasmic domain; P2A, P2A peptide). Figure 9 is a graph showing the expression rate of c-Met CAR or c-Met- CTC28 CAR and CTC28 in CD4 and CD8 T cells. Figure 10 is a graph showing the results of evaluating the in vitro tumor-killing ability of cMet-CTC28 CAR-T cells (CD4-positive CAR-T cells and CD8-positive CAR-T cells). Figure 11 is a graph showing the results of evaluating the secretion capacity of cytokines IFN-Y and IL-2 in cMet CAR-T cells and cMet- CTC28 CAR-T cells. Figure 12 shows the results of evaluating the in vivo efficacy and toxicity of B6 cMet-CTC28 CAR-T cells. Figure 13 is a graph showing the CAR and CTC28 expression rates of Balb / c mouse cMet-CTC28 CAR-T cells. Figure 14 is a graph showing the results of evaluating the in vitro cell killing capacity of cMet CAR-T cells and cMet- CTC28 CAR-T cells. Figure 15 is a graph showing the results of evaluating the in vitro cytokine secretion of cMet CAR-T cells and cMet- CTC28 CAR-T cells. Figure 16 shows the results of evaluating the in vivo efficacy and toxicity of Balb / c cMet-CTC28 CAR-T cells. Figure 17 illustrates the in vivo experimental schedule of cMet CAR-T cells with IL-2 adjuvant therapy. Figure 18 illustrates the results of evaluating the in vivo toxicity of cMet CAR-T cells with IL-2 adjuvant therapy.Figure 19 shows the in vivo experimental schedule for evaluating the on-target off-tumor toxicity of cMet CAR-T cells. Figure 20 is a graph showing the results of evaluating the in vivo toxicity of cMet CAR-T cells. Figure 21 is a graph showing the results of evaluating cMet mRNA expression in mouse organs. Figure 22 shows the cMet CAR and cMet-CTC28 CAR retrovirus structures loaded with luciferase. Figures 23 and 24 show the results of detecting the luminescence of luciferin in GFP-Luc cells, cMet-Luc CAR-T cells, and cMet-CTC28-Luc CAR-T cells using bioluminescence imaging (BLI). Figure 25 shows the infiltration of GFP-Luc CAR-T cells, cMet-Luc CAR-T cells, and cMet— CTC28— Luc CAR-T cells into mouse organs. Figure 26 is a graph showing the results of measuring the concentrations of ALT, AST, and BUN in mouse blood after administration of cMet CAR-T cells. Figure 27 is a graph showing the results of evaluating the in vivo toxicity of cMet CAR-T cells according to the presence or absence of irradiation. Figure 28 is a graph showing the results of measuring the concentrations of ALT and AST in mouse blood after administration of cMet CAR-T cells according to the presence or absence of irradiation. Figures 29 and 30 show the results of confirming the in vivo infiltration and persistence of cMet-Luc CAR-T cells according to the presence or absence of irradiation. Figure 31 is a graph showing the results of evaluating the in vivo toxicity of cMet CAR-T cells using Balb / c nude mice. Figure 32 shows a schedule for evaluating the in vivo toxicity of cMet CAR-T cells after cyclophosphamide treatment. Figure 33 is a graph showing the results of evaluating the in vivo toxicity of cMet CAR-T cells according to the presence or absence of cyclophosphamide treatment.Figure 34 shows the experimental schedule for evaluating the in vivo efficacy and toxicity of cMet CAR-T cells in the absence of lymphodepletion. Figure 35 is a graph showing the results of evaluating the in vivo efficacy and toxicity of Balb / c cMet CAR-T cells in the absence of lymphodepletion. Figure 36 is a graph showing the results of evaluating the in vivo efficacy and toxicity of B6 cMet CAR-T cells in the absence of lymphodepletion. Figure 37 is a schematic diagram showing the structure of mlL- 12f (h, human; m, mouse; (G4S)3, GGGGSGGGGSGGGGS peptide). Figure 38 shows the structure of cMet-iIL12 CAR retrovirus (PGK, PGK promoter; Min IL- 2, minimal IL- 2 promoter; 6xNFAT, six tandem NFAT binding elements; iIL-12, inducible IL- 12). Figure 39 is a graph showing the CAR expression rate of cMet-iIL12 CAR-T cells. Figure 40 is a graph showing the results of evaluating the tumor-killing ability, IL-12 secretion, and IFN-x production of cMet-iIL12 CAR-T cells. Figure 41 is a graph showing the results of evaluating the in vivo efficacy and toxicity of cMet-iIL12 CAR-T cells (arrow: CAR-T cell administration time, day 7). Figure 42 is a schematic diagram showing the structure of cMet- CTC28- iIL12 CAR. Figure 43 is a graph showing the CAR expression rate of cMet-CTC28-iIL12 CAR-T cells. Figure 44 is a graph showing the results of evaluating the in vivo efficacy and toxicity of cMet-CTC28-iIL12 CAR-T cells. Figure 45 is a graph showing the results of measuring ALT, AST, and BUN concentrations in blood after administration of cMet-CTC28-iIL12 CAR-T cells. Figure 46 is a schematic diagram showing the structure of a constitutive IL-12 cMet CAR (T2A, T2A peptide, P2A, P2A peptide). Figure 47 is a graph showing the expression rate of CAR and CTC28 in CAR-T cells.Figure 48 is a graph showing the IL-12 and IFN-γ secretion ability in CAR-T cells. Figure 49 is a graph showing the results of evaluating the cytotoxic ability of CAR-T cells. Figure 50 is a graph showing the results of evaluating the in vivo efficacy and toxicity of Constitutive IL-12 cMet CAR-T cells. Figure 51 shows the results of evaluating the cross-reactivity of the B7H3 antibody clones with human and mouse B7H3 proteins, and shows the ELISA affinity analysis results of the isolated scFv antibody clones for human B7H3 (h4Ig, h2Ig) and mouse B7H3 (m2Ig) (h4Ig, h2Ig: two isoforms of the human B7H3 protein). Figure 52 is a schematic diagram showing the structures of B7H3 CAR and B7H3-CTC28 CAR. Figure 53 is a graph showing the expression rates of CAR and CTC28 in B7H3 CAR, B7H3-CTC28 CAR-T cells. Figure 54 shows the experimental schedule for evaluating the in vivo efficacy and toxicity of B7H3 CAR-T cells. Figure 55 is a graph showing the results of evaluating the in vivo efficacy and toxicity of B7H3 CAR-T cells. Figure 56 is a graph showing the results of evaluating the in vivo efficacy and toxicity of B7H3 CAR-T cells without lymphodepletion. Figure 57 is a schematic diagram showing the structures of B7H3-iIL12 CAR or B7H3-CTC28-iIL12 CAR. Figure 58 is a graph showing the expression rates of CAR and CTC28 in B7H3-iIL12 CAR-T and B7H3-CTC28-iIL12 CAR-T cells. Figure 59 is a graph showing the results of evaluating the in vivo efficacy and toxicity of B7H3-CTC28-iIL12 CAR-T cells.Figure 60 is a schematic diagram showing the structure of a potentiated human CAR gene constructed using cMet scFv antibody (6xNFAT, six tandem NFAT binding element promoter; Min IL-2, minimal IL-2 promoter; h, human; PGK, PGK promoter; L, leader sequence; EC, extracellular domain; TM, transmembrane domain; cyt, cytoplasmic domain; P2A, P2A peptide; CTC28, CTLA4-CD28 chimera). Figure 61 shows the results of measuring the expression of CAR and CTC28 in potentiated human cMet CAR-T cells (CD4-positive CAR-T cells and CD8-positive CAR-T cells) (ChlgY-FITC, FITC-labeled anti-chicken IgY Fab; CTLA4-PE, PE-labeled anti-human CTLA4 antibody; CD4, APC / Cyanine7-labeled anti-human CD4 antibody; CD8, PerCP / Cy5.5—labeled anti-human CD8 antibody). Figure 62 is a graph showing the results of measuring the expression of cell surface cMet in potentiated human cMet CAR-T cells in a human breast cancer cell line (BT20). Figure 63 is a graph showing the results of analyzing the tumor killing capacity of potentiated human cMet CD4 and CD8 CAR-T cells, and measuring the cytotoxicity against BT-20, which is a target cell (T). Figure 64 shows the results of measuring IL-12 expression of human cMet CD4 and CD8 CAR- T cells with enhanced potency. Figure 65 shows the results of measuring IFN- x secretion of human cMet CD4 and CD8 CAR- T cells with enhanced potency. Figure 66 is a schematic diagram showing the structure of a lentiviral vector expressing a human CAR gene, i.e., hB7H3 CAR (hB7H3-BBz) gene, produced using anti-B7-H3 antibody scFv> (L, leader sequence; h, human).Figure 67 is a schematic diagram showing the structure of a lentiviral vector co-conjugated with hB7H3 CAR, inducible IL-12, and CTLA4-CD28 chimeric (CTC28) genes (h, human; PGK, PGK promoter; Min IL-2, cancel IL-2 promoter; 6xNFAT promoter, six tandem NFAT binding elements; P2A, P2A peptide). Figure 68 is a graph showing the CAR and CTC28 expression rates of enhanced human B7-H3 CAR-T cells (UT, untransduced; ChlgY, FITC-labeled anti-chicken IgY Fab; CTLA4-PE, PE-labeled anti-human CTLA4 antibody). Figure 69 is a graph showing the results of evaluating the tumoricidal activity of enhanced human B7-H3 CAR-T cells (Unt, untransduced). Figure 70 is a graph showing the results of evaluating the IL-12 secretion activity of enhanced human B7-H3 CAR-T cells. Figure 70 is a graph showing the results of evaluating the IFN-x secretion activity of enhanced human B7-H3 CAR-T cells. Figure 72 is a schematic diagram showing the structure of a lentiviral vector expressing a CTLA4-CTC28 chimera (CTC28) gene and an inducible IL12 gene together with a human CAR gene produced using a humanized anti-B7H3 antibody scFv>, i.e., a schematic diagram showing the structure of the huB7H3-BBz-CTC28-IL12 gene (6xNFAT, six tandem NFAT binding element promoter; Min IL— 2, minimal IL— 2 promoter; h, human; hu, humanized; PGK, PGK promoter; P2A, P2A peptide; CTC28, CTLA4-CD28 chimera).Figure 73 shows the results of analyzing the expression of CAR and hCTC28 in three types of enhanced humanized B7H3 CD4 and CD8 CAR- T cells (F(ab')2-FITC, FITC-labeled anti-human IgG F(ab')2; CTLA4-PE, PE-labeled anti-human CTLA4 antibody; CD4, APC / Cyanine7—labeled anti-human CD4 antibody; CD8, PerCP / Cy5.5—labeled anti-human CD8 antibody). UT (untransduced) is a negative control group with non-transduced T cells. For convenience, it is denoted as CTC28-IL12# C12. Figure 74 is a graph showing the results of evaluating the tumor killing capacity of enhanced humanized B7H3 CD4 and CD8 CAR- T cells. FIG. 75 is a graph showing the results of evaluating the IL-12 secretion ability of humanized B7H3 CD4 and CD8 CAR-T cells with enhanced potency. FIG. 76 is a graph showing the results of evaluating the IFN- x secretion ability of humanized B7H3 CD4 and CD8 CAR-T cells with enhanced potency. Embodiments for implementing the invention Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples. The present inventors have developed a receptor for proliferation of chemotherapeutic cells called CTLA4-CD28 chimera (CTC28) through previous research (Korean Patent No. 10-1471647). In the present invention, we aimed to verify whether it is possible to create CAR-T cells that are safe and highly effective by producing CAR-T cells that are effective only in tumors while restricting CAR-T cell migration to normal tissues when CAR-T cells loaded with IL-12 or CTC284 IL-12 are administered simultaneously without lymphodepletion. To this end, we used an in vivo model in which lymphodepletion is not performed.0] is required. The existing CAR-T cell preclinical model that verifies the efficacy of CAR-T cells is a model that administers human CAR-T cells to immunodeficient mice inoculated with human tumor cells to observe the anti-tumor effect. However, since this model already lacks lymphocytes such as D cells, it cannot implement a condition in which lymphodepletion is not performed. In addition, there is a disadvantage in that normal tissue toxicity cannot be tested if human CAR-T cells do not recognize mouse antigens in normal mouse tissues. To overcome this disadvantage, the present invention aimed to see whether mouse CAR-T cells exhibit an anti-tumor effect without normal tissue toxicity when mouse CAR-T cells that recognize mouse tumor antigens are administered to normal mice with a normal immune system (so-called syngeneic mouse model). The c-Met antigen, which is well known as an existing tumor antigen, was selected as the tumor antigen. cMet is an antigen known to be overexpressed in various solid tumors, and thus, many studies have been conducted as a target antigen for anticancer treatments such as cMet antibody therapy, cMet inhibitor, and cMet CAR-T cells. However, cMet is known to be expressed in various organs including the liver as a receptor for HGF (Hepatocyte Growth Factor), so there is a possibility of normal tissue toxicity. Existing cMet CAR-T cell studies were conducted using human cMet CAR-T cells in immunodeficient mice, and were models that could only confirm efficacy without evaluating toxicity (Cancer Immunol Res. 2015 3(4):356-67). Therefore, before applying cMet CAR-T cell therapy to actual patients, its safety needs to be evaluated in advance.In the present invention, a strategy was used to increase the possibility of future clinical development of anti-cMet CAR-T cells (hereinafter referred to as cMet CAR-T cells) by discovering cross-reactive anti-cMet antibodies that simultaneously bind to mouse cMet and human c-Met and using them in the production of CAR-T cells. As a result, when cMet CAR-T cells simultaneously loaded with CTC28 and IL-12 were administered to normal mice inoculated with tumors without performing lymphodepletion, an effective anti-tumor effect was observed without serious toxicity. These results were also observed in an experiment using CAR-T cells for another tumor antigen, B7H3. Example 1. Production of mouse cMet CAR-T cells and in vitro and in vivo experiments.

[0012] (1) cMet Antibody Screening In order to secure antibodies for cMet CAR-T cell production, antibodies that simultaneously bind to human cMet and mouse cMet were screened from the scFv library constructed after immunizing chickens with cMet protein, and the structure of the finally selected m4A25 clone is shown in Fig. 1. It was confirmed through ELISA that the m4A25 clone antibody dose-dependently binds to both human cMet and mouse cMet, and the results are shown in Fig. 2. Based on these experimental results, the clone was evaluated as a clone suitable for making a cMet CAR.

[0013] (2) Production of cMet CAR retroviral plasmid After synthesizing the CAR gene using the m4A25 scFv> above, it was cloned into the MSCV retroviral vector (Clontech) to produce the cMet CAR retroviral plasmid. Figure 3 is a schematic diagram showing the structure of mouse cMet CAR (PGK, PGK promoter; m, mouse; h, human; EC, extracellular domain; TM, transmembrane domain; cyt, cytoplasmic domain). To produce ecotrophic retrovirus capable of transducing mouse T cells, Phoenix GP cells were transfected with the cMet CAR retroviral plasmid and the VSV-G envelope expression plasmid (pMD2.G, Addgene plasmid #12259)# using Lipofectamin 3000 (Invitrogen). After 48 hours, the VSV-G pseudotyped retrovirus secreted into the culture supernatant was transduced again into Phoenix Eco cells. After 3 to 5 days, cells stably expressing the CAR protein on the cell surface were isolated using a cell sorter (FACS Aria, Becton Dickinson), and these cells were used as ecotrophic retrovirus production cell lines. The culture supernatant containing the retrovirus secreted from this cell line was concentrated 5 to 10 times using a centrifugal filter device (Amicon Ultra-100 kDa cut-off, Millipore, USA), and then used to produce mouse CAR-T cells.

[0014] (3) Production of mouse cMet CAR-T cells To produce mouse CAR-T cells, spleen and lymph node cells were isolated from normal Balb / C or B6 mice, and then added together with anti-CD28 antibody (37.51, 2 w / mt, BD Biosciences) to a 24-well plate coated with anti-CD3 antibody (145— 2C11, 10^g / m£, BioXcell) to activate each T cell. T cells activated for 24 hours were transduced with retrovirus by centrifugation at 24°C, 2500 rpm for 90 minutes in the presence of 6]ig / ml polybrene (Sigma Aldrich). The retrovirus transduction process through centrifugation was repeated once on the same day. Afterwards, T cells were cultured in the presence of mouse IL-2 (30U / < Gibco) for 48 hours. After washing the retrovirus-transduced T cells twice, fresh culture medium containing mouse IL-2 (20 U / mt) was added and the cells were proliferated for 2-3 days to be used as CAR-T cells. After CAR-T cell production, the expression of cMet CAR on the cell surface was confirmed by flow cytometry (FACS-Canto II, BD Biosciences) after staining the CAR protein on the cell surface with FITC-labeled anti-Chicken IgG Fab fragment (Jackson ImmunoResearch), and the results are shown in Fig. 4. Fig. 4 is a graph showing the CAR expression rate in mouse cMet CAR T cells (blue, non-transduced T cells; red, cMet CAR T cells) (The numbers in the graph indicate the proportion (%) of CAR-positive cells).

[0015] (4) Evaluation of mouse cMet CAR-T toxicity and efficacy Next, in order to evaluate the toxicity and efficacy of cMet CAR-T cells using a mouse syngeneic tumor model, the expression of cell surface cMet in E07714, a breast cancer cell line derived from C57BL / 6 (B6) mice, and 4T1, a breast cancer cell line derived from Balb / c mice, was confirmed by flow cytometry after staining with anti-cMet antibody, and the results are shown in Fig. 5. Fig. 5 is a graph showing the expression of cell surface cMet in E07, a breast cancer cell line derived from C57BL / 6 (B6) mice, and 4T1, a breast cancer cell line derived from Balb / c mice (gray, unstained group; blue, negative control antibody; red, anti-cMet antibody). To confirm whether the manufactured cMet CAR-T cells react with target tumor cells, exhibit cell killing ability, and secrete cytokines, which are indicators of activity, B6 cMet CAR-T cells and Balb / c cMet CAR-T cells were co-cultured with E0771 or 4T1 cells, respectively, in vitro, and then IFN- x secreted into the culture medium was measured by ELISA to confirm the cell killing ability. The results are shown in Fig. 6. Fig. 6 is a graph showing the results of evaluating the in vitro function of mouse cMet CAR-T cells (Untransduced T, T cells not transduced; cMet, cMet CAR T cells). The test results confirmed that both CAR-T cells secreted large amounts of IFN- x, and that they killed target tumor cells in proportion to the number of CAR-T cells. Therefore, the production and in vitro activity of cMet CAR-T cells were successfully confirmed. Afterwards, to confirm the anti-tumor effect and toxicity of cMet CAR-T cells in vivo, E07 group was subcutaneously injected into 5*1 (T5 each) of B6 mice, and on the 6th day, total body irradiation was performed for lymphodepletion.On the 7th day, the following day, B6 cMet CAR-T cells were administered intravenously to 5*1 (T6) animals, and the changes in tumor size and body weight of the animals were measured, and the results are shown in Fig. 7. As a result, it was confirmed that, in contrast to the remarkable in vitro tumor killing effect, tumor proliferation was not significantly inhibited. The initial body weight loss reflecting the toxicity of CAR-T cells did not increase significantly compared to the group that was administered only tumors. Therefore, this result suggested that increasing the efficacy of cMet CAR-T cells was necessary. Example 2. In vivo toxicity evaluation of cMet CAR-T cells in mice with enhanced efficacy.

[0016] To enhance the efficacy of CAR-T cells, the inventors sought to introduce additional function-enhancing genes into CAR-T cells. To this end, they sought to utilize the CTLA4-CD28 chimera protein (CTC28) previously developed by the inventors.

[0017] CTC28 is a fusion protein in which the intracellular inhibitory signaling domain of CTLA4, a T cell inhibitory receptor, is replaced with the intracellular stimulatory signaling domain of CD28, an activating receptor. When CTC28 is artificially expressed in T cells, CTC28 competitively binds to the ligand with CTLA4 originally present in T cells, thereby blocking the inhibitory signal of CTLA4 and at the same time using the CD28 intracellular domain to transmit a T cell activation signal, promoting the activation and proliferation of T cells (Blood, 2012, 119(24):5678 - 87; registered patents (KR10—1471647, US9688740, JP6074435)). Therefore, CAR-T cells in which the CAR protein and the CTC28 protein are simultaneously expressed were produced to confirm whether the function of the CAR-T cells was enhanced. Thus, a retroviral plasmid that simultaneously expresses the mouse cMet CAR gene and the mouse CTC28 gene was constructed, and then a retrovirus was produced using this plasmid. A schematic diagram showing the structure of cMet-CTC28 CAR is disclosed in FIG. 8 (PGK, PGK promoter; m, mouse; EC, extracellular domain; TM, transmembrane domain; cyt, cytoplasmic domain, P2A, P2A peptide). According to the prior research of the present inventors (匕 foot# 2012, 119(24):5678 - 87), the effect of CTC28 in T cell therapy is maximized when CTC28 is transduced into CD4 T cells and CD eight T cells respectively, and these CD4 and CD eight T cells are mixed and administered at a ratio of 1:1. Therefore, in the present invention, the method of separately producing CD four CAR-T cells and CD eight CAR-T cells and mixing and administering them at a ratio of 1:1 was adopted.To this end, CD4 and CD8 T cells were separated from the spleen and lymph node cells of B6 mice by the Magnetic-activated cell sorting (MACS) method, and then c— Met CAR or c— Met— CTC28 CAR retrovirus was transduced into these CD4 and CD8 T cells, respectively. The expression of CAR and CTC28 was confirmed by flow cytometry, and the results are presented in Fig. 9. As a result of the experiment, CAR expression and CTC28 expression were confirmed in both CD4 and CD8 T cells. To examine the in vitro activity of the manufactured cMet-CTC28 CAR-T cells, when co-cultured with E0771 breast cancer cells, cMet- CTC28 CAR-T cells showed a tumor-killing ability similar to that of cMet CAR-T cells in both CD4 and CD8 CAR-T cells (Fig. 10). In addition, the secretion of the active cytokines IFN-Y and IL-2 was also confirmed to be well maintained in vitro, as there was no significant difference between cMet CAR-T cells and cMet-CTC28 CAR-T cells (Fig. 11). Since the induction of T cell function enhancement by CTC28 is mainly observed in vivo, it was expected that the in vitro activity was similar to that of cMet CAR-T cells. Afterwards, in vivo efficacy experiments were conducted according to the schedule in Fig. 7 to confirm whether cMet-CTC28 CAR-T cells exhibit a better anti-tumor effect than cMet CAR-T cells. However, CD4 CAR-T cells and CD8 CAR-T cells were each combined in a 1:1 ratio of 2.5*1(T6) to administer a total of 5*1(T6) CAR-T cells, and tumor proliferation and body weight changes were measured, and the results are shown in Fig. 12. As a result of the test, cMet-CTC28 CAR-T cells showed the effect of inhibiting the growth of some tumors compared to cMet CAR-T cells, but it was confirmed that they showed lethal toxicity by inducing rapid weight loss in mice after 10 days of administration.Therefore, it was found that the increase in CAR-T cell activity by CTC28 not only increased efficacy but also induced toxicity. To confirm whether this phenomenon was the same in Balb / c mice, CAR-T cells were produced from Balb / c mice, and CAR expression was confirmed in both the cMet CAR group and the cMet- CTC28 CAR group in CD4 and CD8 T cells of Balb / c mice, and CTC28 expression was confirmed in the cMet-CTC28 CAR group (Fig. 13). As a result of co-culture with 4T1 breast cancer cells, similar to B6 CAR-T cells, similar tumor killing ability and cytokine secretion ability were confirmed in both the cMet CAR group and the cMet-CTC28 CAR group (Figs. 14 and 15). Next, an in vivo efficacy test of Balb / C CAR-T cells was performed on Balb / c mice inoculated with 4T1 tumors using the same schedule and method as B6 mice, and the results are shown in Fig. 16. As a result of the test, unlike in B6 mice, some anti-tumor effects were observed in the group administered cMet CAR-T cells, but lethal toxicity was observed in which approximately 60% of the mice died. In the group administered cMet-CTC28 CAR-T cells, lethal toxicity was observed, showing much faster weight loss and faster death than in the group administered cMet CAR-T cells. Therefore, it was confirmed that cMet CAR-T cells themselves exhibited toxicity in Balb / C mice, and that the toxicity was aggravated by additional loading on CTC28.

[0018] To confirm whether the increased lethal toxicity of cMet CAR-T cells due to additional loading of CTC28 was limited to CTC28 or whether the increased efficacy of general CAR-T cells increased the toxicity of cMet CAR-T cells, an experiment was conducted in which IL-2, known to increase the proliferation of CAR-T cells in vivo, was additionally administered instead of additional loading of CTC28. That is, after lymphodepletion by radiation was performed on mice inoculated with 4T1 tumors, 50,000 IU of IL-2 was intraperitoneally injected twice a day for a total of 5 days along with administration of Balb / C cMet CAR-T cells (Fig. 17), and the results are shown in Fig. 18. As a result of the test, it was confirmed that in the group that received additional IL-2, mice died more quickly than in the group that did not receive additional IL-2, indicating that enhanced activity of cMet CAR-T cells, in addition to CTC28, can double the toxicity of cMet CAR-T cells. That is, cMet CAR-T cells with enhanced function were confirmed to exhibit lethal toxicity under lymphodepletion in both B6 mice and Balb / C mice, and it was confirmed that the efficacy and toxicity were more prominent in Balb / C mice. Example 3. Evaluation of hepatotoxicity of cMet CAR-T cells cMet is a tumor antigen that is overexpressed in various tumors, but it is also known to be expressed at low levels in normal tissues. Therefore, the in vivo toxicity of cMet CAR-T cells is likely to be on-target off-tumor toxicity in which cMet CAR-T cells recognize and attack cMet expressed in normal tissues. To confirm this, lymphodepletion was performed through total body radiation irradiation on normal Balb / C mice that were not inoculated with cancer cells, and then cMet CAR-T cells were administered to observe whether there was any mouse toxicity (Fig. 19), and the results are shown in Fig. 20.As a result of the test, even though the mice were normal mice without tumors, the cMet CAR-T cell administration group and the cMet-CTC28 CAR-T cell administration group showed toxicity that led to mice death, just like the previous experiment, and faster lethal toxicity was observed in the cMet-CTC28 CAR T group. This CAR-T cytotoxicity was not observed in the non-transduced T cell group that did not express CAR>, so it is a toxicity due to CAR expression, and the result suggests that it may be normal tissue toxicity that occurs when CAR-T cells recognize normal tissues through cMet CAR. If so, in order to confirm which normal tissue cMet CAR-T cells can attack and cause this toxicity, the expression of cMet mRNA in each organ of normal Balb / C mice was measured by qRT-PCR (quantitative RT-PCR), and the results are shown in Fig. 21. The test results showed that cMet mRNA was detected in various organs, and it was expressed at particularly high levels in the kidney and liver. Next, in order to confirm the possibility that cMet CAR-T cells infiltrate these c-Met-expressing organs and cause organ damage, we wanted to track and observe the movement of c-Met CAR-T cells in vivo. To this end, a retroviral plasmid carrying a luciferase protein (Enhanced firefly luciferase, Eff Luc) with high sensitivity to cMet CAR-T cells was constructed, and cMet-Luc CAR T and cMet-CTC28-Luc CAR-T cells expressing luciferase were constructed using this plasmid. Figure 22 shows the structures of cMet CAR and cMet-CTC28 CAR retroviruses loaded with luciferase. As a negative control, GFP-Luc T cells transduced with GFP-Luc retrovirus expressing GFP instead of CAR were used.After injecting these luciferase-expressing cells into mice and administering luciferin, a substrate of luciferase, to the mice, the luciferin product will emit light, and this can be detected using bioluminescence imaging (BLI) equipment, allowing the location of T cells in a living mouse to be identified. After performing lymphodepletion on normal Balb / c mice according to the method of Fig. 19, the group administered GFP-Luc T cells as a control and the groups administered cMet-Luc and cMet-CTC28-Luc T cells were imaged at intervals of several days to confirm the distribution of T cells in vivo, and the results are presented in Fig. 23. As a result of the test, in the case of GFP-Luc T cells, they were observed mainly in the lymph nodes in the early stages after administration, and then spread and proliferated throughout the body, centered around the abdominal cavity. This finding is consistent with the results of previous studies that T cells administered in a body depleted of lymphocytes due to lymphodepletion undergo homeostatic expansion. On the other hand, cMet-Luc CAR-T cells were characteristically observed to accumulate in the liver from day 1, and after reaching the peak of liver accumulation on day 3, they spread throughout the body and then gradually disappeared. cMet-CTC28-Luc CAR-T cells also showed a similar pattern, but showed more liver accumulation on day 3 and showed a pattern of persisting longer in vivo, suggesting that CTC28 increases the in vivo proliferation and survival of CAR-T cells. This was also confirmed by Fig. 24, which shows the results of quantification of bioluminescence. In order to more precisely specify the organs where cMet-Luc and cMet- CTC28-Luc CAR-T cells accumulate, BLI was performed on the organs of mice after luciferin administration on day 3 after administration, and the results are shown in Fig. 25.The test results confirmed that cMet CAR-T cells accumulated mainly in the spleen and liver compared to other organs, consistent with the in vivo tracking results. On the other hand, CAR-T cell infiltration was not observed in the kidney, which had high cMet mRNA levels. The above results strongly suggest the possibility of CAR-T cell-induced hepatotoxicity, as they indicate that cMet CAR-T cells mainly infiltrated into the liver, where cMet expression was high, in the early stage of administration when they showed toxicity. Therefore, the concentrations of ALT (Alanine transaminase) and AST (Aspartate transaminase), which are hepatotoxicity markers, were measured in the mouse blood after CAR-T cell administration, and the results are shown in Fig. 26. As a result of the test, the values ​​of ALT and AST measured on the second day after administration of cMet CAR-T cells and cMet- CTC28 CAR-T cells were both increased in the group administered cMet CAR-T cells compared to the group administered non-transduced T cells as a control group, and in particular, the group administered cMet-CTC28 CAR- T cells was observed to increase even more than the group administered cMet CAR-T cells. On the other hand, it was confirmed that BUN (Blood urea nitrogen), an indicator of kidney toxicity, which is another cMet-expressing organ, did not increase significantly compared to the control group, which was consistent with the result that CAR-T cells did not infiltrate kidney tissue. In summary, it was confirmed that cMet CAR-T cells, when administered to mice, accumulated in the liver with high cMet expression within a few days and showed hepatotoxicity causing liver damage, suggesting that such hepatotoxicity may be the cause of death of mice caused by CAR-T cells. Example 4.Dependence of cMet CAR-T cytotoxicity on lymphodepletion All of the above CAR-T cell administration methods administer CAR-T cells after lymphodepletion, which promotes the proliferation of CAR-T cells in the body. However, systemic inflammation caused by lymphodepletion may act as a factor that increases normal tissue toxicity by triggering CAR-T cell infiltration into normal tissues. Therefore, in order to confirm whether lymphodepletion before CAR-T cell administration induced lethal toxicity by CAR-T cells, cMet CAR-T cells were administered to Balb / C mice without total body irradiation lymphodepletion, and the results are presented in Fig. 27. As a result of the test, in the group that underwent lymphodepletion, all mice died with severe weight loss, as in the above experiments, but in the group that did not undergo lymphodepletion, no weight loss or mouse death was observed. In addition, we tracked the levels of ALT and AST, which are indicators of hepatotoxicity in the blood, and the results are shown in Fig. 28. As a result of the test, a significant increase was observed on the third day after CAR-T cell administration in the group that underwent lymphodepletion, but in the group that did not undergo lymphodepletion, only a small increase was observed on the third day and then a decrease was observed again, confirming that the hepatotoxicity of CAR-T cells was significantly reduced when lymphodepletion was not performed. Next, we confirmed whether not performing lymphodepletion actually alleviated the accumulation of cMet CAR-T cells in the liver through a BLI tracking experiment after cMet-Luc CAR-T cell administration, and the results are presented in Figs. 29 and 30. As a result of the test, it was confirmed that when lymphodepletion was not performed, the hepatic accumulation of cMet-Luc CAR-T cells was significantly reduced, and the kinetics of their subsequent disappearance from the body were also much faster. The above results confirmed that lymphodepletion aggravated the hepatotoxicity and lethal toxicity of cMet CAR-T cells.Additionally, in order to differentiate whether the factor that lymphodepletion worsens the toxicity of cMet CAR-T cells is due to the homeostatic proliferation of CAR-T cells by lymphodepletion in the body or tissue inflammation caused by total body irradiation used in lymphodepletion, CAR-T cells were administered to Balb / C nude mice that originally have a deficiency of T cells in the body, and it was confirmed that there was no weight loss or lethal toxicity caused by cMet CAR-T cells. On the other hand, in nude mice that received total body irradiation, weight loss and death of all mice were observed, confirming that total body irradiation itself, rather than lymphodepletion in the body, mediates the toxicity of CAR-T cells (Fig. 31). In addition, to confirm whether administration of chemotherapy, another method of lymphodepletion, also mediates the toxicity of cMet CAR-T cells, normal Balb / c mice were treated with cyclophosphamide at a concentration of 250 mg / kg instead of irradiation, and cMet CAR-T cells were administered the following day (Fig. 32), and the test results are presented in Fig. 33. In this case, as with irradiation, toxicity such as weight loss and mouse death was observed in the group administered cMet CAR-T cells, unlike the group administered non-transduced T cells. Therefore, it was confirmed that lymphodepletion through total body irradiation or chemotherapy is an important factor that significantly increases the normal tissue toxicity of cMet CAR-T cells. Then, an experiment was conducted to confirm whether cMet CAR-T cells or cMet-CTC28 CAR-T cells with enhanced function could exhibit antitumor efficacy in a situation where lymphodepletion without toxicity was not performed. 4T1 was injected subcutaneously into 5*1 (T5) normal Balb / c mice, and on the 7th day, cMet CAR-T cells and cMet- CTC28 CAR-T cells were injected intravenously into 5*1 (T6) mice (Figure 34).As a result, it was confirmed that the weight loss caused by cMet CAR-T cells and cMet- CTC28 CAR-T cells was minimal, but no anti-tumor efficacy was observed (Fig. 35). Similarly, when B6 mice were inoculated with E07 tumors and B6 cMet CAR-T cells and cMet- CTC28 CAR-T cells were administered in the same manner as in Fig. 32 without lymphodepletion, it was confirmed that no anti-tumor efficacy was observed (Fig. 36). Therefore, although not performing lymphodepletion can prevent normal tissue toxicity of cMet CAR-T cells, an increase in the efficacy of CAR-T cells is required. Example 5. In vivo safety and efficacy evaluation of cMet CAR-T cells expressing inducible IL-12 and CTC28 In previous studies, studies have reported that CAR-T cells exhibit antitumor efficacy even in situations where lymphodepletion was not performed, by loading the IL-12 gene, a cytokine that enhances cytokine activation, together with the CAR gene into CAR-T cells to cause CAR-T cells to secrete IL-12 (Clin. Cancer Res., 2012, 8(6) 672-83; Oncolmmunology, 2015, 4:3, e994446; J. Immunol., 2019, 203(1) 198-207). In particular, in order to minimize the systemic toxicity of IL-12, it has been reported that CAR-T cells are designed to secrete IL-12 only when they recognize the target and a CAR activation signal is transmitted to the cells. Therefore, we sought to determine whether the anti-tumor efficacy could be increased while avoiding the normal tissue toxicity of cMet CAR-T cells by co-loading the inducible IL-12 (iIL12) gene into cMet CAR-T cells.To this end, the mlL-12f gene, in which two subunits of mouse IL-12, p40 and p35, were linked by a flexible linker peptide, was synthesized (Fig. 37), and then cloned into the MSCV retrovirus so that it was positioned downstream of an inducible promoter consisting of six serial NFAT-responsive elements and a minimal IL-12 promoter. The cMet CAR gene was cloned so that it was constitutively expressed under the previously used PGK promoter, and it was designed so that the cMet CAR and inducible IL-12 (iIL12) genes were simultaneously expressed in CAR-T cells through a single retroviral transduction (Fig. 38). Fig. 38 shows the structure of the cMet-iIL12 CAR retrovirus (PGK, PGK promoter; 6xNFAT, six serial NFAT binding element promoter; iIL-12, inducible IL- 12). The cMet-iIL12 CAR vector produced was transduced into B6 mouse T cells to confirm CAR expression, and it was confirmed that cMet CAR was expressed well (Figure 39). The in vitro efficacy of these cMet-iIL12 CAR-T cells was confirmed through a co-culture experiment with E0771 cells, and the results are shown in Figure 40. The IL-12 secretion ability of cMet-iIL12 CAR-T cells was confirmed by detecting IL- 12 protein in the cell culture medium using ELISA. In particular, IL- 12 is known to increase IFN-Y production of T cells, and it was confirmed by ELISA that cMet-iIL12 CAR-T cells showed increased IFN-y production ability compared to existing cMet CAR-T cells. Furthermore, cMet-iIL12 CAR-T cells were confirmed to exhibit tumor-killing potency similar to that of cMet CAR-T cells. Next, we confirmed whether cMet-iIL12 CAR-T cells exhibit anti-tumor effects without toxicity even in vivo without lymphodepletion.According to the same schedule as in Figure 34, E07 group was injected subcutaneously into 5*1(T5) of B6 mice, and cMet-iIL12 CAR-T cells were administered intravenously into 5*1(T6) on the 7th day, and the results are shown in Figure 41. As a result of the test, it was confirmed that the group administered cMet-iIL12 CAR-T cells showed a significant tumor proliferation inhibitory effect, compared to the previous experiment where cMet CAR-T cells had no effect at all. Nevertheless, since no weight loss or death of mice was observed in these CAR-T cell administration groups, it was confirmed that the anti-tumor effect of CAR-T cells could be obtained through additional loading of IL-12 without lymphodepletion. However, in the above experiment, only partial remission was observed, which did not completely remove the tumor, and it was confirmed that additional efficacy enhancement was necessary. Therefore, this When the inventors additionally expressed the CTC28 protein, we wanted to confirm whether non-toxic functional enhancement was possible in a situation where lymphodepletion was not performed. To this end, we constructed a cMet-CTC28-iIL12 CAR retrovirus with inducible IL-12 added to the cMet-CTC28 CAR (Fig. 42) and transduced mouse T cells. We confirmed that CAR-CTC28 was well expressed in cMet-CTC28-iIL12 CAR-T cells (Fig. 43). Next, in order to confirm whether cMet-CTC28-iIL12 CAR-T cells showed a superior anti-tumor effect than cMet-iIL12 CAR-T cells without toxicity, these CAR-T cells were administered to B6 mice inoculated in the E07 group without lymphodepletion, and the results are presented in Fig. 44.As a result of the test, the group administered cMet-CTC28-iIL12 CAR-T cells significantly suppressed tumor proliferation compared to the group administered cMet-iIL12 CAR-T cells, and in particular, complete remission was observed in 4 out of 5 mice administered cMet-CTC28-iIL12 CAR-T cells, confirming an improved long-term survival rate compared to the cMet-iIL12 CAR-T cell administration group. Nevertheless, no weight loss or death was observed in the group administered cMet-iIL12 CAR-T cells, indicating that no increase in toxicity was observed. In order to further confirm the toxicity avoidance ability of the enhanced CAR-T cells, ALT and AST levels, which are indicators of hepatotoxicity in the blood, were measured, and the results are presented in Figure 45. The safety was again proven in the trial results, as only a small, transient initial increase was observed in both the cMet-iIL12 CAR-T cell and cMet— CTC28— iIL12 CAR-T cell groups. BUN, an indicator of renal toxicity, was also confirmed to remain within the normal range in both groups. Therefore, it was proven that cMet CAR-T cells additionally loaded with CTC28 and iIL12 can achieve a safe and potent anti-tumor effect in the absence of lymphodepletion. Example 6. In vitro and in vivo experiments of cMet CAR-T cells expressing constitutive IL-12 and CTC28 Since some previous reports have reported CAR-T cells designed to constitutively express IL-12 rather than inducibly by CAR signaling (Oncolmmunology, 2015, 4:3, e994446), a retrovirus (cMet-IL12) that constitutively expresses IL-12 together with the CAR protein was constructed. In addition, a retrovirus that constitutively expresses IL-12 together with CTC28 and the CAR protein was also constructed.At this time, two types of retroviruses were produced with different designs for the positions of IL- 12 and CTC28 (cMet- CTC28- IL12, cMet- IL12- CTC28), and the difference in efficacy depending on the position of these genes within the retroviral plasmid was confirmed (Fig. 46). These three types of CAR retroviruses were introduced into B6 mouse T cells to confirm the expression of CAR and CTC28, and the results are presented in Fig. 47. From Fig. 47, it was confirmed that CAR and CTC28 were well expressed in all three types of CAR-T cells. In order to confirm the function of the produced CAR-T cells, a co-culture experiment was performed with E07 cells to confirm the secretion ability of IL- 12 and IFN- x and the cell killing ability, and the results are presented in Figs. 48 and 49. As a result of the test, unlike cMet and cMet- CTC28 CAR-T cells that do not express IL-12, it was confirmed that the three newly produced CAR-T cells secrete IL-12, and it was confirmed that the production of IFN-x was significantly increased (Fig. 48). In the case of cell killing ability, it was observed similarly in all CAR-T cells (Fig. 49). In vivo experiments were conducted in B6 mice to see whether CAR-T cells that simultaneously express CTC28 and IL-12 show a superior anti-tumor effect even in cMet CAR-T cells that secrete persistent IL-12, and the results are shown in Fig. 50. As a result, it was confirmed that the inhibition of tumor proliferation was increased in both cMet- CTC28- IL12 {cMet-IL12-CTC28} groups compared to the cMet- IL12 group, as seen in the inducible IL-12 system (left graph in Fig. 50). In terms of toxicity, although an initial temporary weight loss (approximately 15%) was observed, the weight was quickly recovered thereafter, and no deaths due to this were observed, showing a manageable toxicity level (right graph of Figure 50).Therefore, it was confirmed that CAR-T cells expressing not only inducible IL-12 but also persistent IL-12 together with CTC284 can be produced as relatively safe and highly efficacious CAR-T cells. Example 7. Utilization of CTC28-iIL12 in CAR-T cells targeting other solid tumor antigens Next, we sought to determine whether the potentiated CAR-T cell design in which CTC28 and inducible IL-12 are simultaneously expressed can be generally applied to the production of safe and highly efficacious CAR-T cells not only in cMet but also in CARs targeting other antigens. Therefore, a novel antibody that binds to B7H3, which is known to be overexpressed in various solid tumors, was produced. To discover cross-reactive antibodies to mouse B7H3 and human B7H3, chickens were immunized with both mouse and human B7H3 proteins multiple times, and then an scFv phage display library was constructed from RNA from immune organs such as spleen and bone marrow isolated from these chickens. After that, phages that bind to human and mouse B7H3 proteins were selected through multiple biopanning processes, and antibody clones that bind to B7H3 protein were selected through ELISA using crude and purified scFv proteins. As a result, approximately 30 antibody clones that bind to human B7H3 and mouse B7H3 proteins with similar affinity were discovered, and among them, 10 had a Kd value of 10 in ELISA. 9 - IO -10More than 8 high affinity clones of the range were discovered (Fig. 51). Since the B7H3 target antibodies developed by the present inventors are ideal clones that bind to both human and mouse B7H3 with similar affinity, we secured raw materials suitable for the efficacy and toxicity tests in normal mice to be performed in the future in the present invention. Among them, clone #12 was used to construct an anti-B7H3 mouse CAR (hereinafter, B7H3 CAR) gene, which was cloned into a retroviral vector to produce B7H3 CAR and B7H3-CTC28 CAR retroviruses (Fig. 52). Afterwards, it was confirmed that both B7H3 CAR and B7H3-CTC28 CAR were expressed in B6 mouse T cells (Fig. 53). Subsequently, in vivo experiments were conducted to evaluate the efficacy and toxicity of these CAR-T cells. First, in order to confirm whether toxicity was observed under lymphodepletion of B7H3 CAR-T cells, 5*1(T5) E07 cells were injected subcutaneously into B6 mice, and total body radiation was performed for lymphodepletion on the 13th day. On the following day, the 14th, CD4 CAR-T cells and CD8 CAR-T cells were mixed at a 1:1 ratio of 2.5*1(T6) each, and a total of 5*1(T6) CAR-T cells were administered (Figure 54). Tumor proliferation and body weight changes were measured, and the results are shown in Figure 55. As a result of the test, some tumor inhibition effect was observed in the group administered B7H3 CAR-T cells, and a temporary initial body weight loss of about 10% was observed. On the other hand, the B7H3-CTC28 CAR-T cell administration group showed lethal toxicity in which all individuals died along with rapid body weight loss. On the other hand, in an experiment where CAR-T cells were administered without lymphodepletion, no weight loss or lethal toxicity was observed in the B7H3-CTC28 CAR-T cell administration group (Figure 56).However, in this case, no anti-tumor effect was observed in either the B7H3 CAR-T cell administration group or the B7H3-CTC28 CAR-T cell administration group. Therefore, it was confirmed that the CAR-T cells with enhanced efficacy when lymphodepleted also showed lethal toxicity in B7H3-targeted CAR-T cells. Next, in order to confirm whether the non-toxic anti-tumor effect observed in cMet CAR-T cells could be reproduced when inducible IL-12 and CTC28 were loaded together with B7H3 CAR-T cells without lymphodepletion, B7H3 CAR retroviruses loaded with iIL12 alone or iIL12 together with CTC28 were produced, and CAR and CTC28 expression was confirmed (Figs. 57 and 58). Next, as shown in Figure 54, B7H3-iIL12 CAR-T cells and B7H3— CTC28— iIL12 CAR— T cells were administered on the 14th day after E0771 injection without lymphodepletion. As a result, a significant anti-tumor effect was observed in the B7H3-iIL12 CAR-T cell administration group, and in particular, the B7H3-CTC28-iIL12 CAR-T cell administration group showed a more enhanced anti-tumor effect than the B7H3-iIL12 CAR-T cell administration group (complete remission was induced in 4 out of 5 administered mice) (Figure 59). In terms of toxicity, a temporary weight loss of about 10% was observed in both groups, but it soon recovered, showing a tolerable toxicity pattern. Therefore, comprehensively, CAR-T cells co-loaded with CTC28 and IL-12 in the absence of lymphodepletion demonstrated the potential for development into safe and highly efficacious CAR-T cells through two CAR targets. Example 8.In vitro efficacy evaluation of enhanced human cMet CAR-T cells In order to confirm whether the enhanced efficacy effect of the mouse CAR-T cells co-loaded with inducible IL-12 and CTC28 is also realized in human CAR-T cells, human cMet CAR-T cells co-loaded with human inducible IL-12 and human CTC28 were produced and their in vitro efficacy was evaluated.

[0019] (1) Human cMet CAR gene cloning and virus production To produce human cMet CAR-T cells, a human CAR gene using the m4A25 scFv was produced. The m4A25 clone antibody is a cross-reactive anti-cMet antibody that simultaneously binds to human c-Met and mouse cMet, and the human CAR gene was synthesized by linking the human CAR extracellular domain, transmembrane domain, and intracellular domain to the m4A25 scFv. To produce CAR-T cells with enhanced potency, a lentiviral plasmid loaded with human CTC28 and human inducible IL12 was produced along with the CAR gene. The produced CAR gene was cloned into a lentiviral vector to produce a CAR expression lentiviral plasmid. Figure 60 is a schematic diagram showing the structure of a human CAR gene constructed using a cMet scFv antibody (6xNFAT, six tandem NFAT binding element promoter; Min IL-2, minimal IL-2 promoter; h, human; PGK, PGK promoter; L, leader sequence; EC, extracellular domain; TM, transmembrane domain; cyt, cytoplasmic domain; P2A, P2A peptide; CTC28, CTLA4-CD28 chimera). For convenience, m4A25 was named A25. The constructed lentiviral plasmid was transfected into 293T cells together with the envelope and packaging plasmids, and the lentivirus secreted into the culture medium was harvested and concentrated by ultracentrifugation.

[0020] (2) Production of human cMet CAR-T cells

[0021] According to reports that when CD4:CD8 CAR- T cells are administered at a 1:1 ratio, an enhanced anti-tumor effect is observed in wVo (Sommermeyer et al. 2016, Turtle et al. 2016), some commercially available CAR-T supporters (Lisocabtagene maraleucel) are administered after separately manufacturing CD4 and CD8 CAR- T cells. In addition, according to a previous study by the present inventors (Blood (2012) 119(24):5678-87), the effect of CTC28 in T cell therapy is maximized when CD4 T cells and CD8 T cells are each transduced with CTC28 and these CD4 and CD8 T cells are mixed and administered at a 1:1 ratio. Therefore, in the present invention, CD4 CAR- T cells and CD8 CAR- T cells were separately manufactured and their efficacy was analyzed. CD4 T cells and CD8 T cells were separated from lymphocytes collected from normal blood through CD4 and CD8 MACS columns, and T cell activation beads (TransAct, Miltenyi Biotec) were added to activate the T cells for 24 hours. Afterwards, concentrated lentivirus was added to CD4 T cells and CD8 T cells, respectively, and transduced for 2 days. After culturing for approximately 5 days in CD4 and CD8 T cell culture medium containing IL-7 and IL-15, cell surface CAR expression in CD4 CAR-T cells and CD8 CAR-T cells, respectively, was analyzed by flow cytometry. Expression of CAR and CTC28 in human cMet CAR- T cells was analyzed using fluorescently labeled anti-chicken IgY Fab (ChlgY) antibody and fluorescently labeled anti-human CTLA4 antibody, respectively, and the results are shown in Figure 61 (ChlgY— FITC, FITC-labeled anti-chicken IgY Fab; CTLA4-PE, PE-labeled anti-human CTLA4 antibody; CD4, APC / Cyanine7-labeled anti-human CD4 antibody; CD8, PerCP / Cy5.5—labeled anti-human CD8 antibody). As a result of the test, the potentiated human CAR-T cells (cMet A25—BBz—CTC28—IL12) and plain CAR-T cells (cMet A25—BBz) showed 50-60% CAR expression in both CD4 and CD8 T cells, and it was confirmed that cell surface CTC28 was expressed only in the potentiated CAR-T cells. However, the expression levels of CAR and CTLA4 in the potentiated human cMet CD8 CAR-T cells were lower than in CD4 CAR-T cells.

[0022] (3) Confirmation of cMet expression in human breast cancer cell lines Since the efficacy of mouse cMet CAR- T cells was confirmed in mouse-derived breast cancer cell lines through a previous study by the present inventors, we sought to confirm the efficacy of human cMet CAR- T cells in human breast cancer cell lines. BT-20, a triple-negative breast cancer (TNBC) cell line, was selected as the target human breast cancer cell line, and c-Met expression on the BT20 surface was confirmed by flow cytometry using an anti-cMet antibody.

[0023] BT20 cells were treated with in-house purified anti-cMet adaptor (m4A25— hCk— L2— His) at 1 ug / 1 x 10 5 After treatment at 37 °C for 1 hour at a concentration of 10 cells, the cells were treated with a secondary antibody (anti—His—APC) at 4 °C for 30 minutes, and the percentage of positive cells (%) and MFI (Mean Fluorescence Intensity) were analyzed by flow cytometry to confirm the expression of cMet. The commercial antibody, anti-hHGFR / cMet antibody (R&D Systems)#, was used as a control and the results are shown in Figure 62.

[0024] BT-20 showed a cMet expression level of over 98% and a relatively high MFI for both the antibody m4A25, which specifically binds to cMet, and commercial antibodies. Therefore, the in vitro efficacy of human cMet CAR-T cells was confirmed using these tumor cells as target cells.

[0025] (4) Evaluation of in vitro tumor cell killing capacity and activity of potentiated human cMet CD4 and CD8 CAR-T cells

[0026] To compare the in vitro tumor-killing capacity and activity of potentiated human cMet CAR- T cells, each produced from CD4 and CD8 T cells, cytotoxicity against target cells and IL12 and IFN-x secretion capacity were evaluated compared with those of plain human cMet-BBz CAR- T cells. Human cMet CD4 and CD8 CAR- T cells were co-cultured with luciferase-transduced cMet-positive human breast cancer cell line BT-20 for 16 hours, and the tumor-killing capacity was analyzed by measuring luciferase expression in surviving cells. The results are shown in Fig. 63. Fig. 63 is a graph showing the cytotoxicity of human cMet CD4 and CD8 CAR- T cells (E, effector) against BT-20, a target cell (T, target). The test results confirmed that the cytotoxicity was increased in the potentiated human cMet CD4 and CD8 CAR- T cells compared to the untransduced (UT) T cells as a negative control group, and plain CD4 and CD8 CAR-T cells. Since the potentiated CAR- T cells applied an inducible IL-12 platform that allows IL-12 to be expressed only in activated CD4 and CD8 CAR- T cells, the potentiated human cMet CAR- T cells (E) and the target cells BT-20 (T) were co-cultured for 24 hours at an E:T ratio of 1:5. The amounts of IL-12 and IFN-x secreted into the supernatant were confirmed using ELISA. For comparison, plain human cMet-BBz CAR- T cells, untransduced (UT) T cells as a negative control group, and target cells were used. Only in potentiated human cMet CAR-T cells, significant IL-12 expression was observed when co-cultured with tumor cells, BT-20 (Fig. 64). In potentiated CD8 CAR-T cells, IL-12 expression was observed below the detection limit, which is believed to be a result of the low CAR expression level of CD8 CAR-T cells.

[0027] The results of IFN-x analysis in the BT-20 cell line are shown in Fig. 65. A significant increase in the secretion amount was observed in potentiated human cMet CD4 CAR-T cells compared to plain cMet CAR-T cells, and the secretion amount was also increased in potentiated human CD8 CAR-T cells compared to plain CAR-T cells, suggesting that a small amount of IL-12 promoted IFN-x production. This result suggests that the activation of CAR-T cells was significantly enhanced by the additional loading of potentiated CTC28 and IL-12. Example 9. Production of CTC28 and IL-12-loaded CAR-T cells and verification of in vitro activity in human B7H3 CAR-T cells As another example to confirm whether the concept of potentiated CAR-T cells using mouse CAR-T cells can also be implemented in human CAR-T cells, a human CAR gene (hB7H3 CAR; hB7H3-BBz) was produced using three anti-B7H3 scFvs (#12, #2—25, #2—54), including the #12 clone used in mouse CAR-T cells among the newly discovered anti-B7H3 antibody scFvs (Fig. 51). The produced CAR gene was cloned into a lentiviral vector to produce a CAR-expressing lentiviral plasmid (Fig. 66). In addition, for the production of CAR-T cells with enhanced efficacy, a lentiviral plasmid (hB7H3-CTC28-iIL12 CAR) containing human CTC28 and human inducible IL-12 together with the hB7H3 CAR gene was also produced (Fig. 67). For convenience, #12, #2-25, and #2-54 were designated as #12 BBZ-C12, #25 BBz— C12, and #54 BBz— C12, respectively. The produced lentiviral plasmids were transfected into 293T cells together with packaging plasmids, and the lentivirus secreted into the culture medium was harvested and concentrated by ultracentrifugation.Afterwards, T cells were separated from lymphocytes collected from normal blood through a CD4 / CD8 MACS column, and T cell activation beads (TransAct, Miltenyi) were added to activate the T cells for 24 hours, and then concentrated lentivirus was added to transduce them for 2 days. After culturing them for about 5 days in a T cell culture medium containing IL-7 and IL-15, the cell surface CAR expression was analyzed by flow cytometry, and the appropriate expression of CAR and CTC28 was confirmed (Fig. 68). To evaluate the in vitro efficacy of the manufactured potentiated CAR-T cells, the cell killing ability and cytokine secretion ability were confirmed, and the results are presented in Figs. 69-65. As a result, when MDA-MB-231 cells, a human breast cancer cell line, were used as target cells, it was confirmed that the potentiated CAR-T cells showed an effective tumor killing effect similar to the preceding hB7H3-BBz CAR-T cells (Fig. 69). Since the potentiated CAR-T cells were applied with an inducible IL-12 platform that allows IL-12 to be expressed only in activated CAR-T cells, the expression of IL-12 and IFN-x was confirmed using ELISA after co-culture of the potentiated CAR-T cells and the target cells, MDA-MB-231 cells, for 24 hours at an E:T ratio of 1:5. In the three types of potentiated CAR-T cells, significant IL-12 expression was confirmed only when co-cultured with tumor cells, confirming the inducible secretion ability of IL-12 (Fig. 70).

[0028] For IFN- x, similar to the results with mouse CAR-T cells, a significant increase in secretion was observed in the potentiated CAR-T cells compared to the preceding hB7H3 CAR-T cells, suggesting that the activation of CAR-T cells was greatly enhanced by the additional loading of potentiated CTC28 and IL-12 (Fig. 71). Therefore, the feasibility of developing CTC28 and IL-12 co-loaded CAR-T cells, which has been proven in mouse CAR-T cells, was successfully demonstrated in human B7H3 CAR-T cells. Example 10. In vitro efficacy evaluation of potentiated humanized B7H3 CAR-T cells We wanted to confirm in vitro whether this potentiated human B7H3 CAR-T cell design also enhances the efficacy of human B7H3 CAR-T cells using humanized anti-37H3 scFv, which has a higher possibility of practical application.

[0029] (1) Cloning of humanized B7H3 CAR gene with enhanced efficacy and virus production The three anti-B7H3 svFvs (#12, #2-25, #2-54) were each used to construct a lentiviral plasmid loaded with human CTC28 and human inducible IL12 together with the human CAR gene using humanized anti-B7H3 scFvs (z32, z25, zl6). Figure 72 is a schematic diagram showing the structure of a human CAR gene constructed using humanized B7H3 antibody scFv (6xNFAT, six tandem NFAT binding element promoter; Min IL-2, minimal IL-2 promoter; h, human; PGK, PGK promoter; P2A, P2A peptide; CTC28, CTLA4-CD28 chimera). After transfecting 293T cells with the produced lentiviral plasmid together with the envelope and packaging plasmids, the lentivirus secreted into the culture medium was harvested and concentrated through ultracentrifugation.

[0030] (2) Production of humanized B7H3 CD4 and CD8 CAR-T cells with enhanced efficacy As described above, the effect of CTC28 is maximized when CD4 T cells and CD8 T cells are each transduced with CTC28 and these CD4 and CD8 T cells are mixed and administered in a 1:1 ratio. Therefore, in this example, CD4 CAR-T cells and CD8 CAR-T cells were separately produced to analyze their efficacy. CD4 T cells and CD8 T cells were each separated from lymphocytes collected from normal blood through CD4 and CD8 MACS columns, and T cells were activated for 24 hours by adding T cell activation beads (TransAct, Miltenyi Biotec). Afterwards, the concentrated lentivirus was added to CD4 T cells and CD8 T cells, respectively, and transduction was performed for 2 days. After culturing for about 5 days in CD4 and CD8 T cell culture medium containing IL-7 and IL-15, the expression of cell surface CARs in CD4 CAR-T cells and CD8 CAR-T cells was analyzed by flow cytometry, respectively. The expression of CAR and CTC28 in three types of humanized B7H3 CAR-T cells was analyzed using fluorescently labeled anti-human IgG, F(ab')2 antibody and fluorescently labeled anti-human CTLA4 antibody, respectively, and the results are shown in Figure 73 (F(ab')2-FITC, FITC-labeled anti-human IgG F(ab')2; CTLA4-PE, PE-labeled anti-human CTLA4 antibody; CD4, APC / Cyanine7-labeled anti-human CD4 antibody; CD8, PerCP / Cy5.5-labeled anti-human CD8 antibody).The test results showed that three types of potentiated humanized CAR-T cells (z32-BBz-CTC28-IL12, z25-BBz-CTC28-IL12, zl6—BBz—CTC28—IL12) showed similar levels of CAR expression compared to simple humanized B7H3-BBz CAR-T cells (z32-BBz, z25-BBz, zl6-BBz) in both CD4 and CD8 T cells, and that cell surface CTC28 was expressed only in the potentiated CAR-T cells.

[0031] (3) In vitro tumor cell killing capacity and activity evaluation of humanized B7H3 CD4 and CD8 CAR-T cells with enhanced efficacy

[0032] To evaluate the in vitro efficacy of three enhanced humanized B7H3 CAR-T cells, each generated from CD4 and CD8 T cells, cytotoxicity against target cells and IL12 and IFN-x secretion capacity were compared with those of simple humanized B7H3-BBz CAR-T cells.

[0033] B7H3 CD4 and CD8 CAR— T cells were co-cultured with a B7H3-positive human breast cancer cell line (MDA-MB-231) expressing the luciferase gene for 16 hours, and the tumor killing ability was analyzed by measuring the luciferase expression of the surviving cells, and the results are shown in Fig. 74. Fig. 74 is a graph showing the cytotoxicity of B7H3 CD4 and CD8 CAR- T cells (E, effector) against target cells (T, target). The test results confirmed that the cytotoxicity was increased in all humanized B7H3 CAR-T cells compared to the untransduced (UT, untransduced) T cells, which was the negative control. It was confirmed that the potency of the three types of humanized B7H3 and the simple humanized CD4 and CD8 CAR-T cells effectively killed target cells in a similar dose-dependent manner on the cell number. Since the potentiated CAR-T cells are applied with an inducible IL-12 platform that allows IL-12 to be expressed only in activated CD4 and CD8 CAR-T cells, the potentiated humanized CAR-T cells (E) and target cells, MDA-MB-231 cells (T), were co-cultured for 24 hours at an E:T ratio of 1:5, and the amounts of IL-12 and IFN-x secreted into the supernatant were confirmed using ELISA.

[0034] Only in the three types of potency-enhanced B7H3 CD4 and CD8 CAR- T cells, significant IL-12 expression was confirmed when co-cultured with tumor cells, and the results of the IL-12 induction secretion ability are shown in Figure 75.

[0035] The results of the analysis of IFN- x are shown in Fig. 76. According to Fig. 76, a significant increase in the secretion of IFN- x was observed in both enhanced-efficacy humanized B7H3 CD4 and CD8 CAR-T cells compared to simple humanized B7H3 CAR- T cells, suggesting that the activation of CAR- T cells is greatly enhanced by the additional loading of enhanced-efficacy CTC28 and IL-12. In summary, many solid tumor antigens, such as cMet and B7H3, are expressed at increased levels in tumors, but most of them are also expressed at low levels in normal tissues. Therefore, in a situation where the efficacy of CAR-T cells against solid tumors must be increased, antigens expressed in these normal tissues may cause CAR-T cytotoxicity. This toxicity has the problem of being aggravated by lymphodepletion, which is currently commonly performed to enhance efficacy. In the present invention, it was demonstrated that a CAR-T cell system loaded with IL-12 or IL-12 and CTC28 can be used as a CAR-T cell that exhibits excellent efficacy while avoiding toxicity when used in a situation without lymphodepletion.

[0036] [Table 1] Sequences of anti-cMet antibodies

[0037] * In the above table, the underline indicates CDR.

[0038] [Table 2] Sequence of mouse CAR domain

[0039] * In the CAR sequence in the table above, the mCD8a EC sequence is indicated in bold, and the h41BB cyt sequence is indicated in italics.

[0040] [Table 3] Sequence of mouse CTLA4-CD28 chimera

[0041] [Table 4] Sequence of mouse single-chain IL-12

[0042] * In the above table, the underline indicates the (G4S)3 linker sequence.

[0043] [Table 5] Sequence of anti-B7-H3 antibody

[0044] [Table 6] Sequence of human B7H3 CAR The sequence is indicated in italics.

[0045] [Table 7] Sequence of human CTLA4-CD28 chimera I hCD28 cyt I VRSKRSLRLLHSDYMN MTPRRPGPTRKHYQPYAPPRDFAAYRS I 84 I

[0046] *h, human; EC, extracellular domain; TM, transmembrane domain; cyt, cytoplasmic domain

[0047] * The underline in the above table indicates the hCTLA4 TM sequence.

[0048] [Table 8] Sequence of human single-chain IL-12

[0049] * In the above table, the underline indicates the G6S linker sequence.

[0050] [Table 9] Sequence of human cMet CAR

[0051] *h, human; m, mouse; EC, extracellular domain; TM, transmembrane domain; cyt, cytoplasmic domain

[0052] [Table 10] Sequences of humanized anti-B7H3 antibodies

[0053] [Table 11] Sequence of humanized B7H3 CAR domain

[0054] * In the CAR sequences in the table above, the hCD8 EC sequence is indicated in bold, and the h41BB cyt sequence is indicated in italics.

Claims

Scope of the claim

1. A construct comprising (i) a first gene encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain, an extracellular domain, a transmembrane domain, and an intracellular domain, and a second gene encoding IL-12; or (ii) a third gene encoding a fusion protein comprising a CTLA4 (Cytotoxic T Lymphocyte Antigen-4) protein or a domain thereof, and CD28 or a domain thereof (CTLA4-CD28 fusion protein). ［ Claim 1, wherein the antigen binding domain of the chimeric antigen receptor comprises an antibody or antigen binding fragment thereof that specifically binds to one or more antigens selected from the group consisting of: 4-1BB, BCMA, BAFF, B7-H3, B7-H6, CA9, CTAG1B, CEA, cyclin, cyclin A2, cyclin Bl, CCL-1, CCR4, CD3, CD4, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD52, CD58, CD62, CD79A, CD79B, CD80, CD123, CD133, CD138, CD171, CSPG4, CLDN18, CLDN18.2, CLDN6, CTLA-4, c-Met, DLL3, EGFR, tEGFR, EGFRvIII, EPG-2, EPG-40, ephrin B2, EPHA2, estrogen receptor, Fc receptor, FCRL5, FGF23, FBP, FOLR1, FOLR2, GD2, ganglioside GD3, gplOO, GPC3, GPCR5D, GM- CSF, Her2 / neu, Her3, Her4, erbB dimers, HMW— MAA, HBsAg, HLA- Al, HLA-A2, IL-22Ra, IL— 13Ra2, ICOS, IGF-1 receptor, integrin avP6, interferon receptor, IFNx, IL-2R, IL-4R, IL-5R, IL-6R, IL-17RA, IL-31R, IL-36R, kdr, Ll-CAM, LI— CE7 epitope of CAM, LRRC8A, Lewis Y, LAG3, MAGEA1, MAGEA3, MAGEA6, MAGEA1O, MSLN, CMV, MUC1, NKG2D ligand, MART-1, NGF, NCAM, NRP-1, NRP-2, carcinoembryonic antigen, PD-L1, FRAME, progesterone receptor, prostate-specific antigen, PSCA, PSMA, RANKL, ROR1, SLAMF7, survivin, TPBG, TAG72, TRP1, TRP2, and Wilms' tumor 1 (WT1). [

3. A construct according to claim 1 or 2, wherein the antigen binding domain of the chimeric antigen receptor comprises an antibody or antigen binding fragment thereof that specifically binds to c-Met or B7-H3.

4. In any one of claims 1 to 3, the antigen binding domain of the chimeric antigen receptor comprises a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 5, and a heavy chain 55 comprising an amino acid sequence of SEQ ID NO:

6. A heavy chain variable region comprising a CDR3; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9; or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 36, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 41; or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 45, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 46, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 47; And a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 48, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 49, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 50; or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 54, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 56; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 57, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 59; or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 101, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 102, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 103;And a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 104, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 105, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 106; or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 110, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 111, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 112; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 113, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 114, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 115; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 119, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 120, and an amino acid sequence of SEQ ID NO: 121; 56 A construct comprising a heavy chain variable region comprising a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 122; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 122, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 123, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

124. ［

5. A construct according to any one of claims 1 to 4, wherein the intracellular domain of the chimeric antigen receptor comprises an intracellular signaling domain and / or a costimulatory domain.

1. A construct according to any one of claims 1 to 5, wherein the chimeric antigen receptor comprises an amino acid sequence of SEQ ID NO: 11, 61, 68, 75, 91, 126, 133 or 140; or an amino acid sequence having at least 90% identity thereto. ［

7. A construct according to any one of claims 1 to 6, wherein the second gene encoding IL-12 comprises a first coding sequence encoding IL-12 p40 and a second coding sequence encoding IL-12 p35.

8. A construct according to any one of claims 1 to 7, wherein the CTLA4-CD28 fusion protein comprises an extracellular domain of CTLA4 and an intracellular domain of CD28.

9. A construct according to any one of claims 1 to 8, wherein the CTLA4-CD28 fusion protein comprises an amino acid sequence of SEQ ID NO: 24 or 81; or an amino acid sequence having at least 90% identity thereto. ［ Claim 10] In any one of claims 1 to 9, IL-12 comprises an amino acid sequence of SEQ ID NO: 29 or 86; or an amino acid sequence having at least 90% identity therewith; A CTLA4-CD28 fusion protein comprising an amino acid sequence of SEQ ID NO: 24 or 81; or an amino acid sequence having at least 90% identity thereto. ［

11. A construct according to any one of claims 1 to 10, wherein the first gene, the second gene, and the third gene are linked to each other by a 2A peptide sequence or an IRES sequence, or the first gene, the second gene, and the third gene are each operably linked to independent promoters; or two of the first gene, the second gene, and the third gene are operably linked to one promoter, and the remaining gene is operably linked to a separate promoter. 57

12. A vector comprising a construct according to any one of claims 1 to 11.

13. The vector of claim 12, wherein the vector is a viral vector or a non-viral vector.

14. In claim 13, the viral vector is any one selected from the group consisting of a retrovirus, a lentivirus, an adenovirus, a herpes virus, an adeno-associated virus, and a vaccinia virus.

15. An immune cell introduced with a construct according to any one of claims 1 to 11 or a vector comprising the construct.

16. An immune cell comprising a protein expressed from a construct or a vector comprising the construct according to any one of claims 1 to 11.

17. An immune cell comprising (i) a first gene encoding a chimeric antigen receptor comprising an antigen binding domain, an extracellular domain, a transmembrane domain, and an intracellular domain, and a second gene encoding IL-12; or (ii) a third gene encoding a fusion protein comprising a CTLA4 protein or a domain thereof, and CD28 or a domain thereof (CTLA4-CD28 fusion protein).

18. An immune cell expressing (i) a chimeric antigen receptor comprising an antigen binding domain, an extracellular domain, a transmembrane domain, and an intracellular domain, and IL-12; or (ii) a fusion protein comprising CTLA4 protein or a domain thereof, and CD28 or a domain thereof (CTLA4-CD28 fusion protein).

19. An immune cell according to any one of claims 15 to 18, wherein the immune cell is a T cell, an NK cell, an NKT cell, a macrophage, or a combination thereof.

20. A composition comprising a construct according to any one of claims 1 to ii; a vector comprising the construct; an immune cell into which the construct or the vector has been introduced; or an immune cell according to any one of claims 15 to 18.

21. A composition according to any one of claims 1 to ii; comprising the composition. 58 A pharmaceutical composition for treating cancer, comprising a vector; an immune cell into which the construct or the vector has been introduced; or an immune cell according to any one of claims 15 to 18; and a pharmaceutically acceptable carrier.

22. A pharmaceutical composition according to claim 21, wherein the immune cells are administered without lymphodepletion.

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